Methanesulfonamide, N-[2-[(4,6-dimethoxy-1,3,5-triazin-2-yl)carbonyl]-6-fluorophenyl]-1,1-difluoro-N-methyl-

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Aug 2011 - 19 April 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Reference 2

Reference Type:

other: Amendment No. 1

Title:

Unnamed

Year:

2013

Report date:

2013

Reference 3

Reference Type:

other: Amendment No. 2

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japenese Ministry of Agriculture, Forestry and Fisheries, notification 12 Nousan N°8147 (November, 2000)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-[2-(4,6-dimethoxy-1,3,5-triazine-2-carbonyl)-6-fluorophenyl]-1,1-difluoro-N-methylmethanesulfonamide

EC Number:

620-056-5

Cas Number:

874195-61-6

Molecular formula:

C14H13F3N4O5S

IUPAC Name:

N-[2-(4,6-dimethoxy-1,3,5-triazine-2-carbonyl)-6-fluorophenyl]-1,1-difluoro-N-methylmethanesulfonamide

Test animals

Species:

rabbit

Strain:

other: New Zealand White Crl:KBL (NZW) rabbit

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Châtillon-sur-Chalaronne, France
- Age at study initiation: 18±1 weeks old
- Weight at study initiation: between 2.96 and 3.89 kg
- Housing: Individual housing of pregnant females in suspended stainless steel wire mesh cages.
- Diet: 110 C-10 pelleted animal diet from S.A.F.E. (Scientific Animal Food and Engineering, Augy, France) ad libitum.
- Water: Filtered and softened tap water from the municipal water supply, ad libitum.
- Acclimation period: 4 or 5 days prior to commencement of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-21
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod: 16 h light/8 hrs dark ( 5am-9pm)

IN-LIFE DATES: From: 2011-08-04 To: 2012-04-19

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% methylcellulose 400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The appropriate amount of test item was suspended (w/w) in an aqueous solution of 0.5% methylcellulose 400 and stored at approximately 5 °C (±3 °C). Test formulations were prepared 6 times (F1 to F6) during the study. The suspensions were mixed continuously before and during treatment with an electromagnetic stirrer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of the test item in 0.5% aqueous methylcellulose was demonstrated in study SA 08145 at concentrations of 0.5 and 250 g/L for up to 29 days under similar conditions to those of the current study. Homogeneity of the suspensions was checked on the first formulation (F1) for the lowest and the highest concentrations (3.75 and 18.75 g/L). The mean values obtained from the homogeneity check were used as measured concentrations. In addition, the intermediate concentration (7.5 g/L) of the first formulation (F1) and all concentrations of the remaining formulations (F2 to F6) were checked. Data were recorded and analyzed using Empower 2 (Build 2154).
Results: Homogeneity and concentration analysis: 95 to 100% of nominal concentrations, which is within the in-house target range of 90 to 110% of nominal concentration. Therefore dose preparations were considered acceptable for use on this study.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
- Time-mated female rabbits were received from the supplier on GD 1 or GD 2. Nulliparous females were mated with stock males of the same strain and same supplier. The day of mating was designated as gestation Day 0 (GD 0).

Duration of treatment / exposure:

GD 6-28

Frequency of treatment:

daily

Duration of test:

On GD 29, all surviving females were sacrificed (34 days).

Doses / concentrations

Remarks:

Doses / Concentrations:
15, 30, 75 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

animals assigned: 23
animals pregnant at start of treatment: 22, 22, 20, 21 (in the control group, in the 15, 30 and 75 mg/kg bw/d group, respectively)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The range of doses was selected based on the severity of maternal toxic effects obtained in a range-finding study (SA 08261), where groups of 8 presumed-pregnant rabbits were administered the test item by gavage at 0, 25, 75 or 200 mg/kg bw/day, from GD 6-28 inclusive. A dose level of 200 mg/kg bw/day resulted in marked maternal toxicity as evidenced by clinical signs, body weight losses, reduced food consumption and increased liver weight (+21%; p < 0.01). At 75 mg/kg bw/day, there was a slight maternal toxicity as evidenced by a slight reduction of 22% in body weight gain between GD 6-29 (not statistically significant). Mean maternal corrected body weight change (maternal body weight change independent of the uterine weight) was slightly more pronounced than in the controls (-0.27 kg compared to -0.21 kg, not statistically significant) and was slightly outside the range of in-house HCD (-0.08 to -0.25 kg). At necropsy, mean liver weight was unaffected by the treatment. At the macroscopic examination, no treatment-related findings were noted. At 25 mg/kg bw/day, no treatment-related maternal findings were noted.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All cages were checked for dead or moribund animals twice daily, once in the morning and again in the afternoon (except at weekends and public holidays when checking was carried out once daily).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All clinical signs were recorded for individual animals. All animals were examined daily from GD 2-29.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on GD: 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 29

