1,3-diethyl-2-thiourea

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date (Animal Arrival): 10 May 2022
Audited Draft Report Date: September 2023
Final Report Date: January 2024

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Experimental Starting Date (Animal Arrival): 10 May 2022
Audited Draft Report Date: September 2023
Final Report Date: January 2024

Reliability:

1 (reliable without restriction)

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 (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Han Wistar rat (virgin), accepted by regulatory agencies, historical control data available.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Air supply: Filtered, not recirculated.
Temperature: Maintained within the range of 20-24ºC.
Relative humidity: Maintained within the range of 40-70%.
Lighting: 12 hours light : 12 hours dark.
Diet supply: SDS VRF1 Certified (ad libitum). Pelleted diet.
Water supply: Potable water from the public supply (ad libitum).

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Treated at: Constant doses in mg/kg/day.
Controls (Group 1): Vehicle at the same volume dose as treated groups.
Volume dose: 5 mL/kg/day for Groups 1, 2 and 3, Group 4 up until Day 21 (30 May 2022). 3.33 mL/kg/day for Group 4 males from Day 22 (31 May 2022) onwards.
Individual dose volume: Calculated from the most recently recorded scheduled body weight.
Frequency: Once daily, at approximately the same time each day.
Sequence: Groups dosed in ascending order.
Administration: Prior to dosing of each individual animal, the dosing catheter/cannula will be dipped into a container filled with a 5% glucose solution to aid intubation.
Formulation: A daily record of the usage of formulation will be maintained based on weights before and after dosing. Formulations are stirred using a magnetic stirrer before and throughout the dosing procedure. Alternative methods may be used; these will be documented in the study records.
Storage of formulation: Refrigerated temperature (2 to 8°C).
Expected appearance of formulation: Orange opaque suspension

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

3 occasions: Week 1, Week 1 of gestation and final Week.

Duration of treatment / exposure:

Males: Sacrifice during Week 5 - after at least 4 weeks of treatment
Females: Sacrifice on Day 21 of lactation

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Reduced from 75 to mg/kg bw/day during treatment due to severe toxic effects.

No. of animals per sex per dose:

10 animals/sex/dose.

Control animals:

yes, concurrent vehicle

Details on study design:

A 14-day Dose Range Finding (DRF) study was conducted on this substance (Labcorp Study 8468465). Doses of 100, 200 or 300 mg/kg bw/day, in corn oil vehicle, were administrated to groups of 5 male and 5 female Han Wistar rats. An additional group of 3 male and 3 female Han Wistar rats was eventually added, receiving 50 mg/kg bw/day of the test item in corn oil.
Animals dosed at 200 or 300 mg/kg bw/day were terminated early, on Day 10 or 3, respectively. Severe and persistent clinical signs were observed in these animals, along with a body weight loss at 200 mg/kg bw/day associated with reduction in food consumption, and macroscopic findings in the gastrointestinal tract, especially in the stomach (dark, depressions, thickened and distended).
All animals receiving 100 or 50 mg/kg bw/day of the test item survived until completion of the study. At 100 or 50 mg/kg bw/day, limited clinical signs were reported, mostly transient and appearing shortly after dosing of the animals. At 100 mg/kg bw/day, body weight loss in males (-7 g between Days 8 and 15, -14% vs control on Day 15) and females (- 12 g between Days 11 and 15, -4% vs control on Day 15) animals was reported with an associated reduction in food intake, while water intake was increased. One female had macroscopic findings in the stomach consistent with the animals from the higher dose levels.
At 50 mg/kg bw/day, body weight loss was also apparent for males (- 4 g between Days 11 and 15, -5% vs control on Day 15) associated with low food intake, and body weight loss was evident for females (-2 g between Days 11 to 15), although their body weight on Day 15 did not differ from control animals. Water consumption was also increased in both sexes.
Considering that:
- According to the OECD Testing Guideline 422, the highest dose level should be chosen with the aim of inducing toxic effects but not death nor obvious suffering;
- The adverse effects – including local irritation effect in the stomach and reduction in bodyweight – observed in animals exposed to 100 mg/kg bw/day of test item for 14 days, can reasonably be expected to result in severe suffering and/or death when the treatment period is extended, and;
- Limited effects were reported in animals exposed to 50 mg/kg bw/day of the test item for 14 days, so this dose-level may not be sufficient to induce toxic effects.
The most appropriate top dose-level for the OECD Testing Guideline 422 is comprised between 50 and 100 mg/kg bw/day. Therefore, a dose level of 75 mg/kg bw/day is selected as the highest dose-level for the study.
30 and 10 mg/kg bw/day are selected as the intermediate and low dose-levels, respectively.
Treatment at 75 mg/kg/day was not tolerated, with 5 out of 10 females either euthanised for welfare reasons or found dead, and 2 out of 10 males euthanised for welfare reasons. Upon review of the data, the decision was taken to euthanise the remaining 5 females receiving 75 mg/kg/day in order to prevent any further undue suffering or premature deaths for these animals.
The remaining males receiving 75 mg/kg/day were not euthanised, but the dose level received by these males will be reduced to 50 mg/kg/day from Day 22 of dosing (31 May 2022) onwards.

