1,3-bis(2,4,4-trimethylpentan-2-yl)trisulfane

Endpoint summary

Description of key information

Repeated dose toxicity, rats: oral (female), 42 days - NOAEL = 30 mg/kg/day

Repeated dose toxicity: inhalation - no study available

Repeated dose toxicity: dermal - no study available

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

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:

April 11, 2017 - June 6, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The strain of rats employed in this study was chosen since it is widely used in general toxicity studies and reproductive/developmental toxicity studies, its characteristics are well known, and ample background data are available.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Atsugi Breeding Centre, Charles River Laboratories Japan, Inc.
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: 8 weeks
- Weight at study initiation: 422 to 488 g for males and 232 to 284 g for females
- Fasting period before study:
- Housing: plastic cages (W 440 × D 275 × H 180 mm: Hanyu-Seimitsu, Co., Ltd.) with bedding (ALPHA-dri, Shepherd Specialty Papers, Inc., Lot Nos. 11116 and 12216)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3
- Humidity (%): 50 ± 20%
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light): 12/12 (lighting: 07:00 to 19:00)

Route of administration:

oral: gavage

Details on route of administration:

Dose volume was set at 5 mL/kg body weight and the dosing formulation was administered by gavage using a stomach tube.
Administered orally by gavage at 30, 100 or 320 mg/kg/day for 14 days before mating throughout the mating period until the day before necropsy (42 days in total) to males, for 14 days before mating and during the mating and gestation periods until day 13 of lactation (50 to 54 days in total) to females

Vehicle:

other: 0.5% methylcellulose solution containing 0.1% tween 80

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
For each dose concentration, the requisite amount of test article was weighed. An appropriate amount of vehicle was added, ultrasonic irradiation and stirring with a stirrer were carried out to suspend. Vehicle was added to make the specified volume and to prepare the 6, 20 and 64 mg/mL test suspensions (low, middle and high-dose formulations).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Three fractions (10mL each from the upper, middle and lower layers) were collected from each concentration and analyzed by HPLC method. In the results, the percentage to the nominal concentration ranged from 100.5 to 105.6 % and coefficient of variation (CV) ranged from 0.5 to 2.5%.

Duration of treatment / exposure:

42 days for males (14 days prior to mating and 28 days after the start of mating),
50 to 54 days for females in the mating group (14 days prior to mating and throughout the mating and gestation periods until day 13 of lactation) and
42 days for females in the non-mated group.

Frequency of treatment:

Daily

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

320 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12

Control animals:

yes

Observations and examinations performed and frequency:

3 times a day

Statistics:

Bartlett’s test for homogeneity of variance; Homogeneous data were analyzed by Dunnett’s test; Heterogeneous data were analyzed by Steel test. Homogeneous data were analyzed by Student's t-test; Heterogeneous data were analyzed by Aspin-Welch's t-test;
The post-implantation loss index, delivery index, index of external abnormalities, livebirth index and viability index on PND 4 and 13 were analyzed by Wilcoxon’s rank-sum test;
The index of animals with abnormal estrous cycle, copulation index, insemination index,
fertility index, gestation index and sex ratio were calculated from the number of animals with abnormal estrous cycle, number of copulated animals, number of males that impregnated females, number of pregnant females and number of females that delivered liveborns, number of male or female pups which were counted for each group, and then analyzed by Fisher’s exact test;

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Administration Period:
There were no abnormalities in males and females during the administration period including the gestation and lactation periods.

Recovery Period:
No animals showed abnormalities

Grip Strength
Final Week of Administration Period
There were no effects of administration of test article on any test article administration group.

Final Week of Recovery Period
The males in the 320 mg/kg group showed a significantly low value in the forelimbs; however, it was judged to be an incidental significant difference since it was a mild change only in the forelimbs, there were no abnormalities in the final week of administration and there were no related changes in the detailed clinical observation.


Motor Activity
Final Week of Administration Period
There were no changes that are considered to be related to administration of the test article

Final Week of Recovery Period
No animals showed abnormalities

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Administration Period
In females of the mating group, at 320 mg/kg, significant low values were observed in the body weight on GDs 7, 14 and 20 and body weight gain during the gestation period, and a significant low value in body weight gain during the gestation period was observed at 100 mg/kg.
In the males and non-mated females group, there were no changes that are considered to be related to administration of the test article.
Females of the 30 mg/kg mating group showed a significantly low value in body weight on GD 20; however, no significant differences were observed in body weight gain during the gestation period and it was not a dose-dependent change and thus judged to be an incidental significant difference. A significant low value was observed in body weight on LD 4 in the 320 mg/kg group; however, it was judged to be an incidental significant difference as it is a transient slight change and body weight gain during the lactation period is not different from the control group.

