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

Endpoint summary

Description of key information

In the key 28-day repeated dose oral gavage study conducted according to OECD Test Guideline 407 and in compliance with GLP, the NOAEL 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) was 200 mg/kg bw/day in rats (Hita Laboratory, 2000a).

 

The repeated dose toxicity endpoint will be updated upon completion of the planned 90-day OECD Test Guideline study with the read-across substance bis[3-(triethoxysilyl)propyl]polysulfides (draft decision received; DEV-01-2119463597-25-0000-TPE-3), especially in respect of the renal findings (tubular basophilia) identified as in the OECD Test Guideline 407 studies (S2: Hita Laboratory, 2000a; bis[3-(triethoxysilyl)propyl]polysulfides: Hita Laboratory, 2000b) and which was identified as adverse in the OECD Test Guideline 443 (EOGRTS) study with this substance (draft report, Charles River Laboratories, 2022; see Section 5.9.3 (Summary and discussion of reproductive toxicity, Effects on fertility).

No repeated dose data are available for the dermal and inhalation routes.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20.04.1999 to 10.02.2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

full histopathology of required tissues was not carried out.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: Five weeks.
- Weight at study initiation: Males: 139.5-162.9 g. Females: 119.6-144.0 g.
- Fasting period before study:
- Housing: Individually in hanging stainless steel cage with wire mesh floor.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: yes, but period not stated.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): 55 ± 10
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 17.06.1999 To: 29.07.1999

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was weighed accurately and dissolved with olive oil to make a 10 %w/v solution from which the 2.0, 0.4 and 0.08 % w/v solutions were prepared. 10 and 2.0 %w/v solutions were prepared weekly, and the 0.4 and 0.08 % w/v were prepared prior to use.


VEHICLE
- Justification for use and choice of vehicle (if other than water): No data.
- Concentration in vehicle: 10, 2.0, 0.4 and 0.08 %w/v.
- Amount of vehicle (if gavage): No data
- Lot/batch no. (if required): 004RRA
- Purity: No data

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 days (plus a 14 day recovery period with no treatment for the high dose group)

Frequency of treatment:

Daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

200 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:

Six

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a 14 day preliminary study.
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: No data
- Post-exposure recovery period in satellite groups: yes, vehicle only and high dose group recovery groups.
- Section schedule rationale (if not random): No data

Positive control:

None

Observations and examinations performed and frequency:

The day of the initiation of dosing was defined as Day 1, and the day before as Day -1. The week of the initiation of the dosing period was defined as Week 1. Also, the next day of the final dosing was defined as Recovery day 1, and the week of the initiation of the recovery period as Recovery week 1.
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least daily.


DETAILED CLINICAL OBSERVATIONS: No data


BODY WEIGHT: Yes
- Time schedule for examinations: Before dosing on Day -2, then on Days 1, 3, 8, 12, 17, 21, 26 and 28, and during the recovery period on Days 1, 5, 10 and 14. Plus immediately before necropsy for calculation of relative organ weights.


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/rat/day: Yes


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION: No


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the termination of dosing and at the end of the recovery period.
- Anaesthetic used for blood collection: Yes (ether).
- Animals fasted: Yes, overnight (16-20 hours).
- How many animals: All survivors.
- Parameters checked in table 1 were examined. In addition a myelogram was performed as haematological abnormalities were noted in the preliminary toxicity study. Thin bone marrow smears were prepared from the left femurs in the first three animals of each group at the terminal necropsy of the dosing and recovery periods, and examined in the 1000 mg/kg and vehicle control groups on the terminal necropsy of the dosing period. Parameters examined were: myerobrasts, promyelocytes, myelocytes, metamyelocytes, stab neutrophils, segmented neutrophils, eosinophils, basophils, lymphocytes, plasmacytes, megakaryocytes, retoperitheliums, mastocytes, monocytes, proerythroblasts, basoerythroblasts, polychromatic erythroblasts, neuerythroblasts, and myeloblast series/erythroblasts series (M/E ratio).


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the termination of dosing and at the end of the recovery period.
- Animals fasted: Yes, overnight (16-20 hours).
- How many animals: All survivors.
- Parameters checked in table 1 were examined.


URINALYSIS: Yes
- Time schedule for collection of urine: At termination of the dosing and recovery periods.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table 1 were examined.