Positive control:

Not relevant

Observations and examinations performed and frequency:

Clinical Observations: Visually inspected at least twice daily for evidence of reaction to treatment or ill-health
Body Weight: Before dosing on the day that treatment commences, weekly thereafter, twice weekly from Week 4 onwards and on the day of necropsy. Females: Days 0, 4, 7, 11, 14, 17 and 20 after mating and Days 1, 4, 7, 11, 14, 18 and 21 of lactation (the day of necropsy).
Food Consumption: Weekly during treatment.
Water consumption: Weekly during treatment. Measured for a 3-day period in each week.
Sensory Reactivity and Grip Strength / Motor Activity: Week 5 (males). Days 7-9 of lactation.
Estrous Cycles: For 14 days before treatment (all females including spares); animals that fail to exhibit 4-5 day cycles will not be allocated to study. After pairing until mating (for a maximum of 14 days). Females showing no evidence of mating: following completion of the pairing period females will be separated from the male and vaginal smearing continued for up to five days or until the first estrus smear is seen. If a female shows an estrus smear during this period, she will be killed as soon as practically possible and subject to macroscopic examination.
Hematology, Peripheral Blood: At termination.
Blood Chemistry: At termination.
Thyroid Hormone Analysis: At termination.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Statistics:

Included

Remarks on result:

not determinable

Remarks:

Conclusion will be reached once the study has been completed.

Critical effects observed:

not specified

Conclusions:

The objective of the study is to make an assessment of the general systemic toxic potential in Han Wistar rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of 1,3-diethyl-2-thiourea by oral gavage for at least 4 weeks.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

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 (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,3-diethyl-2-thiourea

EC Number:

203-308-5

EC Name:

1,3-diethyl-2-thiourea

Cas Number:

105-55-5

Molecular formula:

C5H12N2S

IUPAC Name:

1,3-diethyl-2-thiourea

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Han Wistar rat (virgin), accepted by regulatory agencies, historical control data available.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Air supply: Filtered, not recirculated.
Temperature: Maintained within the range of 20-24ºC.
Relative humidity: Maintained within the range of 40-70%.
Lighting: 12 hours light : 12 hours dark.
Diet supply: SDS VRF1 Certified (ad libitum). Pelleted diet.
Water supply: Potable water from the public supply (ad libitum).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Treated at: Constant doses in mg/kg/day.
Controls (Group 1): Vehicle at the same volume dose as treated groups.
Volume dose: 5 mL/kg/day for Groups 1, 2 and 3, Group 4 up until Day 21 (30 May 2022). 3.33 mL/kg/day for Group 4 males from Day 22 (31 May 2022) onwards.
Individual dose volume: Calculated from the most recently recorded scheduled body weight.
Frequency: Once daily, at approximately the same time each day.
Sequence: Groups dosed in ascending order.
Administration: Prior to dosing of each individual animal, the dosing catheter/cannula will be dipped into a container filled with a 5% glucose solution to aid intubation.
Formulation: A daily record of the usage of formulation will be maintained based on weights before and after dosing. Formulations are stirred using a magnetic stirrer before and throughout the dosing procedure. Alternative methods may be used; these will be documented in the study records.
Storage of formulation: Refrigerated temperature (2 to 8°C).
Expected appearance of formulation: Orange opaque suspension

Details on mating procedure:

Paired for mating: After minimum of 2 weeks of treatment.
Male/female ratio: 1:1
Duration of pairing: Up to 2 weeks.
Daily checks for evidence of mating Ejected copulation plugs. Sperm within vaginal smear.
Day 0 of gestation: When positive evidence of mating detected.
Male/female separation: Day when mating evidence detected.
Pre-coital interval: Calculated for each female as time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

3 occasions: Week 1, Week 1 of gestation and final Week.

