4,4,13,13-tetraethoxy-3,14-dioxa-8,9-dithia-4,13-disilahexadecane

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No reproductive toxicity data are available for 4,4,13,13-Tetraethoxy-3,14-dioxa-8,9-dithia-4,13-disilahexadecane ("S2"; CAS No. 56706-10-6, EC No. 260-350-7). Thus, the extended one-generation reproductive toxicity study (EOGRTS) for bis[3-(triethoxysilyl)polysulfides (CAS No. 211519-85-6, EC No. 915-673-4; Charles River Laboratories, 2022) is read across for the registered substance.

In the read-across EOGRTS on polysulfides conducted according to OECD Test Guideline 443 and in compliance with GLP, four groups of Wistar Han rats were investigated: F0 (parental), F1 pups, F1 Cohort 1A, and F1 Cohort 1B, with the latter added primarily to assess endocrine and reproductive tissues via necropsy and organ weight measurement, with preserved tissues retained for possible microscopic examination. Based on the findings, the overall study NOAELs were identified as follows:

- Systemic, males: 300 mg/kg bw/day, based on test item-related and adverse kidney histopathology in F0 males at 1000 mg/kg bw/day

- Systemic, females: 100 mg/kg bw/day, based on test item-related and adverse kidney histopathology in F0 females at 300 mg/kg bw/day

- Reproduction: at least 1000 mg/kg bw/day, based on the lack of test item-related reproductive effects in the F0, F1 Cohort 1A, and F1 Cohort 1B animals, and lack of reproductive / developmental effects in the F1 pups.

Link to relevant study records

Reference

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 Dec 2020 (study initiation) to 9 Jul 2021 (in-life completion)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

GLP compliance:

yes

Limit test:

no

Justification for study design:

- Premating exposure duration for parental (F0) animals: 10 weeks per OECD Test Guideline 443
- Basis for dose level selection: Based on a previous OECD Test Guideline 414 dose range finder in which the maternal and foetal NOAELs were at least 1000 mg/kg bw/day
- Inclusion of F1 Cohort 1B: Primarily to primary assess endocrine and reproductive tissues at necropsy, with tissues process through paraffin blocks for possible histopathology depending on the F1 Cohort 1A findings
- Neurotoxicity F1 Cohorts 1A and 1B (treated PNDs 21-90 or 97) Changes in gait and posture, presence of clonic or tonic movements, stereotypy or bizarre behaviour were assessed as part of the detailed clinical observations
- Immunotoxicity F1 Cohort 1A (treated PNDs 21-90): Spleen immunophenotyping was performed at necropsy
- Route of administration: Oral gavage selected since expected route of human exposure
- Species / strain: Han Wistar rat chosen since this strain is an accepted rodent species for non-clinical toxicity testing by regulatory agencies
- Vehicle: Justification not specifically specified, but the test item was shown to be stable and homogeneous in olive oil at 21 °C
- Number of animals: At (F1 cohorts, 20/sex/dose) or slightly higher than (F0 parents, 25/sex/dose) than specified in OECD Test Guideline 443

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK, Margate, Kent, UK
- Females: F0 sexually mature, F1 Cohort 1A and 1B aged PND 21 (assessed for sexual maturation)
- Age at study initiation (dosing): F0 males: 8-9 weeks, F0 females: 7-8 weeks; F1 Cohorts 1A and 1B: PND 21
- Weight at study initiation (dosing): F0 males: 228-342 g, F0 females: 142-204 g; F1 Cohort 1A males: 39-66 g, F1 Cohort 1A females: 33-68 g; F1 Cohort 1B males: 37-69 g, F1 Cohort 1B females: 32-63 g
- Fasting period before study: No
- Housing: For all animals, appropriately sized polycarbonate cages with solid bottoms, appropriate bedding provided
> F0, Cohort 1A, 1B animals: Up to 3/sex/dose together
> F0 males few days prior to mating: Transferred to individual solid bottomed cages
> F0 females for mating: Transferred to male individual cages
> F0 males after mating: Re-housed with their original cages mates until termination from Study Day (SD) 114.
> F0 females after either positive mating sign or at end mating period: Transferred to individual solid bottomed cages and retained in this type of cage until termination LDs 22-24. Appropriate nesting material provided
> F1 pups: Housed with their dams until litter culling on PND 4 (culled pups terminated), with F1 pups selected for F1 Cohorts 1A and 1B on PND 21 housed
- Diet, F0, F1 Cohorts 1A and 1B: Special Diet Services VRF-1, ad libitum except during designated procedures
- Water F0, F1 Cohorts 1A and 1B: Public supply tap water, ad libitum via water bottles except during designated procedures
- F1 pups: Nursing PNDs 0-21
- Acclimation period: Two weeks prior to start of dosing

ENVIRONMENTAL CONDITiONS
- Temperature (°C): 17-36
- Humidity (%): 30-129, excursions outside 30-70 transient, did not affect integrity or outcome of study
- Air changes (per hr): Ten (10) or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1 Dec 2020 To: 9 Jul 2021

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared at least weekly, with the aliquots removed from the freezer and allowed to warm to room temperature and stirred for at least 30 minutes before dosing. The doses were given via syringe with an attached gavage cannula. If a female was found to be in the process of littering or had recently littered at the time of scheduled dose administration, the animal was not dosed on that day.

VEHICLE
- Justification for use and choice of vehicle: Stability and homogeneity testing - Dose selection rationale: Based on OECD Test Guideline 414 dose range finder, with a maternal and foetal NOAELs of at least 1000 mg/kg bw/day (See “Justification for study design” above)
- Rationale for animal assignment: Random
- Fasting period before blood sampling for clinical biochemistry: No
- Dosing concentrations: 0, 25, 75, 250 mg/mL
- Amount of vehicle: Amount needed to derive dosing concentrations, dosing volume of 4 mL/kg for all F0 and F1 cohort groups.

Details on mating procedure:

F0 pairing was on a 1 male to 1 female basis. During the evening (after 5 pm) of Day 71 of dosing, females were housed with their allocated co-group male partner for up to 14 days. The day of detection of a copulatory plug in situ and/or of sperm in the lavage was designated Gestation Day 0. If evidence of mating was not observed by the end of the 14-day pairing period, the female was separated from the male the morning following the last night of pairing and treated as if mating had occurred during that night. F1 Cohort 1A and 1B animals were not mated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For each dose/stratum as relevant, five formulation samples were collected for all groups on Day 1, Week 12 and Week 25, with two samples analysed via ultra-performance liquid chromatography (UPLC) using a validated analytical procedure. The control samples were obtained from the middle stratum, while those for the treated groups were take from the top, middle, and bottom strata.

Duration of treatment / exposure:

- F0, males: 10 weeks prior to mating and continuing throughout and after mating until the day before termination from SD 114
- F0, females: 10 weeks prior to mating and continuing throughout mating and gestation until at least Lactation Day (LD) 21
- F1 pups (PNDs 0-21) were potentially exposed in utero or during lactation
- F1 Cohort 1A: From PND 21 until the day before necropsy (at least PND 90)
- F1 Cohort 1B: From PND 21 until the day before necropsy (at least PND 97)

Frequency of treatment:

Once daily

Details on study schedule:

F1 litter standardisation, PND 4:
- Litter size was standardised to 8 pups per litter (and if possible, comprised of 4 males and 4 females). Any additional pups were selected at random for use in blood sample or necropsy procedures. Culled pups were terminated on PND 4, after (as relevant) any clinical chemistry.

Cohort 1A and 1B selection, F1 PND 21:
- Among the non-culled F1 pups, 40 males and 40 females were selected at random on PND 21 and identified on that day for post-weaning treatment and assessments (F1 Cohorts 1A and 1B), nominally up to 4 males and 4 females from each litter.
- Selected pups were removed from their mother on PND 21 and housed in their new cages
- Unselected pups remained with their dam until maternal (and pup) termination on LDs 22-24.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

- F0: 25/sex/dose
- F1 pups after PND 4 cull, 8 pups//litter, with ideally comprised of 4/sex//litter
- Cohort 1A: 20/sex/dose
- Cohort 1B: 20/sex/dose

Control animals:

yes

Details on study design:

- Dose selection rationale: Based on OECD Test Guideline 414 dose range finder, with a maternal and foetal NOAELs of at least 1000 mg/kg bw/day (See “Justification for study design” above)
- Rationale for animal assignment: Random
- Fasting period before blood sampling for clinical biochemistry: No

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test item related but non-adverse F0 clinical signs were observed at 300 and 1000 mg/kg bw/day, but not at 100 mg/kg bw/day.

There was an increase in the incidence of:
- Ploughing at 1000 mg/kg bw/day
- Salivation at 300 and 1000 mg/kg bw/day

Both of these signs were predominantly observed immediately post-dose, with only a few instances at the 0-1 hour post-dosing timepoint. These findings were most likely due to the taste of the formulations, and thus were considered non-adverse.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths in F0 animals.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related effects on body weight or body weight gain were observed in the F0 animals up to 1000 mg/kg bw/day. Any statistical significance seen was due to individual variation within dose groups.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related effects on food consumption were identified in the F0 animals up to 1000 mg/kgbw/day.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

Water consumption by F0 animals was monitored by visual inspection of the water bottles. No inter-group differences were noted.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no effects on haematology parameters in the F0 animals up to 1000 mg/kg bw/day. All values were within normal biological variation when compared with the control group.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no effects on clinical chemistry parameters in the F0 animals up to 1000 mg/kg bw/day. All values were within normal biological variation when compared with the control group.

