2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Feb - 20 Jul 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Results tables are missing in the translated version of the study report.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 22 Jan 2001

Deviations:

yes

Remarks:

results tables are missing in the translated study report

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 25 Jun 2018

Deviations:

yes

Remarks:

Please refer to "Principles of method if other than guideline".

Principles of method if other than guideline:

Deviations to OECD guideline 414 (2018): Diet not analysed (phytoestrogens); no investigations of thyroid weight and histopathology; reproductive tract of fetuses or cryptorchidism not examined; no comparison between external vs. internal (gonadal) sex morphology; historical control data from the testing facility not included; high dose level (1000 mg/kg bw/day) induced mortality (6/25 animals), therefore exceeding the maximum tolerated dose.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-(2-chloro-4-methanesulfonyl-3-{[(oxolan-2-yl)methoxy]methyl}benzoyl)cyclohexane-1,3-dione

EC Number:

695-022-6

Cas Number:

473278-76-1

Molecular formula:

C20 H23 Cl O7 S

IUPAC Name:

2-(2-chloro-4-methanesulfonyl-3-{[(oxolan-2-yl)methoxy]methyl}benzoyl)cyclohexane-1,3-dione

Test material form:

solid: particulate/powder

Test animals

Species:

rabbit

Strain:

other: Japanese White rabbits (Kbl:JW)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: KITAYAMA LABES Co., Ltd. (Nagano, Japan)
- Age at study initiation: 18 weeks
- Weight at study initiation: 3.10 - 3.74 kg on receipt
- Fasting period before study: No
- Housing: Individually housed in aluminium cages with wire mesh floors (W350 x D480 x H330 mm)
- Diet: Certified solid feed LRC4 (Oriental Yeast Co., Ltd., Tokyo, Japan), ad libitum
- Water: Purified well water, ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: aqueous solution of 0.5% methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing solution was prepared for each dose level by suspending a specified amount of the test substance in an aqueous solution of 0.5% methylcellulose.

VEHICLE
- Concentration in vehicle: 0.002 to 20%
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

All samples were analysed by using a high performance liquid chromatograph (HPLC) LC-2000 Plus (JASCO Corp., Tokyo, Japan) with L-column ODS (Chemicals Evaluation and Research Institute, Japan, Tokyo, Japan).

Prior to the study, a stability study was performed for the low dose group (0.02 mg/mL). The stability of test substance at concentrations of 0.1 mg/mL and 200 mg/mL had already been confirmed in a previous study (SR04179, 2005), in which the test substance was stable for 15 days under 6-hour room temperature and subsequent refrigerated condition. The stability study revealed that the test substance was stable for 14 days after preparation when stored under refrigerated conditions (100% recovery).

Dosing solutions were prepared 4 times with intervals of 7 days during the study period. Analysis of the dose preparations confirmed that they were homogenous and the test substance concentrations were within appropriate ranges.

In the homogeneity study, the coefficient of variation (CV) of the dosing solutions in the low and high dose groups was 0.0% and 1.6%, respectively, demonstrating that the test substance was uniformly distributed in the dosing solutions.

In the concentration analysis, the test substance was detected in the samples from each dosing solution at a range of 97% to 105% of the nominal concentrations, indicating that the preparation of dosing solutions had been properly performed.

Details on mating procedure:

- Impregnation procedure: Artificial insemination

Semen was taken from 2-4 males per day and those with good quality were pooled and diluted with physiological saline at a dilution of 1:10. Each female was injected 0.5 mL of the diluted semen into the vagina. After the injection, the females were given 25 U of hCG (human Chorionic Gonadotropin) through the auricular vein. These mating procedures were repeated on 20 females per day for 5 consecutive days.

- Verification of same strain and source of both sexes: Yes

Duration of treatment / exposure:

22 days (gestation Day 6 to 27)

Frequency of treatment:

Daily, 7 days a week

Duration of test:

Day 6 to 27 of gestation

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.1 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a dose range-finding study (IET 04-0029, 2006), the test substance was administered orally to artificially inseminated Japanese White rabbits (Kbl:JW), eight females per group, once daily from Days 6 to 27 of gestation at doses of 0, 1, 10, 100, and 1000 mg/kg bw/day to examine the potential effects on maternal animals and their fetuses. The study revealed no adverse effects of the test substance on maternal body weights and food consumptions, as well as the number of surviving fetuses and fetal body weights in any of the treated groups. In the skeletal examination, incidences of fetuses with supernumerary ribs and 27 presacral vertebrae in all dose groups were significantly higher than those in the control group. The incidences of fetuses with supernumerary ribs and 27 presacral vertebrae were 52.8% and 39.6%, respectively, in the 1 mg/kg bw/day group, whereas those in the 10 mg/kg bw/day and higher dose groups ranged from 90.2% to 91.0% and from 80.4% to 88.9%, respectively (29.8% and 14.9%, respectively in the control group). None of the malformations observed were thought to be treatment-related in all dose groups. Based on the results described above, a dose level of 0.1 mg/kg bw/day was selected as the low dose level, with the expectation that the incidences of supernumerary ribs and 27 presacral vertebrae in fetuses will be similar to those in the control group, and those of 10 mg/kg bw/day and 1000 mg/kg bw/day were selected as the middle and high dose levels, respectively.
- Rationale for animal assignment: Artificially inseminated females were weighed on Day 0 of gestation (the day of artificial insemination) and distributed to 4 groups in such a way to equalise group mean body weights as closely as possible.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily during the dosing period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were performed on the days of body weight measurement (Days 0, 6, 9, 12, 15, 18, 21, 24, 27 and 28 of gestation)

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 6, 9, 12, 15, 18, 21, 24, 27 and 28 of gestation. Adjusted body weights were calculated by subtracting the gravid uterine weight from the body weight on Day 28 of gestation.

FOOD CONSUMPTION: Yes
- The amount of food supplied and/or unconsumed was determined for each animal on Days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 28 of gestation. Daily food consumption (g/rabbit/day) was calculated for each female by dividing the amount of total food consumption by the number of feeding days.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 28 followed by a gross pathological examination. Organs and tissues were not preserved.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

When no uterine implants were apparent upon gross examination, the uterus was stained with 10% ammonium sulfide solution to detect very early resorptions.

Blood sampling:

- Plasma: No
- Serum: No

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes, half of the animals underwent fresh examination of eyes and brains, the heads of the other half were preserved for observations of eyes, brain, nasal passage and tongue.
-Determination of sex by observation of the internal reproductive organs

After soft tissue examination, these soft tissues (thoracic and abdominal) were removed and preserved in Bouin's solution with the placentas and the examined eyes (half of litter). The heads of the other half of the fetuses was decapitated along with the rima oris and fixed in Bouin's solution. The remaining skeletons were fixed in 70% isopropyl alcohol solution, stained with alizarin red S, and cleared in 70% glycerin for examination of skeletal abnormalities.

Statistics:

See field "Any other information on materials and methods incl. tables".

Indices:

Percent incidences of preimplantation losses were calculated from the following formula:

Percent incidence of preimplantation losses (%) =
((Number of corpora lutea – number of implants) / number of corpora lutea) x 100

The sex ratio was calculated for each group from the following formula:
Sex ratio = total number of male fetuses / total number of surviving fetuses

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No clinical abnormalities were noted in any maternal rabbits in each treated group during the pre-dosing period (Days 0 to 5 of gestation).

During the dosing period (Days 6 to 27 of gestation), five animals at 1000 mg/kg bw/day (one of them was non-pregnant) died between Days 20 to 27 of gestation, and the incidence was significantly higher than that in the control group. These animals showed such clinical signs as soiled fur and red discharge on the tray and one of them aborted before death. At 10 mg/kg bw/day, posterior paralysis was noted in one animal on Day 12 of gestation. This animal was euthanatized because the lesion was determined to non-recoverable. In the control group, one animal aborted on Day 26 of gestation. In addition, loss of fur or crust formation was noted in one or two animals in the control and at 0.1 mg/kg bw/day.

Observations after the termination of administration (Day 28 of gestation) revealed death of an animal (non-pregnant) at 1000 mg/kg bw/day accompanied by the presence of soiled fur and emaciation. Red discharge on the tray was noted in another animal in the same group. There were no abnormal findings in any animals in the control, 0.1 and 10 mg/kg bw/day groups.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality:

mortality observed, treatment-related

Description (incidence):

Five animals at 1000 mg/kg bw/day died between gestation Days 20 and 27. One female at 1000 mg/kg bw/day died on gestation Day 28.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no statistically significant differences in the mean body weights and the adjusted body weight at cesarean section of maternal animals between the control group and the 0.1, 10 and 1000 mg/kg bw/day groups.