Duration of treatment / exposure:

Males: Sacrifice during Week 5 - after at least 4 weeks of treatment
Females: Sacrifice on Day 21 of lactation

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

50 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals/sex/dose.

Control animals:

yes, concurrent vehicle

Details on study design:

A 14-day Dose Range Finding (DRF) study was conducted on this substance (Labcorp Study 8468465). Doses of 100, 200 or 300 mg/kg bw/day, in corn oil vehicle, were administrated to groups of 5 male and 5 female Han Wistar rats. An additional group of 3 male and 3 female Han Wistar rats was eventually added, receiving 50 mg/kg bw/day of the test item in corn oil.
Animals dosed at 200 or 300 mg/kg bw/day were terminated early, on Day 10 or 3, respectively. Severe and persistent clinical signs were observed in these animals, along with a body weight loss at 200 mg/kg bw/day associated with reduction in food consumption, and macroscopic findings in the gastrointestinal tract, especially in the stomach (dark, depressions, thickened and distended).
All animals receiving 100 or 50 mg/kg bw/day of the test item survived until completion of the study. At 100 or 50 mg/kg bw/day, limited clinical signs were reported, mostly transient and appearing shortly after dosing of the animals. At 100 mg/kg bw/day, body weight loss in males (-7 g between Days 8 and 15, -14% vs control on Day 15) and females (- 12 g between Days 11 and 15, -4% vs control on Day 15) animals was reported with an associated reduction in food intake, while water intake was increased. One female had macroscopic findings in the stomach consistent with the animals from the higher dose levels.
At 50 mg/kg bw/day, body weight loss was also apparent for males (- 4 g between Days 11 and 15, -5% vs control on Day 15) associated with low food intake, and body weight loss was evident for females (-2 g between Days 11 to 15), although their body weight on Day 15 did not differ from control animals. Water consumption was also increased in both sexes.
Considering that:
- According to the OECD Testing Guideline 422, the highest dose level should be chosen with the aim of inducing toxic effects but not death nor obvious suffering;
- The adverse effects – including local irritation effect in the stomach and reduction in bodyweight – observed in animals exposed to 100 mg/kg bw/day of test item for 14 days, can reasonably be expected to result in severe suffering and/or death when the treatment period is extended, and;
- Limited effects were reported in animals exposed to 50 mg/kg bw/day of the test item for 14 days, so this dose-level may not be sufficient to induce toxic effects.
The most appropriate top dose-level for the OECD Testing Guideline 422 is comprised between 50 and 100 mg/kg bw/day. Therefore, a dose level of 75 mg/kg bw/day is selected as the highest dose-level for the study.
30 and 10 mg/kg bw/day are selected as the intermediate and low dose-levels, respectively.
Treatment at 75 mg/kg/day was not tolerated, with 5 out of 10 females either euthanised for welfare reasons or found dead, and 2 out of 10 males euthanised for welfare reasons. Upon review of the data, the decision was taken to euthanise the remaining 5 females receiving 75 mg/kg/day in order to prevent any further undue suffering or premature deaths for these animals.
The remaining males receiving 75 mg/kg/day were not euthanised, but the dose level received by these males will be reduced to 50 mg/kg/day from Day 22 of dosing (31 May 2022) onwards.