Endocrine findings:

no effects observed

Description (incidence and severity):

No test item-related effects on T4 concentrations in plasma were observed in the F0 animals up to 1000 mg/kg bw/day. The F0 TSH findings will be added when available.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no effects on urinalysis parameters in the F0 animals up to 1000 mg/kg bw/day. All values were within normal biological variation when compared with the control group.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

For the F0 animals, there were no behaviour changes mentioned in the study results for these parameters: gait and posture, presence of clonic or tonic movements, stereotypy, or bizarre behaviour up 1000 mg/kg bw/day, as assessed as part of the detailed clinical examinations.

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):

In the F0 animals, there were test item-related and adverse effects in the kidney at 1000 mg/kg bw/day in both sexes and at 300 mg/kg bw/day in females, primarily based on the tubular basophilia. The collective renal findings across both doses included tubular basophilia (minimal to moderate), tubular dilatation (minimal to mild), mononuclear cell infiltration (minimal to mild), papillary cellular debris (minimal), mineralisation (minimal to mild), and (in males only and not relevant to humans) minimal to mild accumulation of hyaline droplets in cortical tubules.

Tubular basophilia comprised cortical tubules lined by epithelial cells with cytoplasm that was reduced in amount and/or more basophilic than normal, with nuclei that were larger than normal with dispersed chromatin and with occasional degenerate cells. There also was fibrosis in the basement membranes and surrounding interstitial tissue. Some foci included interstitial mononuclear cell infiltration and/or dilatation of basophilic tubules, that were recorded as separate findings. Papillary cellular debris comprised amorphous or globular, slightly eosinophilic deposits in the lumen of papillary tubules, and/or focal increased cellularity of the epithelium of individual papillary tubules.

Tubular basophilia in the kidney at the mild or moderate grades (considered adverse at mild or higher) occurred only at 1000 mg/kg bw/day in both sexes and at 300 mg/kg bw/day in females. Noting, the average grade of tubular basophilia at 300 and 1000 mg/kg bw/day was higher in females than in males, and papillary cellular debris was more prevalent in females at these doses.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Coagulation findings:
There were no effects on coagulation parameters in the F0 animals up to 1000 mg/kg bw/day. All values were considered to be within normal biological variation when compared with the control group.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No test item-related effects on maternal oestrous cycles were identified for the F0 animals up to 1000 mg/kg bw/day.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No test item-related effects on sperm motility, sperm or spermatid counts, or sperm morphology were seen in the F0 males up to 1000 mg/kg bw/day.

Reproductive performance:

no effects observed

Description (incidence and severity):

No test item-related effects on maternal reproduction performance or maternal care were observed in the F0 animals up to 1000 mg/kg bw/day, as based on male and female reproductive (mating, fertility, and pregnancy) indices, gestation length, female post-implantation loss, and on observed deficiencies in maternal care.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

read-across polysulphides

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: Test item-related and adverse renal histopathology at 1000 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

polysuphides read across

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: Test item-related and adverse renal histopathology from 300 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

polysulphides read across

Sex:

male/female

Basis for effect level:

other: Reproductive function and performance (males / females treated 10 weeks pre-mating until SD 114+ / LD 21)

Remarks on result:

other: Lack of test item-related effects up to 1000 mg/kg bw/day

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

F1 PUPS:
In the absence of mention in the study report results, the reviewer considers that milk was present in the F1 pup stomachs and there were no external gross abnormalities, based on daily observations through PND 4 and then PNDs 7, 14, and 21 up to 1000 mg/kg bw/day of parental F0 treatment.

F1 COHORTS:
Test item-related but non-adverse clinical signs were observed in the Cohorts 1A and 1B animals at 300 and 1000 mg/kg bw/day, but not at 100 mg/kg bw/day.

There was an increase in the incidence of salivation at 300 and 1000 mg/kg/day in the Cohort 1A and Cohort 1B animals.

This clinical sign was predominantly observed immediately post-dose, with only a few instances at the 0-1 hour post-dosing timepoint. The salivation was most likely due to the taste of the formulations, and thus considered non-adverse.

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Description (incidence and severity):

F1 PUPS AND COHORTS:
There were no unscheduled deaths in F1 pups or F1 Cohorts 1A and 1B animals up to 1000 mg/kg bw/day treatment (parental F0 or direct cohort).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Test item-related but non-adverse effects on body weight and body weight gain were noted in male F1 pups at 1000 mg/kg bw/day. The effects in males at 100 and 300 mg/kg bw/day and in females up to 1000 mg/kg bw/day were considered unrelated to the test item.

At 1000 mg/kg bw/day, there was a 7% lower body weight gain for the F0 male pups when compared with the control group, and a 5% lower body weight by LD 21. The study report does not indicate this finding is adverse.

F1 COHORTS:
No test item-related effects on body weight or body weight gain were observed in the F1 Cohort 1A or Cohort 1B animals up to 1000 mg/kg bw/day. Any statistical significance seen was due to individual variation within the dose groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

F1 COHORTS:
No test item-related effects on food consumption were identified in the F1 Cohorts 1A or 1B animals up to 1000 mg/kg/day.

F1 PUPS:
Not relevant (nursing).

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

F1 COHORTS:
Water consumption by F1 Cohort 1A and 1B animals was monitored by visual inspection of the water bottles. No inter-group differences were noted.

F1 PUPS:
Not relevant (nursing).

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

F1 COHORT 1A:
There were no effects on haematology parameters in the F1 Cohort 1A animals up to 1000 mg/kg bw/day. All values were within normal biological variation when compared with the controls.

F1 PUPS AND F1 COHORT 1B:
Not examined.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no effects on clinical chemistry parameters in the F1 Cohort 1A animals up to 1000 mg/kg bw/day. All values were within normal biological variation when compared with the controls.

F1 PUPS AND F1 COHORT 1B:
Not examined.

Urinalysis findings:

no effects observed

Description (incidence and severity):

F1 COHORT 1A:
There were no effects on urinalysis parameters in the F1 Cohort 1A animals up to 1000 mg/kg bw/day. All values were within normal biological variation when compared with the controls.

F1 PUPS AND F1 COHORT 1B:
Not examined.

Sexual maturation:

no effects observed

Description (incidence and severity):

F1 COHORTS:
There were no test-item related effects on sexual maturation or the time to first oestrous cycle in F1 Cohorts 1A or 1B up to 1000 mg/kg bw/day.

F1 PUPS:
Not relevant / examined.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

F1 PUPS:
No test item-related effects on AGD in either sex were observed on PND 1 up to 1000 mg/kg bw/day of parental F0 treatment..

F1 COHORTS:
Not relevant / examined for the F1 Cohort 1A and 1B animals.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

F1 PUPS:
No test item-related effects on nipple retention in male pups were observed on PND 13 up to 1000 mg/kg bw/day of parental F0 exposure.

F1 COHORTS 1A and 1B:
Not relevant / examined for the F1 Cohort 1A and 1B animals.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

F1 PUPS:
No test item-related mean organ weight differences were seen in the F1 pups up to 1000 mg/kg bw/day of parental F0 treatment, as compared to the control group. The kidneys were not required to be weighed in these pups.

F1 COHORT 1A:
Test item-related but non-adverse mean kidney and liver weight findings were noted in females at 300 and 1000 mg/kg bw/day. The findings in males up to 1000 mg/kg bw/day and in females at 100 mg/kg bw/day were assessed as unrelated to the test item.

For the kidney, the mean weight was higher at 300 and 1000 mg/kg bw/day in females, correlating with the minimal or mild tubular basophilia seen histologically (adverse at a mild grade at 1000 mg/kg bw/day). The maximum difference from controls in weight relative to body weight was +15% in females at 1000 mg/kg bw/day.

The mean liver weight was higher at all doses in males and at 300 and 1000 mg/kg bw/day in females, correlating with the hepatocellular basophilia and hypertrophy seen histologically (examined at 1000 mg/kg bw/day). The maximum difference from controls in weight relative to body weight was +15% in females.

The kidney and liver weight findings are not identified as adverse in the study or pathology reports.

Please see Table 16 below.

F1 COHORT 1B:
There were test item-related but non-adverse mean kidney weights in females at 1000 mg/kg bw/day, liver weights in females at all doses, and liver weights in males at 1000 mg/kg bw/day. Male kidney weights up to 1000 mg/kg bw/day and all organ weights, except for the female liver, at 100 and 300 mg/kg bw/day were considered unrelated to the test item.

For the kidney, the mean weight was higher in females at 1000 mg/kg bw/day. The maximum difference from controls in weight relative to body weight was +13% at this dose.

The mean liver weight was higher in females at all doses and in males at 1000 mg/kg bw/day. The maximum difference from controls in weight relative to body weight was +21% in females at 1000 mg/kg bw/day.

These kidney and liver weight findings are not identified as adverse in the study or pathology reports. Noting that, per protocol, F1 Cohort 1B tissue samples were collected and processed to paraffin blocks, but not subject to microscopic examination.

Please see Table 17 below.

Gross pathological findings:

no effects observed

Description (incidence and severity):

F1 PUPS: [no effects observed]
There were no gross abnormalities in the pups culled / necropsied on PND 4 or in the F1 unselected pups necropsied on PNDs 22-24.

