1-[2-fluoro-6-(trifluoromethyl)benzyl]-5-iodo-6-methylpyrimidine-2,4(1H,3H)-dione

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7th August 2018 to 22nd November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-{[2-fluoro-6-(trifluoromethyl)phenyl]methyl}-5-iodo-6-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione

EC Number:

687-893-6

Cas Number:

1150560-54-5

Molecular formula:

C13H9F4IN2O2

IUPAC Name:

1-{[2-fluoro-6-(trifluoromethyl)phenyl]methyl}-5-iodo-6-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Grade SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Beijing Vital River Laboratory Animal Technology Ltd.
- Age at study initiation: (P) - 56-62 days
- Weight at study initiation: (P) Males: 290-340g; Females: 195-235g;
- Housing: Suspension stainless steel cages on cage racks, 2 rats per cage at most, with corn cob bedding. During mating, the rats were housed in mating cages. After mating, females were housed in plastic cages, with a bedding of wood shaving.
- Diet: Sterilised growth and breeding feed from Beijing Keao Xiele Feed Co. ad libitum
- Water: Drinking water ad libitum
- Acclimation period: 10 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25
- Humidity (%): 40-70
- Photoperiod: (12 hrs dark / hrs light 12)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was weighed and placed in a jar, and corn oil added to give the required concentration. The jars were then mixed with the magnetic stirring apparatus for 1 minute. The test item was prepared daily.
VEHICLE
- Amount of vehicle: 5ml/kg bw

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy (GD0)
- After successful mating each pregnant female was caged in a plastic cage

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

2 weeks of mating;
Up to day before scheduled kill, (except during childbirth)

Frequency of treatment:

Once daily in the morning, 7 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Both male and female

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Both male and female

Doses / concentrations 3

Dose / conc.:

800 mg/kg bw/day (nominal)

Remarks:

Female highest dose

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Male highest dose

No. of animals per sex per dose:

14

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked included: appearance, fur, activity, reaction, breathing, posture, excrement and urine.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once per week in conjunction with weighing

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: The food ration was added weekly and food intake weighed the following day.

PARTURITION: Yes
- From GD21, observations made twice daily (morning and afternoon). Any difficulties were recorded.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1offspring (PND0):
stillbirths, live births, postnatal mortality, presence of grossly malformed pups, grossly puny pups.
PND4: signs of behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS: No

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals at the end of the mating period.
- Maternal animals: All surviving animals - PND4 of the last of the parturition.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Number of implantation sites in the uteri were recorded. In addition, the organs of males that failed to sire and females that were non-mated or non-pregnant in low- and mid-dose groups were also examined.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues - testes and epididymus were weighed.

Statistics:

Single factor analysis of variance and if there was significant difference, followed by Dunnett's test. The data from the clinical observations were validated by X2 test. The incidences of gross necroscopy and pathological findings were analysed by the unilateral Fischer's exact probability test.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No deaths or clinical signs were observed in either the males or the solvent control.

300mg/kg bw
One rat was emaciated during lactation - dead PND6.

One rat had soiled perineal region during parturition and lactation dead PND4.
One rat has dehairing on prothorax.

800 mg/kg bw
One rat has dehairing on lower left abdomen.
No other clinical signs.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two females in the 300 mg/kg bw died PND4 and PND6. These deaths were not statistically significant compared to the solvent control

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

800 mg/kg bw
The body weight before parturition (GD20) had a statistically significant decrease compared to control group (p ≤ 0.05)

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

1000 mg/kg bw
Mean food consumption for male rats in week 2 had a statistically significant decrease compared to the control group (p ≤ 0.05) but no significant difference was observed compared to the mean and total food consumption.

100/300 mg/kg bw
During the pre-mating period, the first week and total food consumption female rats had a statistically significant decrease compared to the control group (p ≤ 0.05) but no significant difference was observed comparing the mean and total food consumption (p>0.05). There was no significant difference in food consumption of pregnant rats during gestation and lactation in all dose groups compared with the solvent control (p>0.05).

The increase or decrease in food consumption had no dose dependent adverse effect, so it was considered that the results had no correlation with test item toxicity.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive performance:

no effects observed

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

800 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

2

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive performance

3

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

4

Key result

Dose descriptor:

LOAEL

Effect level:

800 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

5

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No malformation or abnormalities were observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no significant difference in average litter weight in any dose group compared to the control (p>0.05)

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: Developmental toxicity

2

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

800 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: Developmental toxicity

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

800 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

not specified

Applicant's summary and conclusion

Conclusions:

Based on these results, it is concluded that the No Observed Adverse Effect Level (NOAEL) for general toxicity to males for exposure to Methyl Iodouracil is 1000mg/kg bw; the Lowest Observed Adverse Effect Level (LOAEL) for general toxicity to females for exposure to Methyl Iodouracil is 800mg/kg bw; the NOAEL for reproduction toxicity to males for oral exposure to Methyl Iodouracil is 1000mg/kg bw; the NOAEL for general toxicity to females for oral exposure to Methyl Iodouracil is 300mg/kg bw; the LOAEL for development exposure to pups to Methyl Iodouracil is 1000mg/kg bw

Executive summary:

This study was conducted to provide initial information on possible effects on Reproduction/Development toxicity for Methyl Iodouracil following oral exposure in rats, and also to be used as a dose range finding study for other Reproduction/Development studies. This test was designed to be compatible with the Guidelines for the Testing of Chemicals (2nd Edition) No. 421 Reproduction/Development Toxicity Screening Test (2013). 