Positive control:

Not relevant

Examinations

Parental animals: Observations and examinations:

Clinical Observations: Visually inspected at least twice daily for evidence of reaction to treatment or ill-health
Body Weight: Before dosing on the day that treatment commences, weekly thereafter, twice weekly from Week 4 onwards and on the day of necropsy. Females: Days 0, 4, 7, 11, 14, 17 and 20 after mating and Days 1, 4, 7, 11, 14, 18 and 21 of lactation (the day of necropsy).
Food Consumption: Weekly during treatment.
Water consumption: Weekly during treatment. Measured for a 3-day period in each week.
Sensory Reactivity and Grip Strength / Motor Activity: Week 5 (males). Days 7-9 of lactation.
Estrous Cycles: For 14 days before treatment (all females including spares); animals that fail to exhibit 4-5 day cycles will not be allocated to study. After pairing until mating (for a maximum of 14 days). Females showing no evidence of mating: following completion of the pairing period females will be separated from the male and vaginal smearing continued for up to five days or until the first estrus smear is seen. If a female shows an estrus smear during this period, she will be killed as soon as practically possible and subject to macroscopic examination.
Hematology, Peripheral Blood: At termination.
Blood Chemistry: At termination.
Thyroid Hormone Analysis: At termination.
Duration of gestation: Time elapsing between mating and commencement of parturition.
Parturition observations
From Day 20 after mating, animals checked 3 times daily for evidence of parturition. If difficulties observed, progress of parturition process monitored. Numbers of live and dead offspring recorded.

Oestrous cyclicity (parental animals):

Wet smears: Using pipette lavage during the following phases:
• For 14 days before treatment (all females including spares); animals that fail to exhibit 4-5 day cycles will not be allocated to study.
• After pairing until mating (for a maximum of 14 days).
• Females showing no evidence of mating: following completion of the pairing period females will be separated from the male and vaginal smearing continued for up to five days or until the first estrus smear is seen. If a female shows an estrus smear during this period, she will be killed as soon as practically possible and subject to macroscopic examination. NB: The guideline states that “females showing no evidence of copulation are killed 24-26 days after the last day of the mating period”. Reading vaginal smears after separation and confirming failure to mate/non-pregnancy by continuation of cyclicity is considered an enhancement in animal welfare.
• On the day of scheduled termination.
Dry smears: For 15 days before pairing, using cotton swabs.

Sperm parameters (parental animals):

Detailed qualitative examination will be made, taking into account the tubular stages of the spermatogenic cycle. The examination will be conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings will be noted.

Litter observations:

Clinical observations: Observed approximately 24 hours after birth and then daily for evidence of ill-health or reaction to maternal treatment.
Litter size: Daily on Days 1-21 of age.
Sex ratio: Days 1, 4, 7, 14 and 21 of age.
Individual offspring body weights: Days 1, 4, 7, 14, 17 and 21 of age.
Ano-genital distance: Day 1 of age.
Nipple/areolae count: Day 13 of age - male offspring. Representative photographs may be taken if nipples are seen.

Postmortem examinations (parental animals):

Detailed macroscopic examination will be performed for evidence of adverse reaction to treatment. Organs weighed and tissues retained for light microscpy. Any abnormal tissues retained and may be weighed at the discretion of necropsy staff.
For F0 females, the number of uterine implantation sites will also be recorded.

Postmortem examinations (offspring):

F1 offspring culled on Day 4 of age: Externally normal offspring discarded without examination. Externally abnormal offspring identified on despatch to necropsy; examined externally, and retained pending possible future examination.
F1 offspring on Day 21 of age: Thyroid glands retained from two offspring – one male and one female, where possible If insufficient numbers of Day 21 offspring are available for hormone sampling and thyroid retention/preservation, priority is given to hormones, with sample for T4 given first priority. All animals will be subject to an external macroscopic examination; particular attention will be paid to the external genitalia. Abnormalities retained in appropriate fixative.

Statistics:

Included

Reproductive indices:

Fertility index
Gestation index

Offspring viability indices:

Post-implantation survival index
Live birth index
Viability index
Lactation index

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

Remarks on result:

not determinable

Remarks:

Conclusion will be reached once the study has been completed.

Target system / organ toxicity (P0)

Critical effects observed:

not specified

Results: F1 generation

Effect levels (F1)

Remarks on result:

not determinable

Remarks:

Conclusion will be reached once the study has been completed.

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The objective of the study is to make an assessment of the general systemic toxic potential in Han Wistar rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of 1,3-diethyl-2-thiourea by oral gavage for at least 4 weeks.