F1 COHORTS:
No test item-related gross findings were identified for the F1 Cohort 1A and 1B animals up to 1000 mg/kg bw/day. Any findings were generally of the nature commonly observed in this strain of rat at the ages concerned, most were infrequent, and none had a microscopic correlate.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

F1 COHORT 1A:
In the F1 Cohort 1A animals, there were test item-related and adverse effects in the kidney at 1000 mg/kg bw/day in females, primarily based on minimal to mild tubular basophilia (considered adverse at mild or higher). At this dose, the collective kidney findings across both sexes included tubular basophilia (minimal to mild), tubular dilatation (minimal to mild), mononuclear cell infiltration (minimal), papillary cellular debris (minimal), medullary mineralisation (minimal) and, in males only and not relevant to humans, minimal accumulation of hyaline droplets in cortical tubules.

Test item-related increases in tubular basophilia and medullary mineralisation occurred at 300 and 1000 mg/kg bw/day (in a dose level-related fashion). Tubular basophilia was assigned minimal or mild grade (adverse at mild or higher), indicating this finding was less severe than in the F0 animals. Medullary mineralisation comprised basophilic, non-birefringent amorphous deposits in the epithelium of, or between, tubules of the medulla, occasionally involving the outer papilla. The other kidney findings were increased at 1000 mg/kg bw/day only. Tubular basophilia at the mild grade and papillary cellular debris at the minimal grade occurred only in females at 1000 mg/kg bw/day indicating that the kidney findings were more apparent in females, as also evident in the F0 animals.

F1 PUPS AND F1 COHORT 1B:
Not examined, noting that F1 Cohort tissue samples were collected and processed to paraffin blocks for possible microscopic examination.

Other effects:

no effects observed

Description (incidence and severity):

COAGULATION FINDINGS:
F1 COHORT 1A: [no effects observed]
There were no effects on coagulation parameters in the F1 Cohort 1A animals up to 1000 mg/kg bw/day. All values were considered to be within normal biological variation when compared with the controls.

F1 PUPS AND F1 COHORT 1B:
Not examined.

ENDOCRINE FINDINGS:
F1 PUPS AND COHORTS:
No test item-related effects on T4 concentrations in plasma were observed in the F1 pups or in the Cohorts 1A or 1B animals up to 1000 mg/kg bw/day (parental F0 or cohort). The cohort animal TSH findings will be added when available. Samples for TSH analysis were not collected from the F1 pups.

OESTRUS CYCLE FINDINGS:
F1 COHORT 1A:
There were no test item-related effects on maternal oestrous cycles for the Cohort 1A animals up to 1000 mg/kg bw/day.

F1 AND F1 COHORT 1B:
Not examined.

SPERM FINDINGS:
F1 Cohort 1A:
There were no test item-related effects on sperm motility, sperm or spermatid counts, or sperm morphology in the F1 Cohort 1A males up to 1000 mg/kg bw/day.

F1 PUPS AND F1 COHORT 1B:
Not examined.

OVARIAN FOLLICLE COUNTS:
F1 COHORT 1A:
There were no test item-related effects on the ovarian follicle counts in the F1 Cohort 1A females up to 1000 mg/kg bw/day.

F1 AND F1 COHORT 1B:
Not examined.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

F1 COHORTS:
For the F1 Cohort 1A and 1B animals, no behaviour changes were mentioned in the study results for these parameters: gait and posture, presence of clonic or tonic movements, stereotypy, or bizarre behaviour up 1000 mg/kg bw/day, as assessed as part of the detailed clinical examinations.

F1 PUPS:
Not examined, see clinical signs for the F1 pups.

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

F1 COHORT 1A:
No test item-related effects on immunophenotypic expression in the spleen were observed in F1 Cohort 1A animals treated up to 1000 mg/kg bw/day, as compared to the control group. No dose- or sex-dependent effects were observed for any immune cell populations, as expressed as a percentage of total lymphocytes.

F1 PUPS AND F1 COHORT 1B:
Not examined.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

polysulphides read across

Sex:

male/female

Basis for effect level:

other: F1 pup systemic (potentially exposed in utero / during lactation PNDs 0-21)

Remarks on result:

other: Lack of test item-related and adverse effects up to 1000 mg/kg bw/day of parental treatment

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive / developmental

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

polysulphides read across

Sex:

male/female

Basis for effect level:

other: F1 pup reproductive function / developmental (potentially exposed in utero / during lactation PNDs 0-21)

Remarks on result:

other: Lack of test item-related effects up to 1000 mg/kg bw/day of parental treatment.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Generation:

F1 (cohort 1A)

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

polysulphides read across

Sex:

male

Basis for effect level:

other: Systemic (treated PNDs 21-90)

Remarks on result:

other: Lack of test item-related and adverse effects up to 1000 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Generation:

F1 (cohort 1A)

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

polysulphides read across

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: Test item-related and adverse renal histopathology at 1000 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive

Generation:

F1 (cohort 1A)

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

polysulphides read across

Sex:

male/female

Basis for effect level:

other: Reproductive function (treated PNDs 21-90)

Remarks on result:

other: Lack of test item-related effects up to 1000 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Generation:

F1 (cohort 1B)

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

polysulphides read across

Sex:

male/female

Basis for effect level:

other: Systemic (treated PNDs 21-97)

Remarks on result:

other: Lack of test item-related and adverse effects up to 1000 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive

Generation:

F1 (cohort 1B)

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

polysulphides read across

Sex:

male/female

Basis for effect level:

other: Reproductive function (treated PNDs 21-97)

Remarks on result:

other: Lack of test item-related effects up to 1000 mg/kg bw/day

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Reproductive effects observed:

no

Table 14. F0: Summary group mean organ weight data at scheduled euthanasia

	Males				Females
Group:	1	2	3	4	1	2	3	4
Dose (mg/kg/day):	0	100	300	1000	0	100	300	1000
No. animals examined:	25	25	25	25	25	25	25	25
Terminal Body Weight (No. weighed)	(25)	(25)	(25)	(25)	(21)	(23)	(23)	(23)
Absolute value (g)	490	499	478	465	284	277	281	287
Kidney (No. weighed)	(25)	(25)	(25)	(25)	(21)	(23)	(23)	(24)
Absolute value (g)	2.82	2.81	2.74	3.14**	2.16	2.08	2.28	2.76++
% of body weight	0.576	0.567	0.576	0.675**	0.758	0.755	0.812**	0.963**
% of brain weight	132	131	129	147	110	107	117	144
Liver (No. weighed)	(25)	(25)	(25)	(25)	(21)	(23)	(23)	(24)
Absolute value (g)	16.9	18.0	17.7	18.4	15.8	15.6	16.1	17.9**
% of body weight	3.43	3.61	3.69*	3.94**	5.56	5.65	5.74	6.21++
% of brain weight	787	841	831	863	806	805	826	930

Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01

Kruskal-Wallis & Dunn: ++ = p ≤ 0.01

% of brain weight not analysed statistically

Group mean values are rounded to 3 significant figures. Statistical significance is based on unrounded data.

 

Table 15. F0: Summary group test item-related microscopic findings at scheduled euthanasia

	Males				Females
Group:	1	2	3	4	1	2	3	4
Dose (mg/kg/day):	0	100	300	1000	0	100	300	1000
No. animals examined:	25	25	25	25	25	25	25	25
Kidney (No. Examined)	(25)	(25)	(25)	(25)	(25)	(25)	(25)	(25)
Tubular basophilia^a	10	7	20	25	0	2	25	25
Minimal	10	7	20	6	0	2	15	0
Mild	0	0	0	18	0	0	10	17
Moderate	0	0	0	1	0	0	0	8
Mononuclear cell infiltration	13	18	19	25	5	7	25	25
Minimal	13	18	19	24	5	7	25	25
Mild	0	0	0	1	0	0	0	0
Tubular dilatation	6	9	11	18	1	0	3	24
Minimal	5	9	11	16	1	0	3	22
Mild	1	0	0	2	0	0	0	2
Cellular debris, papilla, minimal	0	0	1	5	0	0	17	25
Mineralisation, papilla	1	2	1	8	2	3	5	6
Minimal	1	2	1	7	2	3	5	6
Mild	0	0	0	1	0	0	0	0
Accumulation, cortical, hyaline droplets	0	10	7	17	0	0	0	0
Minimal	0	8	4	9	0	0	0	0
Mild	0	2	3	8	0	0	0	0
Liver (No. Examined)	(25)	(5)	(9)	(25)	(25)	(1)	(1)	(25)
Centrilobular hepatocellular hypertrophy, minimal	0	0	0	4	2	0	0	14
Increased hepatocellular basophilia, minimal	1	0	0	6	2	0	0	12
Jejunum (No. Examined)	(25)	–	–	(25)	(25)	–	–	(25)
Villous vacuolation	0	–	–	21	0	–	–	20
Minimal	0	–	–	12	0	–	–	15
Mild	0	–	–	8	0	–	–	5
Moderate	0	–	–	1	0	–	–	0
Ileum (No. Examined)	(25)	–	–	(25)	(25)	–	–	(25)
Villous vacuolation	0	–	–	2	0	–	–	0
Mild	0	–	–	1	0	–	–	0
Moderate	0	–	–	1	0	–	–	0
Mesenteric lymph node (No. Examined)	(25)	–	–	(25)	(25)	(2)	(2)	(25)
Vacuolation	0	–	–	5	0	0	1	9
Minimal	0	–	–	5	0	0	1	3
Mild	0	–	–	0	0	0	0	5
Moderate	0	–	–	0	0	0	0	1
Gut-associated lymphoid tissue (No. Examined)	(24)	–	–	(23)	(23)	–	–	(23)
Vacuolation, minimal	0	–	–	0	0	–	–	1

