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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 11 - June 6, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

941-787-9

Cas Number:

98222-50-5

Molecular formula:

C16 H34 S3

IUPAC Name:

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

Test material form:

liquid

Details on test material:

Name: DAILUBE IS-30
Chemical Name: 1, 3-bis (2, 4, 4-trimethylpentan-2yl) trisulfane
Use: Lubricant Additive
Molecular Formula: CH3-C(CH3)2-CH2-C(CH3)2-SSS-C(CH3)2-CH2-C(CH3)2-CH3
Molecular Weight: 322 g/mol
Appearance Pale yellow
Physical State: Liquid
Batch Number: L002
Purity: 95.4% by weight
Storage: Room temperature, in the dark
Expiry Date: 2016/8/15

Test animals

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)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% methylcellulose solution containing 0.1% tween 80

Details on exposure:

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 suspe nsions (low, middle and high-dose formulations).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Three fractions (10-mL 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 concent ration 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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

320 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12

Control animals:

yes

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

1) Administration Period
There were no abnormalities in males and females during the administration period.

2) Recovery Period
No animals showed abnormalities.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1) 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.

2) 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):

1) 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:

not specified

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):

1) End of Administration Period
There were no changes that are considered to be related to administration of the test article.
In females of the mating group, a significant low value was observed in the reticulocyte count (RETIC) in the 320 mg/kg group; however, since there is no abnormality in other erythroid parameters, it was considered that there is no toxicological significance. A significant high value was observed in the monocyte count (MONO) of the leukocyte fraction; however, since there is no abnormality in the white blood cell count and related change is not observed in other leukocyte fraction, it is considered to be a change without toxicological significance.
In females of the non-mated group, a significant high value was observed in the mean red blood cell volume (MCV) in the 320 mg/kg group; however, since there is no abnormality in other erythroid parameters, it is considered to be a change without toxicological significance.

2) End of Recovery Period
In male, a significant high value in the neutrophil count (NEUT) of the leukocyte fraction was observed in the 320 mg/kg group, and in females, a significant low mean corpuscular hemoglobin concentration (MCHC) and a significant high value in fibrinogen amount (FIB) were observed in the same dose group. However, since all of the changes described in this section were not observed at the end of the administration period and no other relevant changes were observed, so they were judged to be incidental significant differences.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

1) End of Administration Period
There were no changes that are considered to be related to administration of the test article.
In males, ALP had a significant low value in the groups at 30 mg/kg or more; however, it was considered to be a change without toxicological significance, since no correlation to dose was observed and it was almost within the normal range of the historical background data of the test facility. Glucose was significantly low in the 320 mg/kg group; however, it was judged to be an incidental significant difference, since it was a slight change and within the normal range of the historical background data of the test facility. In addition, a significant low value was observed in total bile acids in the 100 mg/kg group; however, because it is a change unrelated to the dose, it was judged to be an incidental significant difference.
Significantly high values of ALP and total cholesterol and significant low values of chlorine were observed in females of the mating 30 mg/kg group; however, because it is a change unrelated to the dose, it was judged to be an incidental significant difference.

2) End of Recovery Period
In males, a significant low value was observed in the A/G ratio in the 320 mg/kg group; however, since the abnormality was not observed at the end of the administration period and no abnormality was observed in albumin and liver function parameters, it was judged to be an incidental significant difference.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

1) 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.

2) 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.

Behaviour (functional findings):

effects observed, non-treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Examination at the End of Administration Period and Recovery Period
The changes that were considered to be caused by administration of the test article were observed in the kidney (males) and liver (males, females of the mating group and females of the non-mated group).
Kidney: In males at the end of administration, regenerated renal tubules and accumulation of hyaline droplets in the proximal renal tubule was observed at 30 mg/kg or more and granular cast was observed at 100 mg/kg or more. In addition, interstitial cell infiltration was observed at 320 mg/kg with above the frequency of the control group. Regenerated renal tubules, granular cast and interstitial cell infiltration increased in the incidence or extent as the degree of accumulation of hyaline droplets in the proximal renal tubule enhanced. In addition, immunohistochemical staining of α2u globulin carried out in representative animals (Animal Nos: 4001 and 4002) revealed that the hyaline droplet was composed of α2u globulin. On the other hand, immunohistochemical staining of α2u globulin carried out in some female animals (Animal Nos: 1113, 4113 and 4114) did not show positive reaction in the proximal tubular epitheria.
At the end of recovery, the degree or frequency of accumulation of hyaline droplets in the proximal renal tubule, granular cast and interstitial cell infiltration attenuated. Regenerated renal tubules slightly weakened as compared to the end of administration.
Liver: At the end of administration, 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. At the end of recovery, the frequency and extent of hypertrophy of centrilobular hepatocytes decreased in both sexes.
Other than the above, there were some changes in various organs/tissues; however, they were considered to be incidental based on the incidence of their occurrences and their pathological nature.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no statistically significant differences between the control group and any test article administration group in the index of animals with abnormal estrous cycle, in the number of estruses or sexual cycle (estrous cycle).

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

All females had copulated by day 4 of mating, and there were no statistically significant differences between the control group and any test article administration group in the number of days until copulation, copulation index, insemination index or fertility index.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 320 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

30 mg/kg bw/day (nominal)

Organ:

other: body weight gain

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no statistically significant differences between the control group and any test article administration group in the viability index on PNDs 4 and 13.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 100 mg/kg group, there was a significant low value in body weight on PNDs 7 and 13 and body weight gain (between PND 0 and 13) in male pups.
In the 320 mg/kg group, there was a significant low value in body weight on PNDs 7 and 13 and body weight gain (between PND 0 and 13) in male and female pups.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no significant differences between the control group and any test article administration group in T4 in males in the mating group at the end of the administration period or in the pups on PND 13.

Urinalysis findings:

not specified

Sexual maturation:

not specified

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

There were no statistically significant differences between the control group and any test article administration group in the AGD of male or female pups.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There were no significant differences between the control group and any test article administration group.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

significant differences between the control group and any test article administration group.
A significant low value was observed in the autopsy body weight of male pups in the 100 mg/kg group or of both sexes in the 320 mg/kg group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes in any pups.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no significant differences between the control group and any test article administration group in T4 in males in the mating group at the end of the administration period or in the pups on PND 13.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

There were no changes that considered to be related to administration of the test article in the gestation index, gestation length in days, number of implantation sites, post-implantation loss index, delivery index, number of liveborn pups, number of stillborn pups, live birth index and index of external abnormalities.
In the 30 mg/kg group, a significantly low value was recorded in the number of implantation sites; however, it was judged to be an incidental significant difference since it was not dose-related.
In the delivery condition of the pregnant animals, all females delivered during the period from GD 21 to 23, and the treatment of the placenta and amnion was normal. In the lactation condition, there were no abnormalities in the gathering of pups, nesting or lactation behavior in any dam.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

2

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 320 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

30 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

yes

Applicant's summary and conclusion

Conclusions:

The NOAEL on the reproductive-developmental toxicity was judged to be 320 mg/kg for male parent animals and dams and 30 mg/kg for pups.