Polysulfides, di-tert-dodecyl

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No study on fertility is available on TPS32.

An extended one-generation reproduction toxicity study (OECD 443) was proposed.

Effect on fertility: via oral route

Endpoint conclusion:

no study available (further information necessary)

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Polysulfides di-tert-dodecyl did not result in toxicity to the fetus or did not show developmental toxicity after gavage dosing during prgnancy to rats up to 1000 mg/kg bw/day or in rabbit up to 800 mg/kg/day in both developmental toxicity tests (OECD TG 414). 

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

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:

1981

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River France (76410 Saint-Aubin-lès-Elbeuf, France)
- Age at study initiation: 9 weeks old
- Weight at study initiation: mean body weight of 272 g (range: 235 g to 306 g)
- Fasting period before study: no
- Housing: individually in polycarbonate cages
- Diet (ad libitum): A04 C pelleted diet (UAR, France)
- Water (ad libitum): filtered tap wtaer
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was prepared as a dispersion in the vehicle, in order to achieve the concentrations of 10, 50 and 200 mg/ml. The test substance was carefuliy mixed with the required quantity of vehicle using an ultraturrax mixer, for approximately two minutes and then homogenized using a magnetic stirrer.
The test substance preparations were made for up to seven days of treatment, on the basis of available stability data (9 days at +4 °C) and were stored at +4 °C, away from light, pending utilization.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of samples of each dispersion (including the control) prepared for use on the first day of treatment of the first mated females and on the last day of treatment of the last mated females was determined by HPLC with UV detection.

Details on mating procedure:

Females were mated at the breeder's facilities.
The day of the positive mating (assessed by observation of vaginal plug) was designated as day 0 post-coitum (p.c.).

Duration of treatment / exposure:

Gestation day 6 to 15

Frequency of treatment:

daily

Duration of test:

up to gestation day 20

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: based of the results of a four-week toxicity study (C.I.T./study No 12602 TSR): . No toxic effects were noted after four weeks, at dose-levels up to 1000 mg/kg/day.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day for clinical signs and at least twice a day for mortality

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: on days 2, 6, 7, 8, 9, 10, 12, 14, 16 and 20 post-coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
on days 2-6, 6-9, 9-12, 12-16 and 16-20 post-coitum.

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined:
After hysterectomy, the females were subjected to a macroscopic examination of the principal thoracic and abdominal organs.

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 and distribution of dead and live fetuses
In apparently non-pregnant females, the presence of implantation sites was checked using the Salewski staining technique

Fetal examinations:

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

Statistics:

Mean values were compared by one-way analysis of variance and Dunnett's test, values being considered as normally distributed, variances being considered as homogenous. Percentage values were compared by Fischer's exact probability test.

Indices:

Pre-implantation loss; post-implantation loss

Clinical signs:

no effects observed

Description (incidence and severity):

No relevant clinical signs were observed in any female from the control, 50, 250 or 1000 mg/kg/day groups.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Dilated renal pelvis was observed in 1/25 females of the 50 mg/kg/day group (unilateral) and in 2/25 females of the 1000 mg/kg/day group (bilateral). This finding which was observed with a low, and not dose-related incidence and which could be observed spontaneously in female rats of this age and strain, was not attributed to the test substance administration.
The other macroscopic findings encountered did not show any indication of treatment or dose-relationship.