FOOD CONSUMPTION: Yes
- Full feeder weights were measured on GD: 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28
- Empty feeder weights were measured on GD: 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 29
From these records the mean daily consumption was calculated. Food spillage was also noted.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on GD 29
- Organs examined: visceral organs

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
- Number of live and dead fetuses: Yes
- Individual weights of live fetuses: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Visceral examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter

Statistics:

Mean and standard deviation for all maternal, litter and fetal parameters were calculated for each group.
Statistical analyses were performed separately for all pregnant females and for all pregnant females with live fetuses.
Statistical analyses were performed on the following parameters using SAS programs (Version 9.2).
- Maternal endpoints: body weight changes calculated according to interval periods; calculated corrected body weight change (body weight data measured on different days throughout gestation were not statistically analyzed; only descriptive statistics are presented); average food consumption calculated according to interval periods, liver weight
- Litter based endpoints: number of corpora lutea; number of implantation sites; number of resorptions (early, late); pre- and post-implantation loss percentages; fetal body weight (combinded sexes and per sex)
If one or more group variance(s) equaled 0, means were compared using non-parametric procedures.
- Fetal endpoints: fetal sex; fetal death status
If one or more group variance(s) equaled 0, means were compared using non-parametric procedures.
For fetal sex (male vs. female fetuses) and fetal death status (live vs. dead fetuses) endpoints, control group and each exposed group were compared using the Chi-square test for fetal sex parameter, using the Fisher Exact test (2-sided) for fetal death status parameter. Death status was analyzed both using the fetus as the statistical unit and using the litter as the statistical unit. Group means were compared at the 5% and 1% levels of significance.
In addition, statistical analysis was conducted on selected visceral and skeletal fetal observations.

Indices:

Pre-implantation loss
Post-implantation loss
Number of live fetuses per litter
Number of dead fetuses per litter

Historical control data:

Historical control data from studies conducted in-house were referred to in order to allow comparison with concurrent controls.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Mortality
There was no treatment-related mortality throughout the study.
One female in the control group was found dead on GD 7. At the macroscopic examination, the left lobes of the lungs were found collapsed. Thus, the death was attributed to a gavage error.
One animal treated at 75 mg/kg bw/day AE 1887196 aborted on GD 28 after having lost 250 g between GD 20-26 and 330 g between GD 26-28 (early necropsy). Food consumption was reduced between GD 20-26. There were no macroscopic findings at necropsy. In isolation and as there was no severe maternal toxicity noted in the other animals of this dose group, this abortion was not attributed to the treatment.

Clinical observation
At 75 mg/kg bw/day, 12/21 animals had few feces (compared to 6/23 in the controls) on several days.
The other clinical signs were those commonly encountered in pregnant rabbits or were observed with no dose-relationship.

Body weight
Up to the highest dose of 75 mg/kg bw/day, there were no treatment-related effects on mean maternal body weights, body weight gains and maternal corrected body weight change. One animal treated at 15 mg/kg bw/day had total litter resorptions (no live fetuses).

Food consumption
At 75 mg/kg bw/day, the mean food consumption of all pregnant females with live fetuses was slightly reduced by 13% between GD 6-8 (p < 0.05) and by approximately 10 % between GD 8-14 compared to the controls (not statistically significant). Thereafter, mean food consumption was unaffected by treatment. This effect was considered to be treatment-related, though it remained within the range of in-house Historical Control Data (HCD). Up to the dose of 30 mg/kg bw/day, mean food consumption was unaffected by treatment.

Gross pathology and organ weights
The few macroscopic findings observed occurred in isolation and/or with no doserelationship and were thus considered to be incidental.
At 75 mg/kg bw/day, the mean liver weight of pregnant females was increased by 8% when compared to the concurrent control group (not statistically significant). Individual values of liver weight from animals treated at 75 mg/kg bw/day ranged between 73-141 g compared to 73-117 g in the control group. There was no other change in mean liver weights at any dose level.

Histopathology
No microscopic changes were considered to be treatment-related

Cesarean section data
Pregnancy rate was unaffected by treatment. The pregnancy rate was 91% in the 30 and 75 mg/kg bw/day dose groups, 96% in the 15 mg/kg bw/day dose group and 100% in control group. Litter parameters including the number of live fetuses, the number of implant sites per dam, the percentages of pre and post implantation losses, the number of early and late resorptions, the fetal death status, the percentage of male fetuses, and the fetal body weight for combined and separate sexes were unaffected by treatment.
One animal treated at 15 mg/kg bw/day had a total litter loss (resorptions). This finding was considered incidental and not treatment-related.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

>= 75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

>= 75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
External observations (Table 3)
There were no treatment-related malformations or variations noted at the fetal external examination. The few variations observed were considered to be incidental, as they occurred in isolation and were well within the range of in-house HCD.