The study was conducted in SD rats and all animals were SPF grade. Based on the results of the preliminary test for repeated dose oral toxicity, three doses of 1000, 300 and 100 mg/kg bw for males and 800, 300 and 100 mg/kg bw for females were used. A concurrent solvent control was included. There were fourteen male and fourteen female rats in each group. All animals were dosed during the two weeks prior to dosing, the two weeks of the mating period and up to the day before the scheduled kill. The test was terminated four days after the birth of the last litter pup. Clinical observations were made daily, and body weight and food consumption were weighed weekly. The Reproduction/Development parameters were evaluated at the same time. All parental animals were macroscopically examined for any abnormalities and pathological changes. A histopathology examination was carried out on the reproductive organs of rats in the control group and high dose group.

No deaths or clinical toxicity was observed in males in either the solvent control group or any dose groups. In the 300 mg/kg bw, female rat (No. 2205) was emaciated during the lactation period and died 6 days after parturition, a female rat (No. 2208) had soiled perineal region on the day of parturition and during the  lactation period and died on the 4th day after parturition, a female rat (No 2213) had dehairing on the prothorax and a female rat (No 2307) had dehairing on the lower part of left abdomen.

No significant difference in body weight in males was found in any dose group compared to the solvent control group (p>0.05). The body weight of females in the high dose group had a significant decrease compared with the control group during parturition. No significant difference in food consumption between males and females was found in any dose group compared to the solvent control group. 

The mated ratio and reproduction ratio of males in all dose groups had no significant difference compared with the solvent control group (p>0.005). The mated ratio, pregnancy ratio and live birth index of female in all dose groups had no significant difference compared with the solvent control group (p>0.005). There was no significant difference of mating time and gestation time in all dose groups compared with the solvent control group (p>0.005).No pregnant rats died from dystocia and none had all stillbirth pups. These results indicate that there was no adverse affect on the fertility of either males or females. 

The loss of pups in the high-dose groups before and after birth had a statistically significant increase compared with the solvent control group (p<0.01). The live birth index had a statistically significant decrease (p<0.05) and all the pups in four litters were dead within four days of birth. The live birth index in low- and mid-dose had no statistically significant effect. The post implantation loss had no statistically significant increase and the viability index of birth had no statistically significant decrease. The males ratio of the low-dose group had a statistically significant increase compared to the control group (p≤0.05) but no  significant differences were observed in mid- and high-dose groups (p>0.05). Therefore, the results were considered to have no toxicological significance. 

There was no significant difference in average litter weight in all dose groups compared with the solvent control (p>0.05) in PND0 and PND4.  All pups of animals 2306 and 2307 were puny compared to the solvent control group on the same birth day and died berfore PND4. No malformations or significant abnormalities in other live pups were observed from birth to study ending. The gross necroscopy and histopathological examinations carried out on the animals showed no significant difference related to to test item treatment. 

Exposure of rats to various oral doses of Methyl Iodouracil resulted in no deaths. No clinical toxicity observations were made for males in either the solvent control group or any dose groups. Two females in the 300 mg/kg bw group died during the lactation period. Some females had dehairing in the 300 and 800 mg/kg bw groups but the death index and sympton incidence rate had no significant increase compared with the solvent control group and was not dose related, so this was not an adverse effect of the treatment of the test item. At the 800mg/kg bw dose rate, the body weights of females were adversely affected, so it can be concluded that Methyl Iodouracil had general toxicity to parental females at this dose level. 

For Reproduction/Developmental, there was no adverse effects on the fertility of males in the 1000mg/kg bw group. There was no adverse effects on the fertility of females in the 800 mg/kg bw but the loss of pups before and after birth had a statistically significant increase. The growth of some pups were affected, so it is considered that Methyl Iodouracil had induced some effect on pups in the  800mg/kg bw, namely developmental toxicity. 

Based on these results, it is concluded that the No Observed Adverse Effect Level (NOAEL) for general toxicity to males for exposure to Methyl Iodouracil is 1000mg/kg bw; the Lowest Observed Adverse Effect Level (LOAEL) for general toxicity to females for exposure to Methyl Iodouracil is 300mg/kg bw; the NOAEL for reproduction toxicity to males for oral exposure to Methyl Iodouracil is 1000mg/kg bw; the NOAEL for general toxicity to females for oral exposure to Methyl Iodouracil is 800mg/kg bw; the LOAEL for development exposure to pups to Methyl Iodouracil is 1000mg/kg bw;