Recovery Period
There were no significant differences between the 320 mg/kg group and the control group in males or females.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Administration Period
In females of the mating group, at 320 mg/kg, a significant low value or low trend were observed after GD 7, and the total food consumption during the gestation period was significantly low.
In males at 100 and 320 mg/kg and females of the non-mated group at 320 mg/kg, significant low values were observed on Day 2 of administration; however, it was judged to be a change without toxicological significance as it is a transient slight change and body weight during the pre-mating period is not different from the control group.
In females of the mating group, significant low values were observed on Day 2 of administration in the group of 30 mg/kg or more, and in the 100 and 320 mg/kg group, the total food consumption during the pre-mating period showed a significantly low value; however, it was judged to be a change without toxicological significance as it is a transient change and body weight during the pre-mating period is not different from the control group. Significant low values were observed on GD 7 at 30 and 100 mg/kg; however, it was judged to be a change without toxicological significance as it is a transient change and total food consumption during the gestation period is not different from the control group. There was no abnormality during the lactation period.
2) Recovery Period
There were no significant differences between the 320 mg/kg group and the control group in males. In females at 320 mg/kg, a significant high value was observed on day 2 of recovery, and the total food consumption during the recovery period showed a significant high value; however, it was judged to be a change without toxicological significance as body weight during the recovery period is not different from the control group.

Food efficiency:

effects observed, treatment-related

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes that are considered to be related to administration of the test article

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes that are considered to be related to administration of the test article

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Final Week of Administration Period
In males, small round epithelial cells (SREC) increased in urinary sediments in groups of 30 mg/kg and above.
No abnormalities were observed in other qualitative items in males and in qualitative items in females of the non-mated group, and no significant difference was observed in the urine volume between the control group and the test article administered group.

Final Week of Recovery Period
In males, small round epithelial cells (SREC) increased in urinary sediment in the 320 mg/kg group. However, there was a tendency to recover regard with the grade of change.
No abnormalities were observed in other qualitative items in males and qualitative items in females, and no significant difference was observed in the urine volume between the control group and the test article administered group.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

In the kidney, accumulation of α2µ globulin-derived hyaline droplets was observed in proximal renal tubular epithelial cells at 30 mg/kg or more in males, and regenerated renal tubules, granular cast and interstitial cell infiltration was observed with the increasing degree of this hyaline droplet. It is known that the accumulation of α2µ globulin is species-specifically expressed in male rats, which is enhanced by the administration of chemicals and further induces lesions characterized by renal tubular injury. From these facts, the lesions of the kidneys found in this study were considered not to be able to extrapolate to humans who don’t produce α2µ globulin, and were considered to have no toxicological significance. In addition, in males at 30 mg/kg or more, small round epithelial cells increased in the urinary sediment and kidney weight increased. However, both changes were thought to be secondary changes related to the histological findings of the kidney, and were considered to have no toxicological significance.

In the liver, hypertrophy of centrilobular hepatocytes was observed in males at 30 mg/kg or more, females of the mating group at 100 mg/kg or more, and females of the non-mated group at 320 mg/kg. Electron microscopy examination performed in some animals showed an increase of smooth endoplasmic reticulum in hepatocytes of the centrilobular zone. Therefore, the hypertrophy of hepatocytes is considered to be attributable to the induction of drug metabolizing enzymes as an adaptive response of the living body, and was considered to be of no toxicological significance. In addition, males at 320 mg/kg and females of the mating group at 100 mg/kg or more showed an increased liver weight. However, this increase regarded as secondary changes related to the histological findings of the liver was considered to have no toxicological significance.

At the end of recovery, accumulation of hyaline droplets in the proximal renal tubular epithelial cells of the kidney, granular cast, interstitial cell infiltration and the regenerated renal tubule showed recovery or a tendency to recover following the drug withdrawal. Therefore, all the changes in the kidneys due to accumulation of α2µ globulin are considered as reversible.

Recovery of hypertrophy of centrilobular hepatocytes in the liver is also suggests that it is a reversible change. In consideration of extrapolation to humans, it can be concluded that the toxicological effect on specific tissues was not induced up to 320 mg/kg, the maximum dose, in this study.