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 2)
HISTOPATHOLOGY: Yes (see table 2)

Statistics:

Data regarding body weights, food intakes, haematology, clinical chemistry, urinalysis and organ weights were analysed using the Bartlett's test for homogeneity of variance. If the variances were homogeneous at a significance level of 5%, one way analysis of variance was performed. If there was a significant difference in this analysis, the difference between the vehicle group and each of the treatment groups was analysed by the Dunnett's test (equal number of data). If the variances were not homogeneous the Kruskal-Wallis's test was used. If there was a significant difference in this analysis, the difference between the vehicle control group and each of the treatment groups was analysed by the nonparametric Dunnett's test (equal number of data).

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

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:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY: During the dosing period salivation was observed in males of the vehicle control group (8/12), 8 mg/kg bw/day group (3/6), 40 mg/kg bw/day group (5/6), 200 mg/kg bw/day group (6/6) and 1000 mg/kg bw/day group (12/12). In females the salivation was observed in the vehicle control group (4/12), 8 mg/kg bw/day group (3/6), 40 mg/kg bw/day group (3/6), 200 mg/kg bw/day group (5/6) and 1000 mg/kg bw/day group (12/12). Staining around the nose and mouth was observed in 3/12 males and 1/12 females in the high dose group. Hair loss was also observed in the high dose males (2/12). These clinical signs were not observed in the recovery period. Salivation is commonly observed in gavage studies.


BODY WEIGHT AND WEIGHT GAIN: There were no abnormalities in either the dosing or recovery periods.


FOOD CONSUMPTION: There were no abnormalities in either the dosing or recovery periods.


HAEMATOLOGY: There were no abnormalities in males. At termination of dosing there was a decrease in red blood cell count, and an increase in activated thromboplastin time in high dose females. At the end of the recovery period an increase in red blood cell count and a decrease in mean corpuscular haemoglobin was noted in the high dose group.


CLINICAL CHEMISTRY: At the end of the dosing period there was an increase in calcium level in the high dose group males. In high dose females there were increases in GPT and gamma-GTP activities, total cholesterol, total protein, albumin levels, and a decrease in alkaline phosphatase activity. There was also a tendency towards increased GPT activity due to an increase in one animal of the 200 mg/kg bw/day. There were no such findings at the end of the recovery period.


URINALYSIS: There was a tendency towards increased ketone bodies in the high dose groups for males and females, and the 200 mg/kg bw/day group for females at the end of the dosing period. There were no such findings at the end of the recovery period.


ORGAN WEIGHTS: Small testes were observed in one male of the vehicle control group at the end of the recovery period, but these were not analysed statistically as they were considered to be a malformation. In males at termination of dosing, increases in relative liver weights were noted in the 200 and 1000 mg/kg bw/day groups. Decreased absolute testis weights were noted in the 8 and 40 mg/kg bw/day groups, and decreased relative kidney weights were noted in the 200 mg/kg bw/day group. In females there was an increase in absolute liver weight in the 200 mg/kg bw/day group, and an increase in absolute and relative liver weights in the 1000 mg/kg bw/day group. There were no such findings at the end of the recovery period.


GROSS PATHOLOGY: At the end of the dosing period males had enlargement (2/6) of the liver and loss of hair (1/6) in the highest dose group. Females of the highest dose group (4/6) had liver enlargement. At the termination of the recovery period no abnormalities were observed in females, but there was one male of the vehicle control group that had small testes.


HISTOPATHOLOGY: NON-NEOPLASTIC: Table 3 summaries the histopathology findings.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Table 3: Incidence of selected pathologies

