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Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Nov 2012 - 28 Jun 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Guidelines (Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Teratology (2-1-18), Agricultural Production Bureau, dated November 24, 2000).
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
N-[4-(2,4-dihydroxyphenyl)-1,3-thiazol-2-yl]-2-methylpropanamide
EC Number:
807-621-3
Cas Number:
1428450-95-6
Molecular formula:
C13H14N2O3S
IUPAC Name:
N-[4-(2,4-dihydroxyphenyl)-1,3-thiazol-2-yl]-2-methylpropanamide
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
other: RccHan : WIST (SPF)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., NM Horst, Netherlands
- Age at study initiation: 10 weeks (at delivery), 11 weeks (at initiation of pairing)
- Weight at study initiation: 204-255 g
- Housing: in groups of three to five females in Makrolon cages (type 4) with wire mesh tops (during acclimatization), one male with one female in special automatic mating cages (during pairing), mated females were housed individually in Makrolon cages (type 3) with wire mesh tops, sterilized standard softwood was used as bedding material, paper was used as enrichment
- Diet: pelleted standard Harlan Teklad 2018C rodent maintenance diet (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: community tap-water from Füllinsdorf (analysis was performed), ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 300
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dose formulations prepared on the first day of treatment were used only on the day of preparation. During the remaining study period dose formulations were prepared weekly. Dose formulations were prepared by dissolving appropriate amounts of the test material in PEG 300 yielding a final concentration of 12.5, 50 and 200 mg/mL.

VEHICLE
- Lot/batch no.: BCBG8285V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the samples was performed using HPLC coupled to an UV detector. The test substance concentrations in the dose formulations ranged from 88.6% to 100.7% with reference to the nominal and were within the accepted range of ± 20%. The homogeneous distribution of the test substance in the preparations was approved because single results found did not deviate more than 3.9% from the corresponding mean and met the specified acceptance criterion of ≤ 15%. In addition, the test item was found to be stable in application formulations when kept 4 h at room temperature and 8 days in the refrigerator (5 ± 3 °C) due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean value. Furthermore, the absence of the test substance in the vehicle control samples was confirmed.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: After acclimatization, females were housed with sexually mature males with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed.
- M/F ratio per cage: 1/1
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: copulation plug / sperm in vaginal smear referred to as Day 0 of pregnancy
Duration of treatment / exposure:
Day 6-20 of gestation
Frequency of treatment:
daily
Duration of test:
15 days
No. of animals per sex per dose:
22 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous dose range-finding toxicity study in Han Wistar rat, Harlan Laboratories study D58382, using dose levels of 0, 100, 300 and 1000 mg/kg bw/day, resulting in adverse effects at the dose level of 1000 mg/kg bw/day. Effects noted in females at the dose level of 1000 mg/kg bw/day consisted of ruffled fur, reduction in food consumption, body weights and body gains and were considered to be test
item-related. The reduction in fetal body weights at this dose level was also considered to be related to the treatment with the test item and adverse.
At the dose levels of 100 and 300 mg/kg bw/day, reduced food consumption was noted but without a clear dose-dependency. However, as lower food consumption in these dose-groups was already observed prior to the start of the treatment, these differences were finally not considered to be related to the treatment with the test item. There was also a lack of dose-dependency in the slightly reduced body weights and body weight gains at 100 and 300 mg/kg bw/day and consequently, a relation to the test item application was finally considered as not likely.
Based on these results, dose levels of 0, 50, 200, and 800 mg/kg bw/day were selected for the subsequent main prenatal developmental toxicity study in rats.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations which were included: viability / mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: from Day 0 until Day 21 post coitum

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined: Yes (at 3-day intervals)

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: Gross macroscopic examination of all internal organs with emphasis on the uterus and its contents was performed.
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
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
The following statistical methods were used to analyze food consumption, body weights, macroscopical findings, reproduction and skeletal examination data:
- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.
Historical control data:
Corresponding historical control data were included.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
At the dose level of 800 mg/kg bw/day, feces discolored yellow were observed in all females.
Remaining findings were only observed for one day in individual animals and were therefore not
considered to be related to the treatment with the test item. At the high-dose level, reduced
activity and ventral recumbency was noted in one female on day 12 and further three females
had ruffled fur on day 21 or 21 and 22 of the gestation period.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At the dose level of 800 mg/kg bw/day a statistically significant reduction in absolute body
weight (from day 10 to 21 p.c.) and statistically significant reduction in body weight gain (from
day 7 to 21 p.c.) were noted. Corrected body weight gain (body weight gain from day 6 to 21 p.c.
corrected for the gravid uterus weight) was also statistically significantly reduced at the highdose
level.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At the dose level of 800 mg/kg bw/day a statistically significant reduction in food consumption
was noted during the entire treatment period. Mean food consumption from day 6 to 21 of the
gestation period was 16.3 g/animal/day at the high-dose level compared to 25.2 g/animal/day in
the control.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No findings were noted in females during macroscopical examinations at any dose level.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
not examined
Early or late resorptions:
not specified
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
With exception for one female at each dose level 0, 50 and 800 mg/kg bw/day, all females were
pregnant.
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: significant reduced body weight and body weight gain at 800 mg/kg bw/d

Details on maternal toxic effects:
VIABILITY / MORTALITY
All females survived until the scheduled necropsy.

