5-butoxy-2-[4-(4-butoxy-2-hydroxyphenyl)-6-(2,4-dibutoxyphenyl)-1,3,5-triazin-2-yl]phenol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jun 2014- Jan 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

22 Jan 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, 55116 Mainz

Limit test:

no

Test material

Test material information

Constituent 1

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 0008577537
- Expiration date of the lot/batch: 01 Jul 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Guaranteed by the manufacturer
- Stability in the vehicle: Analytical verifications of the stability of the test substance in corn oil at room temperature over a period of 8 days had been verified prior to the start of the study. Given that test substance is completely miscible with corn oil Ph. Eur./DAB, solutions are considered to be homogenous without further analysis.
- Solubility and stability of the test substance in the solvent/vehicle: The test substance was completely miscible with corn oil

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
For the test substance preparations, the test substance was heated up to 72 °C before exposure solution preperation

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Liquid

OTHER SPECIFICS:
- Physical state/Appearance: Solid/ slightly yellow

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI[Han]

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-12 weeks
- Housing: single housing
- Diet (e.g. ad libitum): ad libitum; ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland.
- Water (e.g. ad libitum): ad libitum; potable tap water bottles
- Acclimation period: six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2014-06-23 To: 2014-08-04

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: For the test substance preparation, the test substance was heated up to 72°C. Thereafter the specific amount of test substance was weighed, topped up with corn oil in a calibrated beaker and intensely mixed with a magnetic stirrer (70°C) until it was completely dissolved.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Test item is insoluble in water
- Concentration in vehicle: Adjusted to amount of vehicle
- Amount of vehicle (if gavage): 4ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The results of the analysis of the oily test substance preparations confirmed the correctness of the prepared concentrations. The analytical values of the samples corresponded to the expected values within the limits of the analytical method, i.e. were always above 90% and below 110% of the nominal concentrations

Details on mating procedure:

- Impregnation procedure: The animals were paired by the breeder (“time-mated”)
- Proof of pregnancy: Detection of vaginal plug/sperm

Duration of treatment / exposure:

14 days

Frequency of treatment:

daily

Duration of test:

GD 6-19 / 14 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

60 mg/kg bw/day (nominal)

Remarks:

as low-dose level

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

as mid-dose level

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

as high mid-dose level

Doses / concentrations 4

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

as high-dose level

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A GLP-compliant range finding study was conducted (dosing 300 mg/kg bw/d and 1000 mg/kg bw/d for 14 d)
- As scheduled in the study plan, the high-dose level for this study was 600 mg/kg body weight/day which was intended to be administered to test group 3. Because of a mistake, an incorrect dose level was applied to test group 3; the actually administered dose was 300 mg/kg bw/d throughout the entire study. Therefore, an additional dose group of 600 mg/kg bw/d (test group 5) and a control group (test group 4) were added to the study to cover the dose range as originally specified in the study plan.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Clinical symptoms were checked twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays (GO 0 to 20).

DETAILED CLINICAL OBSERVATIONS: A cage-side examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. If such signs occurred, the animals were examined several times daily (GD 0-20).

Food Consumption: The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

BODY WEIGHT: Yes
- Time schedule for examinations: GO 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: GD 20.
- Organs examined: uterus of the dams (weight, number of corpora lutea, number and distribution of implantation sites); fetuses (gross pathology, soft tissue examination, skeletal examination)

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
- Other: Site of implantations in the uterus

Fetal examinations:

- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [ half per litter ]
- Skeletal examinations: Yes: [ half per litter ]

Examinations of the fetuses after dissection from the uterus:
At necropsy each fetus was weighed, sexed, and external tissues and all orifices were examined macroscopically. The sex was determined by observing the distance between the anus and the base of the genitalia. Furthermore, the viability of the fetuses and the condition of placentae, umbilical cords, fetal membranes, and fluids were examined. The placentas were weighed and their individual weights were recorded. Thereafter, the fetuses were sacrificed by a subcutaneous injection of pentobarbital (Narcoren®; dose: 0.1 mL/fetus). After these examinations, approximately one half of the fetuses per dam were eviscerated, skinned and fixed in ethanol; the other half were placed in Harrison’s fluid for fixation.

Soft tissue examination of the fetuses:
The fetuses fixed in Harrison’s fluid were examined for any visceral findings according to the method of BARROW and TAYLOR. After this examination these fetuses were discarded.

Skeletal examination of the fetuses:
The skeletons of the fetuses fixed in ethanol were stained according to a modified method of KIMMEL and TRAMMELL. Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After this examination the stained fetal skeletons were archived individually.