Parameter	n=6/sex		Dose level (mg/kg bw/day)
		Control		8	40	200	1000	Recovery control	Recovery 1000
Liver enlargement	M	0		0	0	0	2	0	0
	F	0		0	0	0	4	0	0
Testes - small	M	0		0	0	0	0	1	0
	F	-		-	-	-	-	-	-
Skin - Loss of hair	M	0		0	0	0	1	0	0
	F	0		0	0	0	0	0	0
Liver – Centrilobular hypertrophy of hepatocytes	M	0		-	-	-	4/6 (+)	0	0
	F	0		-	-	-	3/6 (+)	0	0
Centrilobular lipid droplets of hepatocytes	M	0		-	-	0	0	0	0
	F	0		-	-	3/6 (+)	0	0	0
Disseminated microgranuloma	M	1/6 (+)		-	-	1/6 (+)	0	1/6 (+)	0
	F	0		-	-	0	2/6 (+)	1/6 (+)	0
Periportal lipid droplets of hepatocytes	M	1/6 (+)		-	-	0	0	0	0
	F	1/6 (+), 1/6(++)		-	-	1/6 (±), 2/6 (+)	3/6 (+), 2/6 (++)	0	0
Periportal microgranuloma	M	0		-	-	0	0	0	0
	F	1/6 (++)		-	-	1/6 (+)	2/6 (+)	0	0
Single cell necrosis of hepatocytes	M	0		-	-	0	0	0	1/6 (+)
	F	0		-	-	0	0	0	2/6 (+)
Kidney – Basophilic tubules	M	0		-	-	0	4/6 (+)	0	1/6 (±), 2/6 (+)
	F	0		-	-	0	1/6 (±)	0	1/6 (±), 2/6 (+)
Increased eosinophilic bodies	M	0		-	-	0	0	0	2/6 (±)
	F	0		-	-	0	0	0	0
Increased hyaline droplets	M	0		-	-	0	1/6 (± ), 1/6 (+)	0	3/6 (±)
	F	0		-	-	0	0	0	0
Subcapsular solitary cyst	M	1/6 (+)		-	-	0	0	0	0
	F	0		-	-	0	0	0	0
Testes – diffuse atrophy of seminiferous tubules	M	-		-	-	-	-	1/1 (+++)	-
	F	-		-	-	-	-	-	-
Leydig cell hyperplasia	M	-		-	-	-	-	1/1 (++)	-
	F	-		-	-	-	-	-	-
Skin – scab formation	M	-		-	-	-	1/1 (+)	-	-
	F	-		-	-	-	1/1	-	-

± very slight, + slight, ++ moderate, +++ severe

Conclusions:

In reliable 28-day repeated dose oral gavage study conducted according to OECD Test Guideline 407 and in compliance with GLP, the NOAEL for S2 was 200 mg/kg bw/day in rats. This was based on histopathological changes in the liver and kidney.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

System:

other: gastrointestinal and urinary systems

Organ:

kidney

liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In the key 28-day repeated dose oral gavage study conducted according to OECD Test Guideline 407 and in compliance with GLP, rats were administered 0, 8, 40, 200, and 1000 mg/kg bw/day of S2, with an olive oil vehicle. In addition, there was a 14-day recovery period for the three lowest doses and the highest tested. In this study no deaths occurred, there were no abnormalities in clinical signs, body weights and food intakes during the dosing period. The S2 NOAEL determined in this study was 200 mg/kg bw/day based on the histopathological changes centrilobular hypertrophy of the hepatocytes and basophilic tubules in the kidney in the 1000 mg/kg males and females, and hyaline droplets in the kidney in the 1000 mg/kg males, and periportal lipid droplets of the hepatocytes in the 1000 mg/kg females. In the recovery test, basophilic tubules in the kidney of male and females, and increased hyaline droplets in the kidney of males remained in the 1000 mg/kg groups. The other treatment-related changes at the termination of the dosing period were not observed after recovery period. The histopathological effects in the high dose group (1000 mg/kg bw/day) are of minor toxicological relevance and did not result in any clinical symptoms. The experience of the lead registrant is that 4 weeks or longer are better to demonstrate reversibility of effects. If a minor toxicological effect is reversible, it is not considered an adverse effect. In summary, this study gives no indication of a severe toxicological concern even in the high dose group. Although the study is conducted according to current OECD test guideline and GLP, it is assigned reliability 2 because a thorough histopathological investigation of required tissues was not carried out.

 

In a 28-day oral supporting study, a substance similar to the registered substance (low purity S2) contained a higher proportion of a trisulfide (S3) isomer (SafePharm Laboratories 2002a). The study was conducted in accordance with Japanese national guidelines for 28-day oral toxicity testing and in compliance with GLP, the test material contained mainly the S2 and S3 constituents, with a small amount of S1 impurity. Dose levels were 0, 61, 305, 1221 mg/kg bw/day. There were no deaths, significant clinical signs or effects on body weight, urinalysis, clinical chemistry and haematological parameters measured. Males treated with 1221 or 305 mg/kg bw/day showed a slight but statistically significant increase in absolute liver weight with a statistically significant increase in relative (to terminal body weight) liver weight noted in both sexes treated with 1221 and 305 mg/kg bw/day. In most cases individual values were within the normal range for rats of the strain and age used, and there were no histopathological correlates. However, the weight increases were apparently dose-related and an association with treatment could not be ruled out. Other isolated findings were considered to be incidental and unrelated to treatment. Treatment-related kidney changes were detected. A higher incidence and marginally greater general severity of groups of basophilic tubules were observed for male rats dosed at 1221 mg/kg bw/day. Although this effect was equivocal it was to some extent confirmed by a similar residual finding among recovery group 1221 mg/kg bw/day male rats. The NOAEL was judged to be 1221 mg/kg bw/day and the NOEL was 61 mg/kg bw/day.