Visceral observations (Table 4)
There were no treatment-related malformations or variations noted at the fetal visceral examination. The visceral malformations observed in a total of 10 fetuses were distributed between the different doses groups including the control (1, 2, 5, 2 malformed fetuses at 0, 15, 30 and 75 mg/kg bw/day groups, respectively).
One fetus from the high dose group, 3 fetuses from the mid dose group, 2 fetuses from the low dose group and one fetus from the controls had cardiac malformations. One fetus from the high dose group and one from the medium dose group had retina fold unilateral. One fetus from the mid dose group had an absence of kidney. Since these findings were observed with no dose-effect relationship and as they were well within the in-house HCD, they were considered to have occurred spontaneously.
The incidences of the visceral variation “Thymic remnant present (uni/bi)” and “Ureter retrocaval (uni)” were higher at 75 mg/kg bw/day than in control group, at both litter and fetal levels. However, since the incidences were well within or at the border of the in-house HCD and since the differences from controls were not statistically significant; these findings were considered not to be treatment-related.
Other variations observed did not occur in a dose-related manner, were within the in-house HCD or occurred as isolated findings and were considered incidental.

Skeletal observations (Table 5)
There were no treatment-related malformations or variations noted at the fetal skeletal examination. Skeletal malformations were observed in a total of 8 fetuses from all dose groups including the control.
One fetus from each treated group had thoracic malformations. Two fetuses in two different litters from the high dose group had lumbar malformations. With the exception of hemivertebrae, each finding on these fetuses was however observed within the range of the in-house HCD. There were 2 cases of 1 lumbar hemivertebra (1.1% fetuses, 10% litters) compared to 1 case (0.5% fetuses, 4.2% litters) in the HCD. Therefore, and in the absence of any other treatment-related malformations in the skeletal axis, these malformations were considered to be incidental.
The incidences of the skeletal variations “Extra ossification point(s) (uni/bi) on 7th cervical vertebra” and “Insertion point(s) (uni/bi) of pelvic girdle on 2nd sacral vertebra” were higher in all treated groups than in the control group, at the litter and/or fetal level. However, as the values were not statistically different from controls, did not occur in a dose-related manner and were well within the in-house HCD, these findings were considered not to be treatment-related.
The incidence of the skeletal variation “13th thoracic rib (bi)” was higher at 30 and 75 mg/kg bw/day than in the control group, at both litter and fetal levels, and statistically significant (p < 0.01) only at the fetal level at the high dose group. However, the incidences were well within the in-house HCD, this finding was therefore considered not to be treatment-related.
Other variations observed did not occur in a dose-related manner, were within the in-house HCD or occurred as isolated findings and were considered incidental.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

>= 75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Table 1: Mean (±SD) Maternal Body Weight Gain (kg) of all pregnant females

Interval	Dose level of AE 1887196 in mg/kg bw/day
	0	15	30	75
Number of pregnant females at start of treatment	22	22(21)	21	20
Pretreatment, GD 3-6:	0.04 ± 0.060	0.05 ± 0.083 (0.05 ± 0.085)	0.04 ± 0.050	0.04 ± 0.058
Treatment, GD 6-8:	0.03 ± 0.034	0.03 ± 0.036 (0.03 ± 0.037)	0.01 ± 0.072	0.02 ± 0.035
Treatment, GD 8-10:	0.04 ± 0.033	0.04 ± 0.034 (0.04 ± 0.035)	0.04 ± 0.026	0.03 ± 0.023
Treatment, GD 10-14:	0.07 ± 0.047	0.09 ± 0.060 (0.09 ± 0.061)	0.08 ± 0.051	0.09 ± 0.041
Treatment, GD 14-18:	0.07 ± 0.051	0.04 ± 0.098 (0.05 ± 0.089)	0.08 ± 0.052	0.05 ±0.065
Treatment, GD 18-22:	0.05 ± 0.044	0.05 ± 0.041 (0.04 ± 0.037)	0.07 ± 0.040	0.04 ± 0.046
Treatment, GD 22-26:	0.05 ± 0.074	0.05 ± 0.061 (0.04 ± 0.059)	0.07 ± 0.046	0.05 ± 0.084
Treatment, GD 26-29:	0.06 ± 0.038	0.08 ± 0.044 (0.08 ± 0.045)	0.08 ± 0.045	0.06 ± 0.051
Treatment, GD 6-29:	0.37 ± 0.129	0.36 ± 0.148 (0.36 ± 0.151)	0.43 ± 0.113	0.36 ± 0.151
Corrected BW gain	-0.17 ± 0.121	-0.16 ± 0.154 (-0.16 ± 0.154)	-0.11 ± 0.158	-0.15 ± 0.147

SD: standard deviation

One animal treated at 15 mg/kg bw/day had total litter loss. For this reason, the figures in parentheses are the means calculated once the data for this female are excluded.

