(1R)-2-[5-(2-halo-3-methoxyphenyl)-3-[2-halo-6-(trihalomethyl)benzyl]- 4-alkyl-2,6-dioxoheterocyclyl]-1-phenylethanaminium salicylate

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 23rd November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

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

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: at least 10 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25
- Humidity (%): 40-70
- Photoperiod: (12 hrs dark / 12 hrs light)

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.:

40 mg/kg bw/day (nominal)

Remarks:

Male and Female

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Male and Female

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Male and Female

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, with handling
- Time schedule: Once per week in conjunction with weighing.
Any abnormality of head/neck including eyes, ears nose and mouth, back, abdomen, anus, skin colour around perineum, muscle tension, any trauma or tumour.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly
Pregnant rats were weighed GD0, 7, 14 and 20.
Prenatal rats and pups weighed PND0 and PND4.
Mother rats weighed once per week in order to adjust the dose.
All animals weighed on the day of the scheduled kill.

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 and non-mated females: 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 cavities. Number of implantation sites in the uteri were recorded.

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):

In the high dose group, one male had a tumour in the right ear and another had dehairing on the whole body. No deaths or clinical signs were observed in any other males.



0, 3, 4 and 9 females in control, low-, mid- and high-doses were found with dehairing, soiled perineal region or tumour. The symptom incidence in the mid- and high-dose groups had a statistically significant increase compared to the control group.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Females: 1,1,1 and 1 were found to have died accidently. There was no statistically significant increase compared to the control group for animal mortality.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males:
250 mg/kg bw
During the pre-mating and mating period, the mean body weight in the third week was significantly decreased compared to control group (p ≤ 0.05)

Females:
250 mg/kg bw
During the pre-mating period, the mean body weight in the first and second week was significantly decreased compared to control group (p ≤ 0.05).
During the gestation period, the body weight of the pregnant rats in the whole gestation period (GD7, 14 and 20) and body weight gain in the 250 and 100 mg/kg bw dose groups were significantly decreased compared to control group (p ≤ 0.05).

During the lactation period, the maternal mean body weight on the 4th day after delivery was significantly decreased compared to the solvent control group (p ≤ 0.05)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was a statistically significantly decrease in food consumption by dams during lactation in all dose groups compared to the solvent control group (p ≤ 0.05)

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive performance:

no effects observed

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

100 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

histopathology: non-neoplastic

2

Key result

Dose descriptor:

LOAEL

Effect level:

250 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

histopathology: non-neoplastic

3

Key result

Dose descriptor:

NOAEL

Effect level:

40 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:

100 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

Results: F1 generation

General toxicity (F1)

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The number of stillbirth pups was significantly increased compared with the control group, namely the live births of pups was statistically significantly decreased compared with the control group (p ≤ 0.05 or p≤ 0.01) and the post-implantation loss in the mid-dose group had a statistically significant increase compared with the control group (p ≤ 0.01). The stillbirth pups in the two dose groups happened mostly in individual litters and the live birth index of pups had no significant decrease in the high-dose groups, so the results above were considered to be non-test item related. However, the number of dead pups in the 4 days post-delivery was significantly increased in all dose groups and the viability index had a statistically significant decrease compared with the control group (p ≤ 0.01). The number of mean live pups in the high-dose groups was significantly decreased compared with the control (p < 0.01)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The average litter weight on PND0 in the mid- and high-dose groups and PND4 in all dose groups  was statistically significantly decreased compared to the control group (p≤ 0.05 or p≤ 0.01) which determined that the test item had adverse effects on viability and growth of pups in all dose groups in the study. 

Effect levels (F1)

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: Developmental toxicity

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

250 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

yes

Relevant for humans:

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 parent males is 100 mg/kg.bw, the Lowest Observed Adverse Effect Level (LOAEL) for general toxicity to males is 250 mg/kg bw; the NOAEL for general toxicity to female rats is 40 mg/kg.bw, the LOAEL is 100 mg/kg.bw. The NOAEL for reproduction toxicity to parent rats for oral exposure to the test item is 250mg/kg bw; the LOAEL for developmental toxicity to pups for oral exposure to the test item is 40mg/kg bw. 

Executive summary:

This study was conducted to provide initial information on the possible effects on Reproduction/Development toxicity for E Stage 3 Intermediate 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 250, 100 and 40 mg/kg.bw were used in the study. 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 mating, 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 the high dose group.

 

During the study, one male in the high-dose group had a tumour in the right ear and dehairing on the whole body. No abnormality symptons were found in any other males and there were no deaths. For females 1,1,1 and 1 were found dead  accidentally and 0, 3, 4 and 9 females in the control, low-, mid- and high-dose groups were found dehaired, with soiled perineal region or tumour. The sympton incidence rate in the mid- and high-dose groups was a statistically significant increase compared with the control group (p≤ 0.05 or p≤ 0.01) and had dose correlation.