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):

The mean number of corpora lutea per animal was similar in the control and treated groups (17.0, 18.2 or 17.7 at 50, 250 or 1000 mg/kg/day vs. 17.8 in controls).
The mean number of implantation sites per animal was similar in the control and treated groups (13.6, 15.1 or 12.8 at 50, 250 or 1000 mg/kg/day vs. 12.3 in controls).
The normal variations observed for the preimplantation loss were considered to be fortuitous since treatment started after implantation.
Accordingly, the post implantation loss was low and similar in the control, 50 or 250 mg/kg/day groups (3.3% or 5.0% of implantation sites at 50 or 250 mg/kg/day vs. 4.5% in controls) and slightly higher at 1000 mg/kg/day (10.7% of implantation sites at 1000 mg/kg/day vs. 4.5% in controls; difference statistically significant: p < 0.05).
This value was slightly outside the range of the historical control data: average = 3.9%, mini = 0.8%, maxi = 7.7% (period: 1995-1996; 202 females evaluated).
Since this difference from controls was mainly attributable to a single individual, and since no other signs of embryo or fetotoxicity were observed, it could not be demonstrated that this single event was treatment-related.

Total litter losses by resorption:

not examined

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of resorptions (early and late) per animal was low and similar in the control and 50 or 250 mg/kg/day groups (0.4 or 0.8 at 50 or 250 mg/kg/day vs. 0.6 in controls, corresponding to a rate of 3.3 or 5.0% of implantation sites, respectively vs. 4.5% in controls). At 1000 mg/kg/day, the mean number of resorptions (early and late) per animal was slightly higher (1.3 vs. 0.6 in controls corresponding to a rate of 10.2% of implantation sites vs. 4.5% in controls; difference statistically significant for the rate: p < 0.05); this difference from controls was due to higher rate of late resorption (6.8% of implantation sites vs. 0.9% in controls; difference statistically significant: p < 0.01) attributed to only one female (13 late resorptions out of 16 implants, female Q24440).

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

No dead fetuses were noted in the control, 50 or 250 mg/kg/day groups.
In the high dose-level group, only one fetus was found dead in the female which presented a high rate of resorptions.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean fetal body weight was similar in the control and treated groups (4.00, 3.95 or 4.07 g at 50, 250 or 1000 mg/kg/day, respectively, vs. 3.91 g in controls).

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of live fetuses per animal was similar in the control and treated groups (13.2, 14.3, or 11.4 at 50, 250 or 1000 mg/kg/day, respectively, vs. 11.8 in controls). The rate of live fetuses was slightly lower in the 1000 mg/kg/day group, when compared to the control group, as a consequence of the higher post-implantation loss.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex-ratio was similar in the control and treated groups, and close to the theoretical value of 50%.

Description (incidence and severity):

n/a

Description (incidence and severity):

n/a

External malformations:

no effects observed

Description (incidence and severity):

No external anomalies or malformations were observed in the fetuses of the 50, 250 or 1000 mg/kg/day groups. In the control group, two fetuses (from the same female) presented malformations.

Skeletal malformations:

no effects observed

Description (incidence and severity):

. Malformations
No skeletal malformations were noted in any fetus of the control, 50 or 250 mg/kg/day group. Fused and distorted lumbar was noted in 1/94 fetuses of the high dose-level group. Since this malformation was noted with a very low incidence and was not associated with any other skeletal anomaly or variation, it was considered to be of spontaneous occurrence.
. Anomalies
The total fetal incidence of skeletal anomalies was similar in all control and treated groups i.e. 36/137 (26.3%) at 50 mg/kg/day, 49/158 (31.0%) at 250 mg/kg/day or 24/94 (25.5%) at 1000 mg/kg/day v.s. 34/109 (31.2%) in controls. No relevant or dose-related differences from controls were noted in any recorded anomalies, and ail were commonly recorded findings in fetuses of this rat strain.
. Variations
The total fetal incidence of skeletal variations was similar in all control and treated groups i.e. 107/137 (78.1%) at 50 mg/kg/day, 120/158 (75.9%) at 250 mg/kg/day or 70/94 (74.5%) at 1000 mg/kg/day v.s. 91/109 (83.5%) in controls. No dose-relationship and no statistically significant difference from controls were noted for the principal variations observed (reduced or absence of ossification of sternebrae).