^a Reported as adverse for males at 1000 mg/kg bw/day and females from 300 mg/kg bw/day

Table 16. F1 Cohort 1A: Summary group mean organ weight data at scheduled euthanasia

	Males				Females
Group:	1	2	3	4	1	2	3	4
Dose (mg/kg/day):	0	100	300	1000	0	100	300	1000
No. animals examined:	20	20	20	20	20	20	20	20
Terminal Body Weight (No. weighed)	(20)	(19)	(19)	(20)	(20)	(20)	(20)	(20)
Absolute value (g)	414	405	396	384	223	219	220	225
Kidney (No. weighed)	(20)	(20)	(20)	(20)	(20)	(20)	(20)	(20)
Absolute value (g)	2.75	2.64	2.55	2.73	1.61	1.61	1.68	1.87**
% of body weight	0.665	0.656	0.643	0.712	0.723	0.738	0.762*	0.828**
% of brain weight	133	129	124	133	85.9	86.8	89.2	99.2
Liver (No. weighed)	(20)	(20)	(20)	(20)	(20)	(20)	(20)	(20)
Absolute value (g)	16.6	17.3	17.0	17.7	8.92	8.89	9.40	10.4**
% of body weight	4.02	4.25*	4.25*	4.59**	4.01	4.06	4.27*	4.62**
% of brain weight	805	842	825	862	475	478	500	554

Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01

% of brain weight not analysed statistically

Group mean values are rounded to 3 significant figures. Statistical significance is based on unrounded data.

Table 17. F1 Cohort 1B: Summary group mean organ weight data at scheduled euthanasia

	Males				Females
Group:	1	2	3	4	1	2	3	4
Dose (mg/kg/day):	0	100	300	1000	0	100	300	1000
No. animals examined:	20	20	20	20	20	20	20	20
Terminal Body Weight (No. weighed)	(20)	(20)	(20)	(20)	(20)	(20)	(20)	(19)
Absolute value (g)	414	422	413	387	226	223	226	226
Kidney (No. weighed)	(20)	(20)	(20)	(20)	(20)	(20)	(20)	(20)
Absolute value (g)	2.68	2.60	2.65	2.66	1.62	1.64	1.67	1.84**
% of body weight	0.649	0.617	0.645	0.686	0.718	0.738	0.738	0.816**
% of brain weight	130	127	130	130	86.5	87.5	88.5	98.1
Liver (No. weighed)	(20)	(20)	(20)	(20)	(20)	(20)	(20)	(20)
Absolute value (g)	16.2	17.2	16.8	17.1	8.77	9.41	9.44	10.6**
% of body weight	3.91	4.07	4.08	4.41**	3.87	4.23*	4.16*	4.66**
% of brain weight	785	840	822	839	467	502	500	565

Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01

% of brain weight not analysed statistically

Group mean values are rounded to 3 significant figures. Statistical significance is based on unrounded data.

Table 18. F1 Cohort 1A: Summary test item-related microscopic findings at scheduled euthanasia

	Males				Females
Group	1	2	3	4	1	2	3	4
Dose (mg/kg/day)	0	100	300	1000	0	100	300	1000
No. animals examined	25	25	25	25	25	25	25	25
Kidney (No. Examined)	(25)	(25)	(25)	(25)	(25)	(25)	(25)	(25)
Tubular basophilia^a	10	7	20	25	0	2	25	25
Minimal	10	7	20	6	0	2	15	0
Mild	0	0	0	18	0	0	10	17
Moderate	0	0	0	1	0	0	0	8
Mononuclear cell infiltration	13	18	19	25	5	7	25	25
Minimal	13	18	19	24	5	7	25	25
Mild	0	0	0	1	0	0	0	0
Tubular dilatation	6	9	11	18	1	0	3	24
Minimal	5	9	11	16	1	0	3	22
Mild	1	0	0	2	0	0	0	2
Cellular debris, papilla, minimal	0	0	1	5	0	0	17	25
Mineralisation, papilla	1	2	1	8	2	3	5	6
Minimal	1	2	1	7	2	3	5	6
Mild	0	0	0	1	0	0	0	0
Accumulation, cortical, hyaline droplets	0	10	7	17	0	0	0	0
Minimal	0	8	4	9	0	0	0	0
Mild	0	2	3	8	0	0	0	0
Liver (No. Examined)	(25)	(5)	(9)	(25)	(25)	(1)	(1)	(25)
Centrilobular hepatocellular hypertrophy, minimal	0	0	0	4	2	0	0	14
Increased hepatocellular basophilia, minimal	1	0	0	6	2	0	0	12
Jejunum (No. Examined)	(25)	–	–	(25)	(25)	–	–	(25)
Villous vacuolation	0	–	–	21	0	–	–	20
Minimal	0	–	–	12	0	–	–	15
Mild	0	–	–	8	0	–	–	5
Moderate	0	–	–	1	0	–	–	0
Ileum (No. Examined)	(25)	–	–	(25)	(25)	–	–	(25)
Villous vacuolation	0	–	–	2	0	–	–	0
Mild	0	–	–	1	0	–	–	0
Moderate	0	–	–	1	0	–	–	0
Mesenteric lymph node (No. Examined)	(25)	–	–	(25)	(25)	(2)	(2)	(25)
Vacuolation	0	–	–	5	0	0	1	9
Minimal	0	–	–	5	0	0	1	3
Mild	0	–	–	0	0	0	0	5
Moderate	0	–	–	0	0	0	0	1
Gut-associated lymphoid tissue (No. Examined)	(24)	–	–	(23)	(23)	–	–	(23)
Vacuolation, minimal	0	–	–	0	0	–	–	1

^a Reported as adverse for females at 1000 mg/kg bw/day

Conclusions:

In the read-across EOGRTS for bis[3-(triethoxysilyl)propyl]polysulfides conducted according to OECD Test Guideline 443 and in compliance with GLP, four groups of Wistar Han rats were investigated: F0 (parental), F1 pups, F1 Cohort 1A, and F1 Cohort 1B, with the latter added to primarily assess endocrine and reproductive tissues via necropsy and organ weights. The F0 and cohort groups were administered the test substance at 0, 100, 300, and 1000 mg/kg bw/day (corn oil vehicle), with the F1 pups potentially exposed in utero or during lactation. Based on the findings, the overall study NOAELs were identified as follows:
- Systemic, males: 300 mg/kg bw/day, based on test item-related and adverse kidney histopathology in F0 males at 1000 mg/kg bw/day
- Systemic, females: 100 mg/kg bw/day, based on test item-related and adverse kidney histopathology in F0 females at 300 mg/kg bw/day
- Reproduction: at least 1000 mg/kg bw/day, based on the lack of test item-related: reproductive effects in the F0, F1 Cohort 1A, and F1 Cohort 1B animals, and reproductive / developmental effects in the F1 pups.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Based on the reliability score 1 OECD Test Guideline 443 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

No reproductive toxicity data are available for S2. Therefore, the EOGRTS in Wistar Han rats for bis[3 -(triethoxysilyl)polysulfides (Charles River Laboratories, 2022) is read across for the registered substance. The read-across hypothesis and justification are presented after the read-across EOGRTS study.

 

EOGRTS STUDY ON POLYSULFIDES:

In the key read-across EOGRTS study with bis[3-(triethoxysilyl)polysulfides conducted according to OECD Test Guideline 443 and in compliance with GLP (Charles River Laboratories, 2022). Wistar Han rats (F0, F1 Cohort 1A, and F1 Cohort 1B) were administered the test substance at 0, 100, 300, and 1000 mg/kg bw/day (corn oil vehicle), with 25 animals/sex/dose in the F0 cohort and 20 animals/sex/dose in the two F1 cohorts. The F1 pups were culled on Postnatal Day (PND 4) to yield litters of 4/sex/litter (as feasible), with the two F1 cohorts selected on PND 21. The treatment durations varied by F0 and cohort group as follows, with F1 pups potentially exposed in utero and during lactation until PND 21:

- F0, males: 10 weeks prior to mating and continuing throughout and after mating until the day before necropsy on Study Day (SD) 114

- F0, females: 10 weeks prior to mating and continuing throughout mating and gestation until at least Lactation Day (LD) 21, necropsied LDs 22-24

- F1 Cohort 1A: From PND 21 until the day before necropsy (at least PND 90)

- F1 Cohort 1A: From PND 21 until the day before necropsy (at least PND 97)

 

For all three adult cohorts, the parameters evaluated included mortality, clinical observations (cageside, pre- and post-dosing, and detailed), body weights, food consumption, water consumption (visual), oestrous cycles, and a complete macroscopic examination. Additional parameters by cohort(s) included:

- F0: Data to determine reproductive indices (mating, fertility, and pregnancy indices), female post-implantation loss, gestation length, and deficiencies in maternal care

- F0 and F1 Cohort 1A, both sexes: Haematology, clinical chemistry, coagulation, urinalysis, serum thyroxine (T4) and/or thyroid stimulating hormone (TSH) levels (TSH results not yet available). At necropsy, a full list of organ / tissues was subject to organ weights, gross pathology, histology and histopathology.