Evaluation criteria for assessing the fetuses:
In the present study the glossary of WISE et al. (1997) and its updated version of MAKRIS et al. (2009) was essentially used to describe findings in fetal morphology. Classification of these findings was based on the terms and definitions proposed by CHAHOUD et al. (1999) and SOLECKI et al. (2001, 2003):

Malformation
A permanent structural change that is likely to adversely affect the survival or health.

Variation
A change that also occurs in the fetuses of control animals and/or is unlikely to adversely affect the survival or health. This includes delays in growth or morphogenesis that have otherwise followed a normal pattern of development. The term "unclassified observation" was used for those fetal findings, which could not be classified as malformations or variations. All fetal findings were listed in tables according to these classifications.

Statistics:

DUNNETT's test:
Food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight

FISHER'S EXACT test (onesided):
Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings

WILCOXON-test (one-sided):
Proportions of fetuses with malformations, variations and/or unclassified observations in each litter

Indices:

sex ratio

Historical control data:

yes, period over 4 years

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Two females of the high-dose group (600 mg/kg bw/d) showed transient salivation at two occasions during the treatment period (GD 14 and GD 17). Salivation persisted in the respective animals only for some minutes after daily gavage dosing (i.e. up to 10 minutes). Such transient salivation shortly after dosing most likely reflects a reaction of the animals to the taste and smell of the test substance. It is not considered to be a sign of systemic toxicity.

Mortality:

no mortality observed

Description (incidence):

There were no substance-related or spontaneous mortalities in any test group (0, 60, 200, 300 or 600 mg/kg bw/d)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The high-dose of the test substance (600 mg/kg bw/d) caused a decrease in body weight gain as well as a significant decrease in the corrected (net) body weight gain. The corrected body weight gain (terminal body weight on GD 20 minus weight of the
unopened uterus minus body weight on GD 6) was statistically significantly lower (about 18% below concurrent control) in test group 5 (600 mg/kg bw/d).

The corrected body weight gain of test groups 1-3 (60, 200 and 300 mg/kg bw/d) revealed no difference of any biological relevance to the concurrent control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In comparison to the respective concurrent control group, the mean food consumption of the high-dose dams (test group 5 - 600 mg/kg bw/d) was slightly, but statistically significantly decreased on GD 10-13 and GD 15-17. If calculated for the entire treatment period (GD 6-19) or study period (GD 0-20), the high-dose dams consumed -6% or -4% less food than the concurrent control group (test group 4).
The mean food consumption of the dams in test groups 1, 2 and 3 (60, 200 and 300 mg/kg bw/d) was generally comparable to the concurrent control throughout the entire study period.
This includes the statistically significantly increased mean food consumption value in test group 2 during pre-treatment on GD 3-6.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The mean gravid uterus weights of the animals of test groups 1, 2, 3 and 5 (60, 200, 300 and 600 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the concurrent control groups revealed no dose-dependency and were assessed to be without biological relevance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No necropsy findings which could be attributed to the test substance were seen in any dam.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

not specified

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no test substance-related and/or biologically relevant differences between the test groups 0-5 (0, 60, 200, 300, 0 and 600 mg/kg bw/d) in the postimplantation loss.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no test substance-related and/or biologically relevant differences between the test groups 0-5 (0, 60, 200, 300, 0 and 600 mg/kg bw/d) in the number of resorptions

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no test substance-related and/or biologically relevant differences between the test groups 0-5 (0, 60, 200, 300, 0 and 600 mg/kg bw/d) in the number of viable fetuses.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There were no test substance-related and/or biologically relevant differences between the test groups 0-5 (0, 60, 200, 300, 0 and 600 mg/kg bw/d) in conception rate.

Other effects:

no effects observed

Description (incidence and severity):

There were no test substance-related and/or biologically relevant differences between the test groups 0-5 (0, 60, 200, 300, 0 and 600 mg/kg bw/d) in the mean number of corpora lutea and implantation sites.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean fetal weights of test groups 1, 2, 3 and 5 (60, 200, 300 and 600 mg/kg bw/d) were not influenced by the test substance and did not show any biologically relevant differences in comparison to the concurrent control groups.

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The apparent skew in the sex distribution of live fetuses in test group 5 (significantly more males than females) is a consequence of a similar skew in the concurrent control group 4 which is almost exactly the reversed image of group 5 (more females than males).

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

External malformations were recorded for one control fetus (0 mg/kg bw/d), this fetus had associated skeletal findings. The total incidence of external malformations in treated animals did not differ significantly from the concurrent control group and was comparable to the historical control data.
One external variation, i.e. limb hyperextension, was detected in test group 2 (200 mg/kg bw/d). This single case was considered to be incidental and not related to treatment.