CLINICAL SIGNS
At 800 mg/kg bw/d, feces discolored yellow were observed in all females. Reduced activity and ventral recumbency was noted in one female on Day 12 and further three females had ruffled fur on Day 21 or 21 and 22 of the gestation period at 800 mg/kg bw/d.

FOOD CONSUMPTION
At 800 mg/kg bw/d, a statistically significant reduction in food consumption was noted during the entire treatment period. Mean food consumption at 800 mg/kg bw/d from Day 6 to 21 of the gestation period was 16.3 g/animal/d compared to 25.2 g/animal/day in the control. No effects on food consumption were recorded up to and including the dose level of 200 mg/kg bw/d. Compared to the values of the control group, mean differences in food consumption during the treatment at the dose levels of 50, 200 and 800 mg/kg bw/day were -3.2%, -4.4% and -35.3%, respectively.

BODY WEIGHTS
At 800 mg/kg bw/d, a statistically significant reduction in absolute body weight (from Day 10 to 21 p.c.) and statistically significant reduction in body weight gain (from Day 7 to 21 p.c.) were noted. Corrected body weight gain (body weight gain from Day 6 to 21 p.c. corrected for the gravid uterus weight) was also statistically significantly reduced at 800 mg/kg bw/d.
No effects on absolute body weights, body weight gain or corrected body weight gain were recorded up to and included the dose level of 200 mg/kg bw/d.

REPRODUCTIVE PERFORMANCE
With exception for one female at each dose level 0, 50 and 800 mg/kg bw/d, all females were pregnant. Mean number of implantation sites per dam was similar at all dose levels 14.7, 14.1, 14.4 and 14.1 at the dose levels of 0, 50, 200 and 800 mg/kg bw/d, respectively.
All reproduction data including post-implantation loss and number of fetuses per dam were not affected by treatment with the test item at any dose level. Mean number of fetuses at termination was 13.5, 12.8, 13.6 and 13.5 at dose levels of 0, 50, 200 and 800 mg/kg bw/d, respectively

GROSS PATHOLOGY
No findings were noted in females during macroscopical examinations at any dose level.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no effects have been observed at this dose level
Key result
Dose descriptor:
NOAEL
Effect level:
800 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Key result
Abnormalities:
not specified

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At the dose level of 800 mg/kg bw/day a statistically significant reduction in fetal body weights
was noted if calculated on both, litter and individual basis.
Reduction in number of live offspring:
not specified
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No effects on fetal sex ratio were noted at any dose level.
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not specified
External malformations:
no effects observed
Description (incidence and severity):
No test item-related findings were observed during external examination of the fetuses at any
dose level.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The increased incidence of findings at the dose level of 800 mg/kg bw/day was due to an
increase in the number of the following variations: bipartite ossification of supraoccipital, fusion
of zygomatic arch in skull, dumbbell ossification of thoracic vertebral body, offset ossification
sites and bipartite ossification in sternebra. These increased incidences were considered to be test
item-related.
All remaining findings occurred in individual animals, or were in the range of historical controls
and were therefore considered to be not related to the treatment.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
Large or slightly large thyroids were recorded in 50 % of fetuses (95% of litters) at this dose
level whereas in the control group this finding was noted in 5% of fetuses (29% of litters).
Therefore this finding was considered to be related to the treatment with the test item.

Remaining findings were considered not to be related to the treatment. With exception of thin
diaphragm and malpositioned testis, findings were noted only in individual fetuses or with an
incidence, which was not correlated with the dose levels. The number of fetuses with thin
diaphragm was higher at the high-dose level. This finding was noted in 21% of fetuses (71% of
litters) compared to 8% of fetuses (43% of litters) in the control group. Values at the high dose
levels were above the historical control values. However, the increase was only slight and
therefore considered as an indication for biological variability and not test item-related. The
malpositioned testes were found in 1% (5%), 4% (24%), 4% (27%) and 6% (33%) fetuses (litters)
in order of ascending dose levels. The incidence of this finding increased slightly with increasing
dose levels but without a clear dose dependency and was therefore considered to be not related to
the test item.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: Disturbance in fetal development at 800 mg/kg bw/d; however, effects were only observed in the presence of adverse maternal toxic effects and were therefore considered to be secondary in nature. No teratogenic effects were found.

Details on embryotoxic / teratogenic effects:
EXTERNAL EXAMINATIONS
No test item-related findings were observed during external examination of the fetuses at any dose level.
In one fetus at the high-dose level multiple findings were noted: small head with a cyst like structure and not visible eyes, ears and mouth. But due to isolated occurrence, this observation was considered to be not related to the treatment with the test item.

BODY WEIGHTS
At 800 mg/kg bw/d, a statistically significant reduction in fetal body weights was noted if calculated on both, litter and individual basis. Mean body weights of male and female fetuses were 3.7 g at the high-dose level and were comparable to 4.9 g in the control group.
No effects on fetal body weights were observed up to and including the dose level of 200 mg/kg bw/d.