- F0 and F1 Cohort 1A, males: Sperm motility, sperm and spermatid counts and morphological analysis

- F1 Cohorts 1A and 1B: Sexual maturation

- F1 Cohort 1A only: Ovarian follicle counts, spleen immunophenotyping

- F1 Cohort 1B only: At necropsy, a subset of organ / tissues (primarily endocrine and reproductive but also, for example, kidney) was subject to organ weights and gross pathology, with tissues collected and processed to paraffin blocks for possible histopathologic examination. Since the in-life phase for this study ended on 21 July 2021 (at approximately the same time as ECHA issued its “Critical aspects for designing and conducting extended one-generation reproductive toxicity (EOGRT) studies under REACH”, processed tissue samples were retained for possible histopathology, but a microscopic examination for the kidney in F1 Cohort 1B animals was not performed (see F1 Cohort 1A kidney results below).

 

In F1 litters or pups, the following parameters were evaluated: numbers of live and dead pups (liver birth index), sex ratio, daily number of live pups (litter size, viability index), daily presence/absence of milk in pup stomachs (as feasible), visible external abnormalities, body weights, anogenital distance (AGD), male nipple retention, serum thyroxine (T4) and/or thyroid stimulating hormone (TSH) levels in subsets of the culled pups necropsy on PND 4 and the unselected pups at necropsy on PNDs 22-24, number of pups surviving to LD 21 (lactation index), external exam prior to necropsy, macroscopic exam for the culled pups and unselected pups, and organ weights for the same subset of unselected pups as the T4 / TSH analysis. 

The systemic findings considered test item-related and adverse (kidney histopathology in the F0 cohort and females in F1 Cohort 1A) are discussed below. For all other parameters in the three cohorts (F0, F1 Cohort 1A, F1 Cohort 1B) and for the F1 pups, there were no effects observed or the findings were considered unrelated to the test item.

 

In the F0 animals, there was adverse histopathology in the kidney of both sexes at 1000 mg/kg/day and of females at 300 mg/kg bw/day, primarily based on the tubular basophilia. The collective renal findings across both doses included tubular basophilia (minimal to moderate), tubular dilatation (minimal to mild), mononuclear cell infiltration (minimal to mild), papillary cellular debris (minimal), mineralisation (minimal to mild), and (in males only and not relevant to humans) minimal to mild accumulation of hyaline droplets in cortical tubules. Tubular basophilia comprised cortical tubules lined by epithelial cells with cytoplasm that was reduced in amount and/or more basophilic than normal, with nuclei that were larger than normal with dispersed chromatin and with occasional degenerate cells. There also was fibrosis in the basement membranes and surrounding interstitial tissue. Some foci included interstitial mononuclear cell infiltration and/or dilatation of basophilic tubules, which were recorded as separate findings. Papillary cellular debris comprised amorphous or globular, slightly eosinophilic deposits in the lumen of papillary tubules, and/or focal increased cellularity of the epithelium of individual papillary tubules. The tubular basophilia in the kidney (mild or moderate; mild and above considered adverse) was identified as adverse in both sexes at 1000 mg/kg bw/day and in females at 300 mg/kg bw/day. Noting, the average grade of tubular basophilia in females at 300 and 1000 mg/kg bw/day was higher in females than in males, and papillary cellular debris was more prevalent in females at these doses.

 

In the F1 Cohort 1A animals, there was adverse histopathology in the female kidney at 1000 mg/kg bw/day, primarily based on tubular basophilia. At this dose, the collective kidney findings across both sexes included tubular basophilia (minimal to mild), tubular dilatation (minimal to mild), mononuclear cell infiltration (minimal), papillary cellular debris (minimal), medullary mineralisation (minimal) and, in males only and not relevant to humans, minimal accumulation of hyaline droplets in cortical tubules.

 

Test item-related tubular basophilia and medullary mineralisation were increased at 300 and 1000 mg/kg/day (in a dose level-related fashion). However, the tubular basophilia did not exceed the mild grade except for females at 1000 mg/kg bw/day, indicating that the changes for this sex and dose were adverse, but less marked than in the F0 animals (up to moderate at 1000 mg/kg bw/day). Medullary mineralisation in both sexes at all doses (no clear dose-response) comprised basophilic, non-birefringent amorphous deposits in the epithelium of, or between, tubules of the medulla, occasionally involving the outer papilla. The other findings in the kidney were increased only at 1000 mg/kg bw/day. Tubular basophilia at the mild grade and minimal papillary cellular debris only occurred in females at this dose, indicating that the kidney findings (apart from male hyaline droplet accumulation) were more apparent in females, as was seen in the F0 animals. Except for the mild tubular basophilia in females at 1000 mg/kg bw/day, the observed renal histology for the F1 Cohort 1A was considered non-adverse.

 

For the F1 Cohort 1B animals, no test item-related and adverse kidney findings (necropsy, organ weights) were observed up to 1000 mg/kg bw/day. As previously indicated, tissue samples including the kidney were collected and processed but, per protocol (prior to July 2021), a renal microscopic examination was not performed for these animals.

 

In summary, the overall study NOAELs in the read-across EOGRTS (OECD Test Guideline 443) for polysulfides for Han Wistar rats in four cohorts (F0, F1 pups, F1 Cohort 1A, F1 Cohort 1A) were identified as follows:

- Systemic, males: 300 mg/kg bw/day, based on test item-related and adverse kidney histopathology in F0 males at 1000 mg/kg bw/day

- Systemic, females: 100 mg/kg bw/day, based on test item-related and adverse histopathology findings in F0 females at 300 mg/kg bw/day

- Reproduction: at least 1000 mg/kg bw/day, based on the lack of: reproductive findings in the F0, Cohort 1A, and Cohort 1B animals and reproductive / developmental findings in the F1 pups.

READ ACROSS 

Read-across hypothesis:

The read-across hypothesis is that the source (polysulfides) and target (S2) substances have similar toxicological properties because:

- The target substance is a constituent of the source substance;

- Other constituents of the source substance are structurally very similar, and their physicochemical properties are very similar;

- All of these constituents are expected to follow a common metabolic pathway;

- The constituents hydrolyse to a common product and to non-common products;

- The non-common hydrolysis products are structurally very similar, and their physicochemical properties are very similar;

- The non-common hydrolysis products follow a common metabolic pathway.

 

This prediction is supported by data on the toxicological properties of the substances, known rapid hydrolysis, and the presence in the molecules of sulphur bridges, which are subject to known metabolism.

 

The target substance (S2) is a monoconstituent substance that consists of two 3-(triethoxysilyl)propyl groups linked by a disulfide group; it is one of the constituents of the source substance (polysulfides). The target substance (S2) and the constituents of the source substance are structurally very similar; each has two common functional groups (3- (triethoxysilyl)propyl) and a linking sulfide group with two, three or four sulphur atoms.

 

Data for developmental toxicity (OECD TG 414) in the rat are available for both the source (polysulfides) and the target substances (S2), and reproductive toxicity data are available for polysulfides. The composition of the substances tested is given in detail in CSR Section 1.4.

 

Therefore, read across of the EOGRTS (OECD Guideline 443) on polysulfides is considered appropriate based on the Annex X information requirements under REACH.

 

Read-across hypothesis:

 

(a) Structural similarity:

 

S2 (target substance) and the constituents of polysulfides (source substance) are structurally similar and hydrolyse to similar substances. See CSR Section 1.4 for discussion of the structures and their hydrolysis. The constituents and major impurities of the source and target substances have two functional groups in common:

- The triethoxysilane, Si(OEt)3, group (there are two of these per molecule, therefore each molecule has six reactive ethoxy groups).

- The (poly)sulfide, CH2SnCH2(n = 1-4), group.

 

Clearly the only difference between the constituents/major impurities is the number of sulfur atoms in the sulfide bridge.

 

In this context, structural similarity has been assessed using the Tanimoto coefficient. The Tanimoto coefficient is a numerical value between 0 and 1. A value of 1 describes an identical fingerprint. The closer the coefficient is to 1, the more similar the substances. The submission substance, S2, has a Tanimoto coefficient of >0.9 for similarity to the other constituents of the polysulfides (S3 and S4), with this number indicating a high degree of structural similarity.

 

(b) Similar physicochemical properties:

 

A data matrix is attached in Section 13 of the IUCLID dossier, and the key physicochemical parameters of the substances are summarised below. The substances all hydrolyse, and the products of hydrolysis are bis(3-(trihydroxysilyl)propylsulfides (with a varying number of sulfur atoms) and ethanol. The key physicochemical properties of the silanol hydrolysis products are also included in the table. Information on the physicochemical properties of the individual constituents can be found in CSR Section 1.4.

 

Due the very similar chemical structures, the substances and constituents are predicted to have near identical physicochemical properties, with high log Kow, low water solubility and low vapour pressure. This includes impurities with Sn (n>4) that are not included in the table.

 

All constituents/major impurities hydrolyse very rapidly (half-life predicted to be approximately 7 seconds) to bis(3 -(trihydroxysilyl)propylsulfides (with a variation in the number of sulfur atoms) and ethanol at pH 2 and 37.5°C (the conditions relevant for oral exposure). The hydrolysis products are predicted to have near identical physicochemical properties, with high water solubility, low log Kow and negligible vapour pressure.