 

During the study, the mean body weights of the third week and the body weight gain of males in the high-dose group was significantly decreased compared with the control group (p≤ 0.05 or p≤ 0.01). During pre-mating period the mean body weight of the first and second weeks and the body weight gain of the females in the high-dose group was significantly decreased compared with the solvent control group (p≤ 0.05 or p≤ 0.01). During gestation period, the body weight of pregnant rats during the whole pregnancy and the body weight gain in mid- and high-dose groups were significantly lower compared with the control group (p≤ 0.05 or p≤ 0.01). During the lactation period, the mothers mean body weights on the fourth day after delivery in the mid- and high-dose groups were significantly lower compared with the control group (p≤ 0.05) and the change in body weight had some dose correlation. During pre-mating and gestation periods, no significant difference was found in mean food consumption in all dose groups compared to the solvent control group. There was a significant difference decrease in food consumption of the dams during the lactation period in all dose groups compared with the control group (p≤ 0.05 or p≤ 0.01). The decrease in food consumption in dams was associated with the decrease in body weight in mid- and high-dose groups and with some dose correlation, so it was considered an adverse effect of the test item. The decrease in food consumption was not asssociated with a significnant decrease in body weight in the low-dose group and had no dose correlation with the other dose groups so it was determined not to be an adverse effect of the test item. 

 

There was no significant difference of the male mating index in all dose groups compared with the solvent control group (p>0.05). There was no significant difference of the females mating index, fertility index, fecundity index and gestation index in all dose groups compared with the solvent control group (p>0.05) and there was no significant difference of mating time and gestation time in all dose groups compared with the solvent control group (p>0.05).

 

For the pups development, the number of still birth pups was significantly increased compared with the control group, namely the live births of pups was statistically significantly decreased compared with the control group (p ≤ 0.05 or p≤ 0.01) and the post-implantation loss in the mid-dose group had a statistically significant increase compared with the control group (p ≤ 0.01). The stillbirth pups in the two dose groups happened mostly in individual litters and the live birth index of pups had no significant decrease in the high-dose groups, so the results above were considered to be non-test item related. However, the number of dead pups in the 4 days post-delivery was significantly increased in all dose groups and the viability index had a statistically significant decrease compared with the control group (p ≤ 0.01). The number of mean live pups in the high-dose groups was significantly decreased compared with the control (p < 0.01)

 

The average litter weight on PND0 in the mid- and high-dose groups and PND4 in all dose groups  was statistically significantly decreased compared to the control group (p≤ 0.05 or p≤ 0.01) which determined that the test item had adverse effects on viability and growth of pups in all dose groups in the study. 

 

No malformation or significant abnormalities were observed in any of the live pups in any group from birth to the end of the study. 

 

The inspection of gross necroscopy and histopathological revealed no significant difference of gross necroscopy and histopathological examination found to be related to the test item treatment.

 

Exposure of rats to oral doses of E Stage 3 Intermediate resulted in no test item related animal deaths or toxicity symptoms for parental males in the 250 mg/kg.bw groups, but body weight gain was adversely affected so it was considered that the test item had general toxicity to parent males at 250 mg/kg.bw dose level. The incidence of symptoms for parental females had a significant increase compared with the control group at 100 and 250 mg/kg.bw dose level and the body weight and food consumtion of females in different periods were adversely affected, so it was considered that the test item had general toxicity to parent females at 100 mg/kg.bw dose level.

For fertlity and development, there was no adverse effects on the fertility of males and females at 250 mg/kg.bw dose level, so it was considered that E Stage 3 Intermediate had no fertility toxicity to rats at 250 mg/kg.bw but there was some adverse effects on growth and development of pups at 40, 100 and 250 mg/kg.bw dose levels, such as the decrease of the viability index and mean litter weight, so it was considered that E Stage 3 Intermediate had developmental toxicity for pups at the 40 mg/kg.bw dose level and above. 

 

Based on these results, it is concluded that the No Observed Adverse Effect Level (NOAEL) for general toxicity to parent males is 100 mg/kg.bw, the Lowest Observed Adverse Effect Level (LOAEL) for general toxicity to males is 250 mg/kg bw; the NOAEL for general toxicity to female rats is 40 mg/kg.bw, the LOAEL is 100 mg/kg.bw. The NOAEL for reproduction toxicity to parent rats for oral exposure to the test item is 250mg/kg bw; the LOAEL for developmental toxicity to pups for oral exposure to the test item is 40mg/kg bw.