Visceral malformations:

no effects observed

Description (incidence and severity):

. Malformations
No fetal soft tissue malformations were noted in any fetus of any treated group. The two control fetuses malformed at external examination presented several internal soft tissue malformations.
. Anomalies
The litter and the fetal incidences of the few recorded anomalies were similar in control and treated groups, without dose-relationship and close to the higher limit or in the range of the historical control data. Consequently, they were not considered to be treatment-related, but to be of a spontaneous nature.

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

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Developmental effects observed:

no

Table 1 : pregnancy status

 

 

Conclusions:

Test substance administered daily by the oral route at 50, 250 or 1000 mg/kg/day to pregnant Sprague-Dawley female rats during organogenesis did not show any signs of toxicity in the pregnant female and did not produce any embryotoxicity, fetotoxicity or teratogenic effects. At 1000 mg/kg/day, only a slight increase in post-implantation loss was noted, as a contribution of a single female: it could not be demonstrated that this single event was treatment-related. The No Effect Level is defined as 1000 mg/kg/day in terms of maternotoxicity, embryofetotoxicity and teratogenic effects.

Executive summary:

In an OECD TG 414 study, three groups of 25 mated female rats received di-tert-dodecyl polysulfides (TPS 32), by oral gavage at the dose levels of 50, 250 or 1000 mg/kg/day, each day from day 6 to day 15 post-coitum inclusive. Simultaneously, a group of 25 mated females was given the vehicle alone (0.5% carboxymethylcellulose) under the same conditions and acted as a control group.Clinical signs including (including evidence of abortion/resorption) and mortality were checked daily. Food consumption and body weight were recorded at designated intervals during pregnancy. On day 20 post-coitum, females were killed. The gravid uterus was weighed and fetuses were removed by hysterectomy. Females were examined macroscopically. Litter parameters were recorded: number of corpora lutea, implantation sites, resorptions, dead and live fetuses. The live fetuses were weighed, sexed, submitted to an external examination and then to soft tissue or skeletal examinations.

No clinical signs and no unscheduled deaths were observed in any group. No females aborted or presented total resorption in any group. The food consumption and body weight gain of the pregnant females from all treated groups were similar to those of controls. No treatment-related macroscopic findings were observed, in any group.

The post-implantation loss was similar in the 0, 50 and 250 mg/kg/day groups. In the 1000 mg/kg/day group, a slightly increased post-implantation loss (represented mainly by late resorptions, observed in one female) was observed: it could not be demonstrated that this single event was treatment-related. No treatment-related effects were observed on the number of live fetuses per animal, the fetal body weight or the sex-ratio.No treatment-related external, soft tissue and skeletal anomalies or malformations were observed in any group. 

The No Effect Level is defined as 1000 mg/kg/day in terms of maternotoxicity, embryofetotoxicity and teratogenic effects.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

800 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

The study is considered as reliable with a klimisch score of 1.

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

Developmental toxicity study in rat (1997)

In an OECD TG 414 study, three groups of 25 mated female rats received polysulfides, di-tert-dodecyl by oral gavage at the dose levels of 50, 250 or 1000 mg/kg/day, each day from day 6 to day 15 post-coitum inclusive (Richard, 1997). Simultaneously, a group of 25 mated females was given the vehicle alone (0.5% carboxymethylcellulose) under the same conditions and acted as a control group. Clinical signs including (including evidence of abortion/resorption) and mortality were checked daily. Food consumption and body weight were recorded at designated intervals during pregnancy. On day 20 post-coitum, females were killed. The gravid uterus was weighed and fetuses were removed by hysterectomy. Females were examined macroscopically. Litter parameters were recorded: number of corpora lutea, implantation sites, resorptions, dead and live fetuses. The live fetuses were weighed, sexed, submitted to an external examination and then to soft tissue or skeletal examinations. No clinical signs and no unscheduled deaths were observed in any group. No females aborted or presented total resorption in any group. The food consumption and body weight gain of the pregnant females from all treated groups were similar to those of controls. No treatment-related macroscopic findings were observed, in any group. The post-implantation loss was similar in the 0, 50 and 250 mg/kg/day groups. In the 1000 mg/kg/day group, a slightly increased post-implantation loss (represented mainly by late resorptions, observed in one female) was observed: it could not be demonstrated that this single event was treatment-related. No treatment-related effects were observed on the number of live fetuses per animal, the fetal body weight or the sex-ratio. No treatment-related external, soft tissue and skeletal anomalies or malformations were observed in any group. The No Effect Level was higher than 1000 mg/kg/day in terms of maternotoxicity, embryofetotoxicity and teratogenic effects.