 

(c) Similar toxicokinetics:

 

Based on the predicted physicochemical properties, it can be inferred that the substances in the sulfidosilane group behave similarly with respect to adsorption, distribution and excretion. The toxicokinetic properties of S2, reaction mass S2/S3 and polysulfides have been predicted based on the physicochemical properties and estimated using pharmacokinetic or toxicokinetic (PBTK) prediction models. See Section 5.1.3 for more information.

 

(d) Similar toxicity:

 

The toxicological properties of the other constituents of polysulfides are predicted to be similar to those of S2. This is based on common functionality and supported by similarity in structure, physicochemical characteristics, toxicokinetics, and toxicity data for the endpoints with data for both substances (acute toxicity, repeated dose toxicity, and developmental toxicity; reproductive toxicity data for polysulfides also discussed).

 

Acute:

Acute oral toxicity studies are available for S2, low purity S2, and polysulfides. Acute dermal toxicity studies are available for low purity S2 and for polysulfides. Acute inhalation toxicity studies are available for polysulfides. No mortalities or systemic effects were observed with any of these substances in any of the studies, at doses greater than or equal to 2000 mg/kg bw. Furthermore, there were no effects in an acute inhalation study with the polysulfides. Together these results indicate that the sulphidosilanes have a low acute toxicological profile.

 

Repeated dose, systemic:

Four relevant 28-day oral toxicity studies are available, one on the monoconstituent substance S2 (CAS 56706-10-6), one on the multiconstituent substance low purity S2, and two on the polysulfides (CAS 211519-85-6). Summaries of these studies can be found in Section 5.6.3, and in Table 5.9.4. An OECD Test Guideline 408 study is planned, and will be contracted following ECHA study authorisation, with a specific aim of further investigating the systemic renal histopathology identified as adverse in the read-across polysulfides EOGRTS.

 

Reproductive (polysulfides only):

As discussed above, there were no test item-related reproductive (fertility or developmental) findings in the EOGRTS for polysulfides up to 1000 mg/kg bw/day (Charles River Laboratories, 2022)

 

In the repeated dose studies for polysulfides, there were no adverse effects for any of the reproductive organs examined. One polysulfide study included histopathological examination of prostate, testes and epididymis (Hita Labs., 2000b), while the other examined testes and ovaries (Degussa AG, 1983). No abnormalities were seen in reproductive organs in either of these studies. The polysulfides tested included S2 at between 12 and 25%, 30-35% s (S3) and 20-30% tetrasulfied (S4). These results are consistent with the lack of reproductive findings in the polysulfies EOGRTS.

 

Developmental:

The oral prenatal developmental toxicity studies on S2 (BSL Bioservice, 2016) and polysulfides (BSL Bioservice, 2013) demonstrates that neither test substance induces maternal toxicity or foetal developmental toxicity up to a dose of 1000 mg/kg bw/day. The polysulfide EOGRTS findings support developmental conclusion (NOAEL of 1000 mg/kg bw/day). Although maternal toxicity was seen in the EOGRTS for polysulfides (at 100 mg/kg bw/day), the result likely reflects the much longer exposure duration (10 weeks prior to mating, through mating and gestation, and until at least LD 21) versus (GDs 5-19 in the rat S2 developmental study, GDs 7-28 in the rabbit developmental study for polysulfides).

 

The repeated dose toxicity studies are considered to be reliable with restrictions, but adequate for assessment of repeated dose toxicity.

 

The studies clearly show that S2 and polysulfides have a very similar toxicological profile. In addition, the substances and their breakdown products are not classified for any human health hazard.

 

Consideration of possible mixture effects:

When considering the toxicity of mixtures, it is necessary to consider the possibility that the toxicity of the mixture is greater than would be expected based on the sum of the constituents (synergistic effects). The sulfidosilane substances polysulfides, S2/S3 reaction mix and low purity S2 are multiconstituent substances. However, synergistic effects are considered unlikely based on the high degree of similarity between the different constituents. The structural similarity of the constituents makes it probable that any toxicity would be exerted by each constituent of the multiconstituent substances with the same mode of action. This would make the effects additive, rather than synergistic. Evidence of this can be seen in the 28 day repeated dose toxicity studies where both the type of effects (adaptive changes in liver and kidney) and the effect levels seen in studies on S2, low purity S2 and polysulfides are very similar.

 

If mixture effects did occur for the polysulfides, this would probably make the polysulfides more toxic than the target substance (S2). It is very unlikely that mixture effects would lead to a reduction in toxicity. Therefore, the proposed read-across is considered to be conservative.

 

Justification of read-across conclusions:

The structural similarities between the source and the target substances and the similarities in their breakdown products presented above support the read-across hypothesis. Adequate, reliable and available scientific information indicates that the source and target substances and their abiotic degradation products follow similar metabolic pathways resulting in identical products of metabolism, and therefore have similar toxicity profiles.

 

The target substance is a constituent of the source substance (12-25%). Information on hydrolysis indicates that, under the conditions in the stomach in oral toxicity studies, source and target substances are subject to rapid hydrolysis to ethanol, and to silanol hydrolysis products that differ only in the number of sulfur atoms in the sulfide bridge. Thus, systemic exposure following oral administration will be predominantly to these breakdown products.

Information on sulfur metabolism indicates that the substances and the silanol hydrolysis products of source and target substances will follow similar biotransformation pathways producing identical metabolites.

 

Repeated dose, systemic:

In the 28-day studies available, the following results were observed:

- S2: Mild systemic toxicity, NOAEL 200 mg/kg bw/day based on liver and kidney adaptive changes.

- Low purity S2: Mild systemic toxicity, NOAEL 1221 mg/kg bw/day based on liver and kidney adaptive changes.

- Polysulfides: Mild systemic toxicity, NOAELs of 200 and 2309 mg/kg bw/day based on liver and adaptive changes.

 

Although adverse renal findings were not reported in these subacute studies, the systemic kidney histopathology was considered adverse in the read-across EOGRTS with polysulfides, likely due to its much longer exposure durations (113 days at a minimum versus 28 days). A 90-day OECD Test Guideline 408 study is planned,and will further investigate the renal histopathology as seen in the EOGRTS.

 

Developmental:

A prenatal developmental toxicity study (oral administration in rat; NOAEL ≥1000mg/kg bw/day) is available for the target substance, S2. In the light of the information on the basis for read-across between the source and the target substance presented above, the results from the study are considered as adequate to fulfil the information requirement of Annex IX, 8.7.2 applying to the other substances in the sulfidosilane group.

 

A prenatal developmental toxicity study (oral administration in rat; NOAEL ≥1000 mg/kg bw/day) has also been performed with the source substance, polysulfides. Prenatal developmental toxicity for substances with the sulfidosilane functional group would be mediated by systemically available chemicals (i.e., primarily the breakdown products of the substances), and thus the justification for read-across presented above is relevant for prenatal developmental toxicity. In the light of the information on the basis for read-across between the source and the target substance presented above, the results from the study are considered as adequate to fulfil the information requirement of Annex IX.

 

The developmental findings in the read-across EOGRTS with polysulfides (NOAEL ≥1000 mg/kg bw/day) supports the prenatal toxicity findings, and fulfils the information requirement of Annex X.

Effects on developmental toxicity

Description of key information

The key study for prenatal developmental toxicity of 4,4,13,13-Tetraethoxy-3,14-dioxa-8,9-dithia-4,13-disilahexadecane (“S2”; CAS No. 56706-10-6, EC No. 260-350-7) in the rat was conducted according to OECD Test Guideline 414 and in compliance with GLP (BSL Bioservice, 2016). No adverse effects on the dams or fetuses were reported, and consequently the NOAEL for this study was at least 1000 mg/kg bw/day S2. 

 

Similarly, the key read-across prenatal developmental toxicity study in the rabbit for the related substance bis[3-(triethoxysilyl)polysulfides (“polysulfides”; CAS No. 211519-85-6, EC No. 915-673-4) was conducted according to OECD Test Guideline 414 and in compliance with GLP (Charles River laboratories, 2021). No effects attributable to treatment noted at any dose, and the NOAEL established for maternal as well as developmental toxicity is therefore at least 1000 mg/kg bw/day.

 

In the read-across EOGRTS (OECD Test Guideline 443) for polysulfides, the lack of developmental findings were consistent with the OECD Test Guideline 414 studies, and also resulted in a reproductive (developmental) NOAEL of at least 1000 mg/kg bw/day. See Effects on fertility, Description of key information above.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23/06/2015 to 05/10/2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany.
- Age at study initiation: Females: 11-12 weeks old; Males: 12-13 weeks old.
- Weight at study initiation: Females: 200 - 244 g; Males: 304 - 351 g.
- Fasting period before study:
- Housing: The animals were kept individually in type III H, polysulphone cages on Altromin saw fibre bedding (except during the pre-mating period when females were kept in groups of two animals and during mating period when two females were paired with one male)
- Diet (e.g. ad libitum): Ad libitum.
- Water (e.g. ad libitum): Ad libitum.
- Acclimation period: At least five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 29-25
- Humidity (%): 45-65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18/06/2015 To: No data.

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item plus further vortexing for 2-3 minutes. Homogeneity of the test item in the vehicle was maintained by vortexing the prepared formulation thoroughly before every dose administration. The test item formulation was prepared freshly on each administration day before the administration procedure.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected based on the test item characteristics and testing guideline.
- Lot/batch no. (if required): BCBM3643V.
- Purity: No data.
- Concentration in vehicle: 0, 25, 75 and 250 mg/l.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for analysis of concentration of test item in the dosing formulations were taken on the first (treatment day 1), third and last week of the study for all doses (12 samples in total). Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparation. Samples were taken in the first (treatment day 1), third and last week of the study (18 samples in total). These samples were either measured within 6 hours after arrival at the analytical department on the day of sampling (week 3 and last week samples) or were stored under appropriate conditions (-15 to -35°C) for 16 days until analysis (week 1 samples).