 

Two 28-day oral studies are also available for the related substance polysulfides which is a reaction mass containing principally the S2 (the registered disulfide, at 15-60%), S3 (the trisulfide, at 25-40%) and S4 (the tetrasulfide, at 5-30%) as constituents.

In the first supporting 28-day repeated dose toxicity study with polysulfides, four dose groups (8, 40, 200 and 1000 mg/kg bw/day), a 14-day recovery group (1000 mg/kg/day) and a vehicle control group were examined. In this study no deaths occurred and there were no adverse findings in clinical observations, body weights and food intakes during the dosing period. At the end of the treatment period there was an increase in liver weights in males and females, and an increase in kidney weights in males in groups dosed with 1000 mg/kg bw/day. Centrilobular hepatocyte hypertrophy in males and females, and basophilic renal tubules in males were observed at the end of the administration period in the 1000 mg/kg bw/day group. There was also an increase in acidophilic bodies and hyaline droplets observed in males in the 200 mg/kg bw/day group. The basophilic renal tubules in males, and increased relative liver weight in females in the highest dose group were still present at the end of the recovery period. However, the effects in the liver appeared to be reversible, as weights were reducing and histopathological changes were not apparent by the end of the recovery period.

 

Therefore, the majority of the treatment-related changes present at the termination of the dosing period were not observed after the 14 -day recovery period. The histopathological effects observed in the high dose group (1000 mg/kg bw/day) are of minor toxicological relevance and did not result in any clinical symptoms.

 

Thus, the NOAEL determined for polysulfides in this study was 200 mg/kg bw/day based on the enlarged liver and histopathological changes (centrilobular hypertrophy) of the hepatocytes in the 1000 mg/kg bw/day males and females in the 1000 mg/kg bw/day females (Hita Laboratory, 2000).

 

A second supporting study with polysufides was conducted according to OECD Test Guideline 407 and in compliance with GLP, with polysulfides administered to Wistar rats (10/sex/dose) for 28 days by oral gavage (Degussa Institute of Toxicology, 1983). Twenty (20) animals were treated with 2309 mg/kg bw/day (2.15 mL/kg) of test substance, and 20 were control animals that received an equal volume of tap water. Following 28 days of treatment, 10 animals from each group were observed for an additional 14 days without any further treatment (recovery groups). There were no deaths or clinical findings. Nor were there any adverse treatment-related effects on food consumption, body weight, reflexes, haematology, clinical chemistry, gross pathology, histopathology and organ weights. The polysulfides NOAEL was therefore at least 2309 mg/kg bw/day.

 

A limitation of both polysulfide studies was the lack of full histopathology on the 32 tissues required by OECD TG 407. The substance in the studies with polysulfides contained up to 25% of S2. The other constituents are the structurally-similar analogue substances S3 and S4. These studies are included to support read-across justification for reproductive toxicity. See discussion in the reproductive toxicity section endpoint summary (CSR section 5.9) for a detailed justification of the read-across approach. The repeated dose 28 day studies on S2 and related substances (low purity S2 and polysulfides) show similar target organs and NOAELs indicating that the toxicity profiles are comparable. The histopathological effects which were recorded in the high dose groups in all available studies were of minor toxicological relevance and did not result in any clinical symptoms. Thus, overall, these studies give no indication of a severe toxicological concern up to 1000 mg/kg bw/day.

 

The repeated dose toxicity endpoint will be updated upon completion of the planned 90-day OECD Test Guideline study with the read-across substance bis[3-(triethoxysilyl)propyl]polysulfides (draft decision received; DEV-01-2119463597-25-0000-TPE-3), especially in respect of the renal findings (tubular basophilia) identified as in the OECD Test Guideline 407 studies (S2: Hita Laboratory, 2000a; bis[3-(triethoxysilyl)propyl]polysulfides: Hita Laboratory, 2000b) and which was identified as adverse in the OECD Test Guideline 443 (EOGRTS) study with this substance (draft report, Charles River Laboratories, 2022; see Section 5.9.3 (Summary and discussion of reproductive toxicity, Effects on fertility).

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 does not require classification for repeated dose toxicity according to Regulation (EC) 1272/2008.