SEX RATIO
No effects on fetal sex ratio were noted at any dose level.

VISCERAL ABNORMALITIES AND VARIATIONS
At 800 mg/kg bw/d, a higher number of variations was observed. Large or slightly large thyroids were recorded in 50 % of fetuses (95% of litters) at this dose level whereas in the control group this finding was noted in 5% of fetuses (29% of litters). Therefore this finding was considered to be related to the treatment with the test item.
Remaining findings were considered not to be related to the treatment. With exception of thin diaphragm and malpositioned testis, findings were noted only in individual fetuses or with an incidence, which was not correlated with the dose levels. The number of fetuses with thin diaphragm was higher at the high-dose level. This finding was noted in 21% of fetuses (71% of litters) compared to 8% of fetuses (43% of litters) in the control group. Values at the high dose levels were above the historical control values. However, the increase was only slight and therefore considered as an indication for biological variability and not test item-related. The malpositioned testes were found in 1% (5%), 4% (24%), 4% (27%) and 6% (33%) fetuses (litters) in order of ascending dose levels. The incidence of this finding increased slightly with increasing dose levels but without a clear dose dependency and was therefore considered to be not related to the test item.

BONE AND CARTILAGE ABNORMALITIES AND VARIATIONS
During skeletal examination of the fetuses, findings were observed in 18% of examined fetuses (in 67% litters) in the control group compared to 40% of examined fetuses (in 95% litters) at 800 mg/kg bw/d.
The increased incidence of findings at 800 mg/kg bw/d was due to an increase in the number of the following variations: bipartite ossification of supraoccipital, fusion of zygomatic arch in skull, dumbbell ossification of thoracic vertebral body, offset ossification sites and bipartite ossification in sternebra. These increased incidences were considered to be test item-related.
All remaining findings occurred in individual animals, or were in the range of historical controls and were therefore considered to be not related to the treatment.

OSSIFICATION AND SUPERNUMERARY RIBS
At 800 mg/kg bw/d, an increase in incidence of fetuses with incomplete ossification of cranium bones and sternebrae, increased numbers of non-ossified cervical vertebrae and limb bones, as well as increased number of supernumerary ribs (rudimentary) was observed. These differences were statistically significant, if calculated on both, litter and individual basis. Therefore these findings were considered to be test item-related.
The remaining statistically significantly changes on a litter basis did not follow a dose dependency pattern and were therefore considered to be not related to the treatment.

ADDITIONAL CARTILAGE VARIATIONS
At 800 mg/kg bw/d, an increased frequency of branched xiphoid cartilage and an increased frequency of interrupted individual costal cartilages were noted. Both changes were statistically significant, if calculated on both, litter and individual basis and therefore considered to be test item-related. Changed (both, increased or decreased) numbers of fetuses with interrupted costal cartilages were also noted in low- and mid-dose groups. But these changes were statistically significant only, if calculated on an individual basis and they did not follow a dose dependency and were therefore considered to be not test item-related.
The remaining cartilage changes in the high-dose group were minor and statistically significant only if calculated on an individual basis and therefore considered not to best item related.

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: prenatal development
Key result
Dose descriptor:
NOEL
Effect level:
> 800 mg/kg bw/day
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. Body weights (mean values in g)

 

Dose level (mg/kg bw/day)

0

50

200

800

Day 0

228 ± 9

228 ± 11

226 ± 10

230 ± 13

Day 7

256 ± 10

255 ± 11

251 ± 10

257 ± 13

Day 14

288 ± 13

288 ± 13

284 ± 11

268 ± 18**

Day 21

374 ± 20

369 ± 20

368 ± 25

309 ± 29**

** (p ≤ 0.01) significantly different from controls (Dunnett-Test)

Applicant's summary and conclusion

Conclusions:
Based on these results, the NOEL (No Observed Effect Level) for maternal toxicity was 200
mg/kg bw/day.
The NOEL for prenatal developmental toxicity was 200 mg/kg bw/day. Effects on the fetal
development were only observed in the presence of adverse maternal toxic effects and were
therefore considered to be secondary in nature.
The NOEL for teratogenicity was 800 mg/kg bw/day, the highest dose level investigated.
Executive summary:

The purpose of this study was to investigate W630 (Sample ID: 26480) for its potential to induce

effects in the pregnant Han Wistar rat and in the developing embryo and fetus following

exposure from day 6 post coitum (implantation) to day 20 post coitum (the day prior to

Caesarean section).

Four groups of 22 mated females per group were treated by gavage with the test item once daily

at dose levels of:

Group 1: 0 mg/kg body weight/day (control group)

Group 2: 50 mg/kg body weight/day

Group 3: 200 mg/kg body weight/day

Group 4: 800 mg/kg body weight/day

A standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body

weight was used. Control animals were dosed with the vehicle alone (PEG 300).

All females were sacrificed on day 21 post coitum and the fetuses were removed by Caesarean

section.