The mean body weight gains in the 0.1 and 10 mg/kg bw/day groups were comparable to those in the control group during the study period. At 1000 mg/kg bw/day, the mean body weight gains were significantly lower than those in the control group on gestation Day 15 and thereafter.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean food consumption in the 0.1 and 10 mg/kg bw/day groups was comparable to that in the control group during the study period. At 1000 mg/kg bw/day, the mean food consumption of females was suppressed slightly after the initiation of treatment and the values during gestation Days 6 to 9 and 15 to 18 were significantly lower than those in the control group.

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

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

not examined

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

not examined

Description (incidence and severity):

Not applicable.

Haematological findings:

not examined

Description (incidence and severity):

Not applicable.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not applicable.

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

not examined

Description (incidence and severity):

Not applicable.

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The mean gravid uterine weights in the 10 and 1000 mg/kg bw/day groups were comparable to those in the control group. Although the mean gravid uterine weight in the 0.1 mg/kg bw/day group was significantly higher than that in the control group, this is likely to be due to a slightly increased fetal number in this group and is considered to be unrelated to the test substance treatment.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Animals subjected to necropsy during the study included six females that died at 1000 mg/kg bw/day, one female at 10 mg/kg bw/day that was euthanatized due to posterior paralysis and one female in the control group that aborted. Among them, two females out of 6 at 1000 mg/kg bw/day were non-pregnant and were excluded from the evaluation. The necropsy revealed 1 - 3 cases of soiled fur in the perinasal/perianal regions, hair bolus in the stomach, distention of the stomach, watery contents in the caecum, spots or red in color of the lungs and haemorrhage in the uterine horn in four dead females at 1000 mg/kg bw/day. In addition, one female euthanatized at 10 mg/kg bw/day had dislocation in the lumbar vertebra and one aborted female in the control group exhibited watery contents in the caecum and haemorrhage in the uterine horn.

No gross abnormal findings were observed in any animals euthanatized on Day 28 of gestation.

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Not applicable.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Maternal developmental toxicity

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

Of the six animals that died at 1000 mg/kg bw/day, one aborted before death.

In the control group, one animal aborted on Day 26 of gestation.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in the mean numbers of corpora lutea, implants and the mean percent incidences of preimplantation losses between control and treated groups.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

Two females in the control group, three females at 0.1 mg/kg bw/day, one female at 10 mg/kg bw/day and one female at 1000 mg/kg bw/day had no grossly observable conceptus at cesarean sectioning. However, the staining of these uteri with 10% ammonium sulfide solution revealed that all these females except for one at 10 mg/kg bw/day group were pregnant because of the presence of staining-positive implantation vestiges.

All the data obtained from females with no grossly observable conceptus were excluded from the calculation of group mean values. Consequently, the numbers of maternal animals with live fetuses at cesarean sectioning were 22, 22, 23 and 18, respectively, in the control, 0.1, 10 and 1000 mg/kg bw/day groups.

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Not applicable.

Changes in number of pregnant:

no effects observed

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Details on maternal toxic effects:

Maternal toxicity was noted in the 1000 mg/kg bw/day group, in which six animals (including two non-pregnant animals) died between gestation Days 20 to 28 after showing a decrease in food consumption and/or a stop of feeding with decreased body weights. Five of them (including two non-pregnant animals) also exhibited such clinical findings as soiled fur and/or red discharge on the tray and one aborted on the day of death. The mean body weight gains on gestation Day 15 and thereafter, as well as the mean food consumption during gestation Days 6 to 9 and 15 to 18 in this group were significantly lower than those in the control group.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: No adverse effects seen at this dose level.

2

Key result

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

mortality

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean weights of female fetuses were comparable to those in the control group. The mean weights of male fetuses at 10 and 1000 mg/kg bw/day were significantly lower than that in the control group.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

The mean numbers of live fetuses at 10 and 1000 mg/kg bw/day were comparable to that in the control group. The mean number of live fetuses at 0.1 mg/kg bw/day was significantly higher than that in the control group. However, this was not thought to be toxicologically significant.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Description (incidence and severity):

Not applicable.

Changes in postnatal survival:

not examined

Description (incidence and severity):

Not applicable.

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal observations revealed that one and two fetuses in the control and 0.1 mg/kg bw/day groups, respectively, had fused sternebrae, one fetus each exhibited absent rib (12th left), supernumerary lumber vertebra (right), and absence of lumbar arch (right) and centrum (4th) in the control group, and two and one fetus in the control and 1000 mg/kg bw/day groups, respectively, had misaligned caudal centrum. However, these abnormalities are considered to be unrelated to the test substance treatment since their incidences were low.