Developmental toxicity study in rabbit (CRL 2021c)

In a developmental toxicity study, performed in accordance with GLP and OECD guideline 414, three groups of 22 time-mated New Zealand White rabbits, received the test item daily by gavage from GD 6 to 28 inclusive at 100, 300 or 800 mg/kg bw/day. In the preliminary study, the dose level of 1000 mg/kg bw/day exceeded the Maximal Tolerated Dose (MTD) in pregnant rabbits and was associated with one premature euthanasia for ethical grounds and two abortions and following bodyweight loss and severe decrease in food consumption. A constant dosage volume of 1.5 mL/kg bw/day was used. One additional group of 22 females received the vehicle alone (corn oil) under the same experimental conditions. Test item concentrations were checked in formulations given in the first and last weeks of the study. The animals were checked daily for mortality and clinical signs. Body weight was measured regularly and food consumption was recorded daily. On GD29, surviving females were euthanized and given a macroscopic post-mortem examination. Hysterectomy was performed and the numbers of corpora lutea, implantations, early/late resorptions and live/dead fetuses were recorded. The fetuses were weighed, examined for external abnormalities, and sexed by internal examination of the gonads. Detailed soft tissue and skeletal/cartilage examinations were also performed.

At hysterectomy on GD29, there were 21/22, 20/22, 20/22 and 20/22 pregnant dams in the groups treated at 0, 100, 300 or 800 mg/kg bw/day, respectively.

There were no test item-related effects on mortality or clinical signs.

A body weight loss was observed between GD 6 and 12 (-136g vs. -17 g in controls, p<0.001 between GD 6 and 9) at 800 mg/kg bw/day. This finding was associated with lower, mostly with statistical significance, mean food consumption between GD 6 and 14 (down to -62%, p<0.001, between GD 8 and 9) when compared with controls.  As these test item-related findings were transient and did not impact the mean body weight or mean carcass weight, they were not considered to be adverse. There were no test item-related effects on mean body weight, mean body weight change or mean food consumption at 100 or 300 mg/kg bw/day.

No effects were noted on mean gravid uterus weight or mean carcass weight at any dose level. Mean net body weight change from GD 6 tended to be lower than controls at 800 mg/kg bw/day (-351g vs. -297 g in controls). There were no test item-related effects on mean net body weight change at 100 and 300 mg/kg/day.

No test item-related effects were noted at hysterectomy or macroscopic post-mortem examination at any dose level, and there were no effects on mean fetal body weight and sex ratio.

Minor external abnormalities were noted from 300 mg/kg bw/day [i.e. polyhydramnios (10% and 5% of litters affected at 300 and 800 mg/kg bw/day, respectively, vs. none in controls and 5% in Historical Control Data (HCD)], along with abnormal color of amniotic fluid at 800 mg/kg bw/day). Minor findings of delayed ossification in fetuses were noted with high litter and/or fetal incidences at 800 mg/kg bw/day [i.e. short supernumerary 13th rib (75% of litters affected vs. 61.9% in controls and 68.4% in HCD), unossified hindpaw median phalanx (10% of litters affected vs. none in controls and 5.6% in HCD), and/or unossified pubis (15% of litters affected vs. none in controls and 10.5% in HCD)]; a relationship to the test item could not be ruled out.