Two unclassified external observations, i.e. polyhydramnios and placentae fused, were recorded for two fetuses of test group 2 (200 mg/kg bw/d). These findings were not considered to be biologically relevant.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Some skeletal malformations were detected in test groups 0, 2 and 3 (0, 200 and 300 mg/kg bw/d) affecting the skull, sternum, vertebral column (with or without involving the ribs) and humerus. One control fetus of test group 0 showed multiple skeletal malformations and had associated external findings. All these findings were single cases, most of them can be found in the historical control data. An association with the treatment is not assumed.

Additionally, some isolated cartilage findings without impact on the respective bone structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the vertebral column, the sternum and ribs and did not show any relation to dosing. However, the incidence of notched cartilage between basisphenoid and basioccipital was significantly increased in test group 2 (200 mg/kg bw/d) and the incidence of branched rib cartilage was significantly increased in test group 1 (60 mg/kg bw/d). However, this findings showed no dose-dependency and were therefore assessed to be without biological relevance.

Visceral malformations:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Fetal soft tissue variations
Three soft tissue variations were detected, i.e. short innominate, dilated renal pelvis and dilated ureter. These variations were neither significantly different from the concurrent control nor dose-dependently present. Therefore, they were not considered to be biologically relevant

Weight of the placentae
The mean placental weights of test groups 1, 2, 3 and 5 (60, 200, 300 and 600 mg/kg bw/d) were comparable to the concurrent control groups. The statistically significantly higher mean placental weights of test group 2 were well within the historical control range (0.47 g [0.35 g - 0.99 g] and therefore were assessed to be of no biological relevance.

Details on embryotoxic / teratogenic effects:

No effects

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: abscence of adverse effects for the tested doses

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Tab. 2: Mean maternal body weight change during gestation

	group 0 - 0 mg/kg bw	group 1 - 60 mg/ kg bw	group 2 - 200 mg/kg bw	group 3 - 300 mg/kg bw	group 4 - 0 mg/kg bw	group 5 - 600 mg/kg bw
days 6 - 6	31.0 g	31.5 g	33.8 g	31.7 g	34.7 g	31.2 g *
days 6 - 19	83.5g	83.7 g	83.7 g	80.7 g	87.6 g	79.4 g *
days 0 - 20	126.7 g	128.0 g	130.9 g	125.7 g	135.9 g	124.8 g *

Dunnett-test, two-sided, * p< = 0.05

Tab. 3: Individual fetal external malformations

test group	Dam No, Fetus No, Sex	Findings
0 (0 mg/kg bw)	4, 07, male	short trunk, thread-like tail
1 (60 mg/kg bw)	none
2 (200 mg/kg bw)	none
3 (300 mg/kg bw)	none
4 (0 mg/kg bw)	none
5 (600 mg/kg bw)	none

   

 Tab. 4: Total fetal external variations

		group 0 - 0 mg/kg bw/d	group 1 -60 mg/kg bw/d	group 2 -200 mg/kg bw/d	group 3 -300 mg/kg bw/d	group 4 -0 mg/kg bw/d	group 5 -600 mg/kg bw/d
Litter	N	23	25	25	25	25	24
Fetuses	N	228	253	229	249	263	255
Fetal incidence	N (%)	0.0	0.0	1 (0.4)	0.0	0.0	0.0
Litter incidence	N (%)	0.0	0.0	1 (4.0)	0.0	0.0	0.0
Affected fetuses/litter	Mean %	0.0	0.0	4.0	0.0	0.0	0.0

Tab. 5: Total external unclassified observations

		group 0 - 0 mg/kg bw/d	group 1 -60 mg/kg bw/d	group 2 -200 mg/kg bw/d	group 3 -300 mg/kg bw/d	group 4 -0 mg/kg bw/d	group 5 -600 mg/kg bw/d
Litter	N	23	25	25	25	25	24
Fetuses	N	228	253	229	249	263	255
Fetal incidence	N (%)	0.0	0.0	2 (0.9)	0.0	0.0	0.0
Litter incidence	N (%)	0.0	0.0	2 (8.0)	0.0	0.0	0.0
Affected fetuses/litter	Mean %	0.0	0.0	4.4	0.0	0.0	0.0

                                                                         

Tab. 6: Total fetal soft tissue variations

		group 0 - 0 mg/kg bw/d	group 1 -60 mg/kg bw/d	group 2 -200 mg/kg bw/d	group 3 -300 mg/kg bw/d	group 4 -0 mg/kg bw/d	group 5 -600 mg/kg bw/d
Litter Fetuses	N N	23 109	25 121	25 107	25 119	25 127	24 121
fetal incidence	N (%)	6 (5.5)	3 (2.5)	3 (2.8)	4 (3.4)	5 (3.9)	2 (1.7)
litter incidence	N (%)	6 (26)	3 (12)	2 (8.3)	4 (16)	5 (20)	2 (8.3)
affected fetuses / litter	mean %	5.4	3.0	2.2	3.4	3.7	2.1