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2
- Length of cohabitation: No data.
- Matings were continued until 100 'sperm positive' females were obtained.
- Further matings after two unsuccessful attempts: No data.
- Verification of same strain and source of both sexes: yes.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.

Duration of treatment / exposure:

15 days (gestation days 5-19)

Frequency of treatment:

Daily

Duration of test:

15 days

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

25 'sperm positive' females.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were based on the results of a dose-range finding study. The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once per day. Twice daily all animals checked for mortality and morbidity. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once before initiation of pairing to ensure that the body weights were within + 20% variation. The sperm positive females were weighed on gestations days 0, 5, 8, 11, 14, 17 and 20.

FOOD CONSUMPTION: Gestations days 5, 8, 11, 14, 17 and 20.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20. The animals were examined macroscopically for structural abnormalities or pathological changes.

Ovaries and uterine content:

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

Fetal examinations:

- External examinations: Yes: all fetuses in each litter.
- Soft tissue examinations: Yes: half fetuses per litter.
- Skeletal examinations: Yes: half fetuses per litter.
- Head examinations: Yes: half fetuses per litter (from soft tissue examination).

Statistics:

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

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
One animal (of the control group) was euthanised on study day 19 due to littering before the scheduled sacrifice. This was considered as incidental in nature. There were no deaths in the test groups. Low incidences of the slight clinical sign alopecia were noted in isolated females of the dose groups and the control group. This finding was considered incidental. Moving the bedding was noted in five females of the high dose group on a few treatment days. Salivation was observed in one single female of the high dose group on one occasion. These transient findings were noted shortly after administration and were considered to be signs of discomfort without toxicological relevance.

Body weight gain was not affected by treatment. Some animals in the mid and high dose groups had a statistically significantly reduced food consumption, but as this did not impact on body weights it was not considered to be an adverse finding.

Successful mating resulted in 20/25 pregnancies in the low dose group, 22/25 in the mid dose group and 22/25 in the high dose group compared to 19/25 pregnancies in the control group. Low pregnancy rate (no. of pregnancies / no. of females mated or sperm positive x 100) of 76 % in the control group was considered to be a biological variation.

Prenatal parameters such as group mean terminal body weight, gravid uterus weight, adjusted maternal weight, number of corpora lutea, implantation sites, early and late resorptions, and percent pre- and post-implantation loss were not affected by treatment.

No gross pathological changes related to treatment were observed during the macroscopic examination of the females of any group.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The number of live fetuses, male and female fetuses, sex ratio, number of fetuses in each uterine horn were unaffected in the dose groups when compared to the control group. No dead fetuses were noted in any of the groups. Therefore overall there were no statistically significant differences for prenatal data.

There were no test substance-related effects of toxicological relevance for the total number of fetuses, number of male and female fetuses, mean fetus weight, total litter weight and male and female litter weight. Therefore overall there were no statistically significant differences for litter data.

Fetal examinations did not reveal any dose-related adverse findings. All findings were similar to controls and concluded to be incidental.

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse findings for developmental toxicity

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

In a Prenatal Developmental Toxicity study (BSL Bioservice, 2016) conducted to OECD Test Guideline 414 and in compliance with GLP 4,4,13,13-tetraethoxy-3,14-dioxa-8,9-dithia-4,13-disilahexadecane did not induce maternal toxicity or developmental toxicity at doses up to 1000 mg/kg bw/day. Therefore the NOAEL for this study was at least 1000 mg/kg bw/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30th of April 2020 to 25th September 2020

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:

2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

2008

GLP compliance:

yes

Remarks:

Body weights of Day 0 post-coitum (i.e. the day of mating) reported were collected non-GLP by the Supplier.

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Age at study initiation: 18-20 weeks
- Weight at study initiation: 2981-4167 g
- Fasting period before study: no
- Housing: randomly housed individually in cages with perforated floors equipped with water bottles
- Diet : approximately 25 g of pelleted diet on day of arrival and 140-160 g per day except during designated procedures. Pressed hay was also provided during the study period.
- Water : ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-21°C
- Humidity (%): 40-70%
- Air changes (per hr): 10 x / hour or greater with 100% fresh air
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

IN-LIFE DATES: Not specified

Route of administration:

oral: gavage

Vehicle:

arachis oil

Remarks:

dried and de-acidified

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was administered to Group 4 (1000 mg/kg bw/day) as a solution as received. An adequate amount of the test item was dispensed into daily aliquots, which were stored in a refrigerator set to maintain 4°C. The aliquots were removed from the refrigerator and allowed to warm to room temperature for at least 30 minutes before dosing. Test item dosing formulations (w/w) for Groups 1 (Control), 2 (100 mg/kg bw/day) and 3 (300 mg/kg bw/day) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared at least weekly as a suspension, filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item. Any residual volumes were discarded.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure. These trials were not performed as part of this study and these preparations were not used for dosing. Raw data of these trials will be retained by the Test Facility.
- Concentration in vehicle: 0, 100, 300 and 1100 mg/mL. Test item concentration for Group 4 was corrected for specific gravity (factor 1.1)
- Amount of vehicle (if gavage): 1 and 0.91 for Group 4, as it was dosed with pure test item, the dose volume was calculated as dose level (mg/ kg bw/day) divided by corrected test item concentration (mg/mL).
- Lot/batch no. (if required): not specified
- Purity: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During the first week of the treatment (13 July 2020), dose formulation samples were collected for analysis. For concentration analysis specifically, all groups were sampled, whereas for homogeneity, groups 2 and 4 were analysed. All samples to be analyzed were transferred (at room temperature protected from light) to the analytical laboratory at the Test Facility. Residual samples were discarded after completion of the sample analysis. Analyses were performed by using a validated analytical procedure (Test Facility Study No. 20224626).
For concentration analysis, duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory. Concentration results were considered acceptable (i.e. mean accuracies between 90% and 110%). No test item was detected in the Group 1 formulation.
For homogeneity analysis, duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory. Homogeneity results were considered acceptable (i.e. coefficient of variation ≤ 10%).
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20224626) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Test Facility Study No. 20224626.

Details on mating procedure:

- Impregnation procedure: females were mated at the supplier and are at Day 1 post-coitum on arrival
- M/F ratio per cage: N/A
- Length of cohabitation: N/A
- Proof of pregnancy: Not specified
- Any other deviations from standard protocol: Not specified

Duration of treatment / exposure:

7 days a week from Day 7 to Day 28 post-coitum inclusive

Frequency of treatment:

Once daily

Duration of test:

28 days

Dose / conc.:

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

Remarks:

Group 1= Control Group

Dose / conc.:

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

Remarks:

Group 2

Dose / conc.:

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

Remarks:

Group 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Group 4

No. of animals per sex per dose:

22 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of the dose range finder (Test Facility Reference No. 20224629).
Dosing of the DRF was initiated on 06 May 2020. The in-life phase of the DRF was completed on 28 May 2020. Four groups of six females were exposed at doses of 0, 300, 600 and 1000 mg/kg bw/day for days 7 to 28 post-coitum inclusive by oral gavage.
No mortality occured during the study period, no toxicologically significant clinical signs were noted up to 1000 mg/kg bw/day, body weights and body weight gain were considered normal. Food consumption before or after correction for body weight was comparable between all groups over the study period. Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment with the test item. One control female (Female No. 6) and one female treated at 1000 mg/kg bw/day (Female No. 21) were non-pregnant. All other females were found to be pregnant with viable fetuses. A relatively low mean number of implantation sites was noted in females of the control group and at 300 mg/kg bw/day (both below the range of historical control data), whereas a normal mean number of implantation sites was observed at 600 and 1000 mg/kg bw/day. Accordingly, a relatively low mean litter size was noted in controls and at 300 mg/kg bw/day (below the range
of historical control data), whereas mean litter sizes at 600 and 1000 mg/kg bw/day were within normal limits.
Litter incidence of pre-implantation loss was relatively high in females of the control group, at 300 and 600 mg/kg bw/day (mean outside the range of historical control data) and within the normal range in females at 1000 mg/kg bw/day. No test item-related changes for the number of post-implantation loss were observed. Post-implantation loss was relatively high in females treated at 1000 mg/kg bw/day, however, a relatively high post-implantation loss was also observed in concurrent controls. The high post-implantation loss observed in controls was due to a higher number of late resorptions in one female only, whereas the number of late resorptions in females treated at 1000 mg/kg bw/day was higher in 4/5 females. Mean number of early resorptions was higher at 1000 mg/kg bw/day compared to controls (no statistical significance reached and mean values within the range of historical control data). Mean number of late resorptions was increased at 1000 mg/kg bw/day (above the range of historical control data). In controls, a relatively high mean number of late resorptions was noted as well, which was due to a high number of late resorptions in one control female only. The number of corpora lutea were considered unaffected by treatment with the test item. Mean combined fetal body weights were unaffected by treatment up to 1000 mg/kg bw/day. External examinations revealed no external malformations or variations in this study. Based on the results of this dose range finder, selected dose levels for the Main study were 100, 300 and 1000 mg/kg bw/day.
- Rationale for animal assignment : randomised

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were performed at least once daily, beginning on Day 7 post coitum and lasting up to the day prior to necropsy. During the dosing period, these observations were performed post-dose. Cage debris was examined to detect abortion or premature birth

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were individually weighed on Days 7, 9, 12, 15, 18, 21, 24, 27 and 29 post coitum.