For skeletal variations, the incidences of supernumerary rib and 27 presacral vertebrae in the 10 and 1000 mg/kg bw/day groups were significantly higher than those in the control group. These findings are considered to be related to the treatment of test substance since similar changes were also found in the preliminary range-finding study. Significantly lowered incidence of cervical rib was seen in the 10 mg/kg bw/day group, but was not considered to be toxicologically significant. In addition, some fetuses in all groups including the control exhibited incomplete ossification of interparietal bones, misaligned sternebra, extra sternebral ossification site (between the 5th and 6th sternebrae), incomplete ossification of cervical arch (1st, right), incomplete ossification of cervical centrum (3rd or 4th), lumbosacral transitional vertebra and bipartite ossification of caudal centrum. However, there were no statistically significant differences in the incidences of these findings between the control and any of the treated groups.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

In the visceral observations, one fetus each in the control group exhibited cutis aplasia on the head, narrowed pulmonary artery accompanied by membranous ventricular septum defect, narrowed right subclavian artery accompanied by membranous ventricular septum defect and hydronephrosis, narrowed left common carotid artery and lobation anomaly of the liver. Abnormalities found in the 10 mg/kg bw/day group were one case each of malpositioned kidney, thickened peritoneum around the kidney and fusion of the intestine and bladder. In the 1000 mg/kg bw/day group, one fetus showed misshapen cerebellum and another had a small spleen. However, these abnormalities are considered to be unrelated to the test substance treatment since their incidences were low.

Malpositioned testis was observed in 0, 1, 5 and 6 fetuses in the control, 0.1, 10 and 1000 mg/kg bw/day groups, respectively, and the incidence of this finding in the 10 mg/kg bw/day group was significantly higher than that in the control group. However, the relationship between the increased incidence of malpositioned testis and the test substance treatment is unclear because this abnormality was not found in the rangefinding study in rabbits, nor in the previous teratogenicity study (M-400385-01-2, 2006) and two-generation reproductive toxicity study (M-399182-01-2, 2007) in rats. Additionally, malpositioned testis have been seen in control groups at low incidence in previously conducted teratogenicity studies using the same strain of rabbit, and the frequency seen in the present study (3.0 and 3.7%, in the 10 and 1000 mg/kg bw/day, respectively) falls within the range (0 - 4.4%) published in literature for this strain of rabbit (Nakatsuka et al., Cong. Anom., 37 (1): 47-138, 1997).

Visceral variations observed in this study included malpositioned left common carotid branch, malpositioned right subclavian branch, malpositioned internal thoracic branch and thymic remnant in the neck. There were no statistically significant differences between the control and any of the treated groups in the incidences of these findings.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Details on embryotoxic / teratogenic effects:

Reduced fetal weights and increased skeletal variations at 10 and 1000 mg/kg bw/day. None of the findings in the study were considered severe enough to be indicative of a teratogenic effect.

Effect levels (fetuses)

1

Key result

Dose descriptor:

NOAEL

Effect level:

0.1 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects seen at this dose level.

2

Key result

Dose descriptor:

LOAEL

Effect level:

10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

other: Increased incidence of skeletal variations

Fetal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: supernumerary rib

skeletal: vertebra

Description (incidence and severity):

For skeletal variations, the incidences of supernumerary rib and 27 presacral vertebrae in the 10 and 1000 mg/kg bw/day groups were significantly higher than those in the control group.

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

10 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

References

Toshio Nakatsuka et al., Japan Pharmaceutical Manufacturers Association (JPMA) survey on background control data of developmental and reproductive toxicity studies in rats, rabbits and mice. Cong. Anom., 37 (1): 47-138, 1997.

Applicant's summary and conclusion

Conclusions:

The study was performed under GLP conditions and according to OECD 414. The test substance caused reduced body weight gain and food consumption, adverse clinical findings and mortality in maternal animals at 1000 mg/kg bw/day, therefore the maternal NOAEL was 10 mg/kg bw/day. Reduced fetal weights in males and increased skeletal variations in males and females were seen at 10 and 1000 mg/kg bw/day, therefore the fetal NOAEL was 0.1 mg/kg bw/day. It was concluded that none of the findings were indicative of a teratogenic effect.