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

effects observed, treatment-related

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 320 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

30 mg/kg bw/day (nominal)

Conclusions:

Based on these results, the repeated oral administrations of DAILUBE IS-30 under the conditions of this study caused suppressed body weight gain during the gestation period in females at 100 mg/kg or more. Therefore, it was judged that the no-observed-adverse-effect level (NOAEL) for the repeated dose toxicity of the test article was 320 mg/kg for males and 30 mg/kg for females.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

30 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was conducted (Gotemba Laboratory, 2017, Study R-1200) to assess the general toxicity and reproductive toxicity of Dailube IS-30 in the rat. The study was conducted in accordance with OECD 422 guideline and in compliance with GLP.

 

Dailube IS-30 was suspended in 0.5% methylcellulose solution containing 0.1% tween 80 and administered by oral gavage at 30, 100 or 320 mg/kg/day to twelve male and female Sprague-Dawley strain SPF rats. In addition, further two groups of 10 female rats, control group at dosage levels of 0 and 320 mg/kg group as the non-mated group. Males rats were administered the test substance for total of 42 days, females in the mating group were administered the test substance for total of 50 to 54 days, whereas females in the non-mate group for total of 42 days. Moreover, for some animals in the 0 and 320 mg/kg groups (5 males in the mating group and 5 females in the non-mated group), a 14-day recovery period was provided after the 42-day administration to examine the reversibility of the toxic changes. The animals in the control group received the vehicle, 0.5% methylcellulose solution containing 0.1% tween 80, alone in the same manner.

 

No test article-related toxicologically meaningful changes were noted in detailed clinical observation, manipulative tests, grip strength, motor activity, urinalysis, hematological examination, blood chemistry examination, T4 concentration in blood, organ weight, or macroscopic and histopathological examinations.Change in body weight and food consumption, as well as suppression of body weight gain with a decrease in food consumption during the gestation period was observed at 320 mg/kg in females of the mating group and a tendency of suppressed body weight gain at 100 mg/kg during the gestation period. These were considered to be due to the influence of administration of the test article.

 

Test article-related changes were observed in the kidney (males) and liver (males, females of the mating group and females of the non-mated group). Accumulation of α2u globulin-derived hyaline droplets was observed in the kidney of male rats. It is known that the accumulation of α2u globulin is species-specifically expressed in male rats, hence the lesions of the kidneys found in this study were considered not to be able to extrapolate to humans who don’t produce α2u globulin and were considered to have no toxicological significance. In the liver, hypertrophy of centrilobular hepatocytes was observed in males at 30 mg/kg or more, females of the mating group at 100 mg/kg or more, and females of the non-mated group at 320 mg/kg. The observed hypertrophy of hepatocytes is considered to be attributable to the induction of drug metabolizing enzymes as an adaptive response of the living body and was considered to be of no toxicological significance. At the end of recovery, the frequency and extent of hypertrophy of centrilobular hepatocytes decreased in both sexes.

 

Based on these results, the repeated oral administrations of DAILUBE IS-30 under the conditions of this study caused suppressed body weight gain during the gestation period in females at 100 mg/kg or more. Therefore, it was judged that the no-observed-adverse-effect level (NOAEL) for the repeated dose toxicity of the test article was 320 mg/kg for males and 30 mg/kg for females.

Justification for classification or non-classification

As a change observed in the parent animal, suppression of body weight gain during pregnancy was observed in the female administered 100 mg/kg or more. But body weight suppression was not observed up to the maximum dose during pre-mating and lactation periods in female animals.
In the kidney, accumulation of a2u globulin-derived hyaline droplets was observed in proximal renal tubular epithelial cells in male animals, and regenerated renal tubules, granular cast and interstitial cell infiltration was observed with the increasing degree of this hyaline droplet. It is known that the accumulation of a2u globulin is species-specifically expressed in male rats, which is enhanced by the administration of chemicals and further induces lesions characterized by renal tubular injury l), l)_ From these facts, the lesions of the kidneys found in this study were considered not to be able to extrapolate to humans who don't produce a2u globulin, and were considered to have no toxicological significance. In addition, in male animals, small round epithelial cells increased in the urinary sediment and kidney weight increased. However, both changes were thought to be secondary changes related to the histological findings of the kidney, and were considered to have no toxicological significance.
In the liver, organ weight was increased and hypertrophy of centrilobular hepatocytes was observed in male and female animals. These changes were considered to attributable to the induction of drug metabolizing enzymes as an adaptive response of the living body, and were considered no toxicological significance.

In conclusion, there was no finding that toxicologically significant changes will have affected the function or morphology of a tissue/organ, or has produced serious changes to the biochemistry or hematology of the organism and these changes are relevant for human health.

GHS classification was follows.
Because the data is conclusive but not sufficient for classification, it is judged that GHS classification of STOT-RE is not classified.