FOOD CONSUMPTION : Yes
- Food consumption was quantitatively measured daily from Day 3 post-coitum onwards.

WATER CONSUMPTION : Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected. Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined: All animals (including animals found dead or sacrificed before planned necropsy and females with early delivery) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution).

Ovaries and uterine content:

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

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [half per litter]

Statistics:

Means, standard deviations (or % coefficient of variation or standard error, when deemed appropriate), ratio, percentages, numbers, and/or incidences were reported as appropriate by dataset.

Clinical signs:

no effects observed

Description (incidence and severity):

No test item-related clinical signs were noted up to 1000 mg/kg bw/day.
Clinical signs noted were erected fur, fur loss, scabs, broken teeth, absent nail or digits on left forepaw, lesions, flaking, swelling, and/or discoloration of the skin, red material present and/or decreased or liquid feces output among some females treated at 0, 100, 300 and/or 1000 mg/kg bw/day. These clinical signs occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In total, two females were sacrificed in extremis during the study period, due to circumstances unrelated to treatment with the test item.
Female No. 11 (control) was sacrificed on Day 27 post-coitum, its death was considered related to the gavage procedure. Blood was noted on the dosing tube and breathing difficulties and dark red eyes were noted after dosing was completed on Day 26 post-coitum (registered in the study daybook). Since the following day (Day 27 post coitum), the animal’s condition was deteriorated including labored breathing, decreased activity and a pale appearance (pallor), it was decided to sacrifice this animal for animal welfare reasons. Macroscopic abnormalities were observed in the thoracic cavity and lungs, confirming complications during dosing. In addition, the thymus had a gelatinous consistency. At necropsy, this female was pregnant with live fetuses.
Female No. 80 (1000 mg/kg/day) was sacrificed for animal welfare reasons on Day 19 post coitum, due to a severe skin lesion with ulceration (ventral cervical), body weight loss of 6% between Days 15 and 18 post-coitum, and reduced food and/or water consumption over Days 16-19 post-coitum, resulting in decreased feces production (Days 18 and 19 post-coitum). Macroscopic examination revealed no other abnormalities than scabs and red discoloration of the skin. This female was not pregnant.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weights, body weight gain and weight gain corrected for gravid uterus weight were considered to have been unaffected by treatment with the test item.
The higher mean body weight at 300 mg/kg bw/day when compared to concurrent controls observed from Day 15 post-coitum onwards (not statistically significant on Days 18 and 21 post-coitum) was considered to be related to the relatively high initial mean body weight of animals in this group. Body weight gain over the complete treatment period (i.e. Days 7 to 29 post-coitum) was comparable in all groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test item-related changes in food consumption were recorded.
Any statistically significant changes in food consumption over Days 9-27 in females treated at 100, 300 and 1000 mg/kg bw/day were considered to be caused by the relatively low food consumption observed in the control group, rather than to be related to treatment.

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment with the test item.
The recorded macroscopic findings were within the range of background gross observations encountered in rabbits of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Pre-implantation loss was higher in all treatment groups when compared with concurrent control (13.9, 19.9 and 13.2% at 100, 300 and 1000 mg/kg/day, respectively vs. 6.4% in the control group). As no dose-related response was observed, the mean number of implantation sites was within normal range and no differences were observed in the incidence of early resorptions, this difference in pre-implantation loss was considered not to be related to treatment with the test item.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

The number of corpora lutea, implantation sites, viable and dead fetuses, early and late resorptions, and post-implantation loss were considered unaffected by treatment with the test item.

Dead fetuses:

no effects observed

Description (incidence and severity):

One female of the control group and two, three and five females of the groups treated at 100, 300 and 1000 mg/kg bw/day, respectively, were not pregnant at necropsy. All other females (21, 20, 19 and 17 for the control, 100, 300 and 1000 mg/kg bw/day, respectively) were found to be pregnant with viable fetuses.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

One female of the control group and two, three and five females of the groups treated at 100, 300 and 1000 mg/kg bw/day, respectively, were not pregnant at necropsy. All other females (21, 20, 19 and 17 for the control, 100, 300 and 1000 mg/kg bw/day, respectively) were found to be pregnant with viable fetuses. For one of the five females at 1000 mg/kg/day that were non pregnant at necropsy, corpora lutea were observed indicating a successful mating. For this female, it cannot be confirmed whether implantation did not occur or if implantations were lost very early during pregnancy. If implantation did not occur, this female would have 100% pre-implantation loss, resulting in a mean pre-implantation loss of 18% at 1000 mg/kg/day.

Other effects:

no effects observed

Description (incidence and severity):

A relatively high mean number of corpora lutea was observed for females at 300 mg/kg/day, whereas the mean number of implantations was within normal range for all groups, and which contributed to the higher pre-implantation loss for this group.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: No adverse general and developmental effects in maternal animals

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no test item-related effects on fetal body weights (both sexes combined) noted by treatment with the test item up to 1000 mg/kg bw/day.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment with the test item up to 1000 mg/kg bw/day.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no test item-related effects on litter size of any group.
Mean litter sizes were 9.3, 8.2, 8.7 and 8.8 live fetuses/litter for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

Changes in postnatal survival:

not specified

External malformations:

no effects observed

Description (incidence and severity):

External malformations or variations were not observed in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related effects on skeletal morphology following treatment with the test item up to 1000 mg/kg bw/day.
Skeletal malformations occurred in 7 (6), 7 (5), 6 (5) and 2 (2) fetuses (litters) at 0, 100, 300 and 1000 mg/kg bw/day.
The most common malformation was a supernumerary lumbar vertebra that was observed in 4 (3), 4 (2) and 3 (2) fetuses (litters) of the control, 100 and 300 mg/kg bw/day groups, respectively, and not at 1000 mg/kg bw/day. The group distribution does not point towards a treatment relationship and therefore, these extra lumbar vertebrae were considered not to be related to treatment with the test item.
At 1000 mg/kg bw/day, the remaining malformations observed in two fetuses included fused ribs and thoracic arches (Fetus No. 75-R10) and fused ribs, a thoracic hemivertebra and an absent cervical vertebra (Fetus No. 86-L7).
At 300 mg/kg bw/day, the other malformations observed in two fetuses included supernumerary thoracic arch with rib (Fetus No. 50-L3) and fused caudal vertebrae (Fetus No. 66-R9).
Remaining malformations observed at 100 mg/kg bw/day affected the vertebral column in two fetuses and the sternum in one fetus. These included an absent cervical centrum (Fetus No. 40-L1), a lumbar vertebra missing (Fetus No. 44-L2), and severely fused sternebrae (Fetus No. 26-L2).
In summary, malformations affecting the vertebral column and/or rib region other than supernumerary lumbar vertebra, were noted in 2 (2) fetuses (litters) of all treated groups. Because this is not indicative for a treatment relationship and as also two control fetuses (Fetus Nos. 4-L2 and 7-R13) had a malformation in the same region, these vertebral and/or rib anomalies were considered chance findings. Due to the single occurrence, the sternal malformation at 100 mg/kg bw/day was considered not to be related to treatment with the test item.
Apart from one fetus with incomplete ossification of various skull bones (Fetus No. 8-L4) and one fetus with multiple malformations of the thoracic centrum (Fetus No. 17-R4), no other skeletal malformations were observed in the control group.
All skeletal variations occurred in the absence of a dose-related incidence trend, infrequently and/or in control fetuses only. Therefore, they were not considered related to treatment with the test item.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects on visceral morphology following treatment with the test item up to 1000 mg/kg bw/day.
Visceral malformations were observed in one fetus each at 100 and 1000 mg/kg bw/day and in two fetuses of the control group.
In the group treated at 1000 mg/kg bw/day, Fetus No. 75-R10, had a cardiovascular abnormality consisting of a persistent truncus arteriosus, right-sided aortic arch and ventricular septal defect. Fetus No. 23-R9 at 100 mg/kg bw/day had a retroesophageal right subclavian artery. Due to the single occurrence of these findings, they were considered chance findings and considered not related to treatment with the test item.
Viscerally malformed fetuses of the control group either had a narrow aortic arch and dilatation of the pulmonary trunk (Fetus No. 1-L5) or a severe dilatation of both lateral ventricles (Fetus No. 21-R6) which was observed at fixed head examination.
All visceral variations that were noted were considered unrelated to treatment with the test item as they occurred in the absence of a dose-related trend, infrequently and/or in control fetuses only.

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects in fetuses

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

In a prenatal developmental toxicity study in rabbits conducted according to OECD Test Guideline 414 and in compliance with GLP, the NOAEL for maternal toxicity and developmental toxicity was concluded to be at least 1000 mg/kg bw/day based on no observed adverse effects in pregnant New Zealand White female rabbit treated orally by gavage from days 7 to days 28 post-coitum as well as in any of the fetuses.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

XX

Justification for classification or non-classification

Based on the available data, 4,4,13,13 -tetraethoxy-3,14-dioxa-8,9-dithia-4,13-disilahexadecane is not classified for reproductive (fertility or developmental) toxicity according to Regulation (EC) No. 1272/2008.