Misshapen cartilage of caudal vertebra centrum was also noted at 800 mg/kg bw/day (15% of litters affected, vs. 4.8% in controls and 5.3% in HCD).

There were no test item-related variations at soft tissue or malformations at external, soft tissue or skeletal examination at any dose level.

In conclusion, under the experimental conditions and results of this study, the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 800 mg/kg bw/day, based on the absence of adverse effects on dams at this dose level. The NOAEL for embryo-fetal development was considered to be 800 mg/kg bw/day, based on the absence of adverse effects and test item-related malformations in litters at this dose level.

Preliminary study in rabbit (CRL 2021b)

The objective of this preliminary study was to evaluate the potential toxic effects of the test item on the pregnant female and on embryonic and fetal development, following daily oral administration (gavage) to pregnant female rabbits from implantation to the day before scheduled hysterectomy [Days 6 to 28 post-coitum (p.c.) inclusive], in order to select dose levels for a further main study.

Three groups of eight time-mated female New Zealand White rabbits received the test item at 100, 300 or 1000 mg/kg bw/day by the oral route (gavage), once daily from Days 6 to 28 p.c. One group of eight time-mated females received the vehicle alone (corn oil) under the same experimental conditions and acted as control group. A constant dosage volume of 1.5 mL/kg bw/day was used. All animals were checked daily for mortality, clinical signs and food consumption. Body weights were recorded on Days 2, 5, 6, 9, 12, 15, 18, 21, 24, 27 and 29 p.c.

On Day 29 p.c., the animals were euthanized and a macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed. At hysterectomy, the numbers of corpora lutea, implantations, uterine scars, early and late resorptions and live/dead fetuses were recorded. The fetuses were weighed and examined for oral cavity and external abnormalities. The placenta of each fetus was weighed. All fetuses were then discarded.

At hysterectomy on Day 29 p.c., there were seven, five, eight and four pregnant females with live fetuses at 0, 100, 300 and 1000 mg/kg bw/day respectively.

At 1000 mg/kg bw/day, three dams were prematurely euthanized for ethical reasons or abortion, following body weight loss (-9 to -12%) and a severe decrease in food consumption (down to 5 g/day). The only clinical sign observed at this dose was thin appearance in five dams including the three dams prematurely euthanized. Body weight losses were noted in all dams at the beginning of the treatment period (mainly for the interval of Days 6-9 p.c.: -147 g vs. -19 g in controls, p<0.001). This finding correlated with decreased food consumption over the first week of the treatment period (down to -74% vs. controls, p<0.05, for the interval of Days 8-9 p.c.), but there were no biologically relevant effects on mean body and carcass weights.

At 300 or 100 mg/kg bw/day, there were no test item-related premature deaths, clinical signs or effects on mean body and carcass weights, mean body weight change or mean food consumption.

There were no test item-related macroscopic observations in dams or effects on mean gravid uterus weight, mean hysterectomy data and placental weight at any dose level. There were no effects on fetuses (fetal body weight, external examination).

Based on the results of this study, the high dose level of 1000 mg/kg bw/day exceeded the Maximal Tolerated Dose (MTD) in pregnant rabbits and was therefore considered to be too high to be selected for a future main prenatal developmental toxicity study in rabbits (i.e. associated with two abortions and premature euthanasia due to severe decrease in food consumption).

 

Toxicity to reproduction: other studies

Description of key information

In an OECD TG 408 study, male and female Sprague-Dawley rats administered 0, 100, 300, or 1000 mg/kg bw/day of polysulfides, di-tert-dodecyl by daily gavage for 90 days. No effects on reproductive organ weights or histopathology were observed at 1000 mg/kg bw/day, the highest dose tested.

 

Justification for classification or non-classification

According to the available data and criteria of Regulation no. 1272/2008, no classification is warranted for reproductive and developmental toxicity.