 

Table 2: Cesarean Section Observations

Observation	Dose Level of AE 1887196 (mg/kg bw/day)
	0	15	30	75
Maternal data:
No. Animals assigned	23	23	23	23
No. Animals pregnant	23	22	21	21
Pregnancy rate, %	100	96	91	91
No. Animals non-pregnant	0	1	2	2
Uterine data at scheduled sacrifice:
Total No. corpora lutea (c)	256	253	242	226
Total No. Implantations (c)	229	217	203	194
Total No. Litters (c)	22	22	21	20
Total No. live fetuses (c)	207	196	190	178
Total No. dead fetuses	6	9	4	8
Total No. early resorptions (c)   Total No. late resorptions (c)   Litters with total resorptions (c,d)	13     3     0	12 (7)     0     1	8     1     0	6     2     0
Mean fetal weight, combined sexes [g]	39.36 ± 4.17	38.87 ± 4.17	41.37 ± 2.86	37.84 ± 3.30
Sex ratio, % male	44.0 ± 21.6	47.2 ± 17.2	49.4 ± 19.3	54.0 ± 20.8
Preimplantation loss per litter, %	10.55 ± 13.07	13.46 ± 15.49 (14.10 ±15.58)	15.65 ± 15.17	14.16 ± 10.51
Postimplantation loss per litter, %	9.2 ± 8.8	10.8 ± 21.4 (6.6 ± 8.1)	5.9 ± 8.0	9.1 ± 16.0

(c) statistical analysis was not conducted on this endpoints

(d) Also includes litters with dead fetuses only or dead fetuses and resorptions

The figures in parentheses are the means calculated once the data for the animal having total litter loss are excluded.

 

Table 3: External examinations

Dose level [mg/kg bw/day]	0	15	30	75	Historical control range	0	15	30	75	Historical control range
Observations	Number of litters examined					Number of fetuses examined
	22	21	21	20		207	196	190	178
	Number of litters affected (Percentage of litters affected)					Number of fetuses affected (Percentage of fetuses affected)
Malformations Omphalocele	0 (0.0)	0 (0.0)	0 (0.0)	1 (5.0)	(0.0-4.5)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.6)	(0.0-0.5)

 

 

Table 4: Visceral examinations

Dose level [mg/kg bw/day]	0	15		30		75	HCR	0	15	30	75	HCR
Observations	Number of litters examined							Number of fetuses examined
	22	21		21		20		207	196	190	178
								Number of heads examined
								97	92	90	85
	Number of litters affected (Percentage of litters affected)							Number of fetuses affected (Percentage of fetuses affected)
Variations
#Thymic remnant present (uni/bi)	11 (50.0)	7 (33.3)	6 (28.6)		12 (60.0)		(20.8 - 84.2)	25 (12.1)	10 (5.1)	8 (4.2)	25 (14.0)	(3.8 - 23.9)
#Ureter (uni): retrocaval	2 (9.1)	0 (0.0)	1 (4.8)		2 (10.0)		(0.0 - 18.2)	3 (1.4)	0 (0.0)	1 (0.5)	5 (2.8)	(0.0 - 2.3)

# : Statistical analysis was conducted on this observation

HCR: historical control range

 

Table 5: Summary of skeletal malformations

Dose level [mg/kg bw/day]	0	15				30		75		0	15		30		75
	Number of litters examined									Number of fetuses examined
	22		21		21				20	207		196		190		178
	Number of litters examined									Number of heads examined
	22		21		21				20	97	92		90		85
Malformations	Number of litters affected									Number of fetuses affected
One thoracic vertebra : hemivertebra. One rib (uni) : absent	1	0		0			0			1	0		0		0
Two ribs (uni) : fused. Two thoracic centrum : hemicentrum and fused	0	1		1			0			0	1		1		0
One lumbar vertebra : hemivertebra. One lumbar hemicentrum and one lumbar centrum : fused	0	0		0			2			0	0		0		2
Caudal and all sacral vertebrae : fused. Caudal vertebrae : absent	1	0		0			0			1	0		0		0
All caudal vertebrae (except 1st): fused	0	0		1			0			0	0		1		0