Reaction products of 2,5-dimercapto-1,3,4-thiadiazole, sodium salt, with 1-octanethiol and hydrogen peroxide

Endpoint summary

Description of key information

Repeat dose toxicty via the oral route

Key study

In a study conducted in accordance with the standardized guidelines OECD 422, under GLP conditions the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity was concluded to be 330 mg/kg/day (Envigo, 2018).

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:

16 January 2017 to 04 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same 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

Limit test:

no

Specific details on test material used for the study:

- Purity: > 99% (UVCB)
- Description: Amber liquid

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Duration of acclimatization:
- Males: six days prior to the commencement of treatment
- Females: 20 days prior to the commencement of treatment

Age of the animals at the start of treatment:
- Males ranged from 69 to 76 days old
- Females ranged from 83 to 90 days old

Weight range of the animals at the start of treatment:
- Males 326 to 386 g
- Females 232 to 295 g

Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.

- Diet: SDS VRF1 Certified pelleted diet, ad libitum
- Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 24ºC
- Humidity: 40 to 70%
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated
- Lighting: 12 hours light : 12 hours dark

Animal Replacement:
Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Replacement before treatment:
- Irregular estrous cycle: Two females

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

The required amount of test item was weighed into a suitable container and mixed, by magnetic stirring, with approximately 50% of the final volume of vehicle. Further amounts of vehicle were added and mixed to achieve the required volume. The formulation was magnetically stirred until visibly homogenous.

A series of formulations at the required concentrations were prepared in ascending order by dilution of individual weighings of the test item.

The frequency of preparation was weekly.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 1 and 300 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix. 15 days stability was confirmed when stored refrigerated (2 to 8°C) or 1 day when stored at room temperature (15 to 25°C).

The mean concentrations of the test material in formulations analyzed for the study were within applied limits +10/-15% for both GC and HPLC, confirming accurate formulation.

Duration of treatment / exposure:

Males: Two weeks before pairing up to necropsy after minimum of five weeks.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 13 of lactation.

Frequency of treatment:

Once daily at approximately the same time each day.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

330 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

The dose selection was based upon a range-finding study (please see RSS section 7.5.1 Supporting study, Envigo, 2017 (range-finding)).

Observations and examinations performed and frequency:

Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:

F0 males Week 1 - daily
Week 2 onwards - once each week

F0 females Week 1 - daily
Week 2 - once
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 6 and 12

Detailed observations were recorded at the following times in relation to dose administration:
Pre-dose observation
One to two hours after completion of dosing of all groups
As late as possible in the working day

Sacrifice and pathology:

All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Time of Necropsy:
F0 males After Week 5 investigations completed.
F0 females whose litter died before Day 13 On or after day the last offspring died.
F0 females Day 14 of lactation (following terminal blood sampling).
Day 15 of lactation (following terminal blood sampling) - animals 54 (Group 2), 72 (Group 3) and 47 (Group 4).
F1 offspring Selected offspring for thyroid hormone analysis - Day 4 of age.
Scheduled kill - Day 13 of age.

The organs weighed, tissue samples fixed and sections examined microscopically.

Statistics:

Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non-significant. For some parameters, including gestation index and stage of estous cycle at termination the similarity of the data was such that analyses were not considered to be necessary.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No test item related clinical signs were apparent in the male animals at all dose levels. On Day 15 of the treatment period excessive chewing, piloerection and partially closed eyelids were evident in all females receiving 1000 mg/kg/day after the dosing procedure.

Mortality:

no mortality observed

Description (incidence):

Five adult females (1 each in the low and mid dose groups and 3 in the high dose group) were sacrificed early (4 out of 5 on post-natal day 1 or 2) during the course of the study due to pup loss according to protocol.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Effects on body weight were evident for animals receiving 1000 mg/kg/day. When compared to control, male animals receiving 1000 mg/kg/day had a reduced overall mean bodyweight gain from Week 0 to 5 (74% of Control gain). Mean bodyweight gain for females receiving 1000 mg/kg/day during Week 2 of study was reduced when compared to Controls, and this was reflected in the overall Week 0 to 2 bodyweight gain.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Male animals receiving 1000 mg/kg/day consumed less than Control in Week 1 of treatment (90% of Control). In Week 2 and 4 of treatment consumption was comparable to Control.

Female animals receiving 1000 mg/kg/day consumed slightly less than Control in Week 1 of treatment (92% of Control). In Week 2 of treatment females receiving 1000 mg/kg/day consumed 8 grams more than Control (107% of Control).

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Both males and females receiving 1000 mg/kg/day had visually consumed more water than the other treated groups and Control.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The mean absolute and body weight adjusted liver and spleen weights were higher than Control in male animals receiving 1000 mg/kg/day. The mean absolute and body weight adjusted spleen weights were higher than Control in female animals receiving 1000 mg/kg/day

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Dark livers were seen in females sacrificed early (three in the 1000 mg/kg/day group and one in the 330 mg/kg/day group).

Dark spleens were seen in several males and females treated at 1000 mg/kg/day, two females treated at 330 mg/kg/day and one female treated at 100 mg/kg/day.

Dark kidneys were seen in females sacrificed early (3 in the 1000 mg/kg/day group and one in the 330 mg/kg/day group).

The incidence and distribution of all other findings were considered to be unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The microscopic examination of F0 adult animals revealed changes related to treatment with the test item in the liver, kidneys, spleen, thyroid and adrenal, but none of these changes was considered adverse.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Main study adult males showed that there was a trend of increasing mean plasma TSH concentrations with increasing doses of the test item compared to the concurrent control, however the individual TSH concentration ranges overlapped among the three test item treated groups. The increase seen for adult terminal males receiving 1000 mg/kg/day was statistically significant.

The test item may affect the homeostatis of thyroid hormones.

Key result

Dose descriptor:

NOAEL

Effect level:

330 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: general systemic toxicity

Key result

Critical effects observed:

not specified

Conclusions:

The No Observed Adverse Effect Level (NOAEL) for general systemic toxicity was concluded to be 330 mg/kg/day.

Executive summary:

The study was conducted in accordance with the standardized guidelines OECD 422, under GLP conditions to assess the potential systemic toxicity in rats, including an assessment of endocrine disruptor relevant endpoints, with administration of the test item by oral administration for at least five weeks.

 

Three groups of ten male and ten female rats received the test item at doses of 100, 330 or 1000 mg/kg/day in corn oil by oral gavage administration. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation or Day 14 of lactation for 3 animals. A similarly constituted Control group received the vehicle at the same volume dose as the treated groups. During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, water consumption, hematology (peripheral blood), blood chemistry, thyroid hormone analysis, organ weight and macroscopic pathology and histopathology investigations were undertaken.

 

Administration of the test item to adult CD:Crl rats at doses ≤ 330 mg/kg/day was generally well tolerated. There were minimal dose observations, no test item-related signs observed during the detailed physical examination and arena observations, and no effects on sensory reactivity, grip strength, motor activity and food consumption.

 

Test item-related findings observed at ≤ 330 mg/kg/day were minimal and low incidence and therefore were not considered adverse. Considering the incidence, severity and significance of the test item-related clinical signs, poor body weight performance, increased water consumption, decreased serum T4 and increased plasma TSH that were evident in the adult animals receiving 1000 mg/kg/day, the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity was concluded to be 330 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

330 mg/kg bw/day

Study duration:

subacute

Species:

rat