Tab. 7: Individual fetal skeletal malformations

test group	Dam No, Fetus No, Sex	Findings
0 (0 mg/kg bw)	4, 07, male	fetus with multiple skeletal malformations
1 (60 mg/kg bw)	none
2 (200 mg/kg bw)	72-10 F 72-12 M	shortened humerus shortened humerus
3 (300 mg/kg bw)	89-05 F93-10 F	misshapen basioccipital, severely malformed vertebralcolumn and/or ribs, cleft sternumshortened humerus
4 (0 mg/kg bw)	none
5 (600 mg/kg bw)	none

Tab. 8: Total fetal skeletal malformations

		group 0 - 0 mg/kg bw/d	group 1 -60 mg/kg bw/d	group 2 -200 mg/kg bw/d	group 3 -300 mg/kg bw/d	group 4 -0 mg/kg bw/d	group 5 -600 mg/kg bw/d
Litter Fetuses	N N	23 119	25 132	25 122	25 130	25 136	24 134
fetal incidence	N (%)	1 (0.8)	0.0	2 (1.6)	2 (1.5)	0.0	0.0
litter incidence	N (%)	1 (4.3)	0.0	1 (4.0)	2 (8.0)	0.0	0.0
affected fetuses / litter	mean %	0.9	0.0	1.3	1.6	0.0	0.0

Tab. 9: Total fetal skeletal variations

		group 0 - 0 mg/kg bw/d	group 1 -60 mg/kg bw/d	group 2 -200 mg/kg bw/d	group 3 -300 mg/kg bw/d	group 4 -0 mg/kg bw/d	group 5 -600 mg/kg bw/d
Litter Fetuses	N N	23 119	25 132	25 122	25 130	25 136	24 134
fetal incidence	N (%)	116 (97)	128 (97)	120 (98)	126 (97)	134 (99)	134 (100)
litter incidence	N (%)	23 (100)	25 (100)	25 (100)	25 (100)	25 (100)	24 (100)
affected fetuses / litter	mean %	97.4	97.1	98.5	96.9	98.5	100.0

Tab. 10: Occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter)

For a better overview, all skeletal variations with statistically significant differences between the control and the treated groups were compiled in the table below. All incidences were expressed on a fetus per litter basis and any statistically significant differences, which were outside historical control ranges were marked in bold and italics types.

Finding	group 0 - 0 mg/kg bw/d	group 1 -60 mg/kg bw/d	group 2 -200 mg/kg bw/d	group 3 -300 mg/kg bw/d	group 4 -0 mg/kg bw/d	group 5 -600 mg/kg bw/d	HCDMean %(range)
Incomplete ossification of parietal; unchanged cartilage	9.2	10.0	11.0	7.3	3.2	10.2*	11.1(6.4 – 17.1)
Incomplete ossification of sternebra; unchanged cartilage	92.8	87.5	94.5	84.4	90.6	96.3*	84.7(69.3 – 94.7)
Incomplete ossification of sternebra; unchanged cartilage	36.3	43.7	39.3	45.0	36.2	50.7*	52.7(29.5 – 76.9)

HCD = Historical control data; % = per cent * = p ≤ 0.05 (Wilcoxon-test [one-sided]) ** = p ≤ 0.01 (Wilcoxon-test [one-sided])

As can be seen from the table above, the rate of incompletely ossified sternebra was statistically significantly increased and slightly outside the historical control range in the test group 5 (600 mg/kg bw/d). This minor change may represent a slight delay of ossification which did not affect morphology, as the underlying cartilage model was completely intact. As can be seen from the historical background data, increased incidences of such incomplete or nonossifications of skeletal elements are routinely quantified and are among the most frequently noted skeletal variants in control populations of this Crl:WI(Han) rat strain. This indicates that these findings reflect species-specific anatomic variation at the time around birth without any detrimental effects on further development. As the incidence in this study is still in the order of magnitude of the background rate the finding is neither considered as biologically relevant nor as adverse. Concerning the other statistically significant differences, no dose dependency was observed and all values were clearly inside the historical control range, thus, an association with the test substance and a toxicological relevance is not assumed.

Tab. 11: Total unclassified cartilage observations

		group 0 - 0 mg/kg bw/d	group 1 -60 mg/kg bw/d	group 2 -200 mg/kg bw/d	group 3 -300 mg/kg bw/d	group 4 -0 mg/kg bw/d	group 5 -600 mg/kg bw/d
Litter	N	23	25	25	25	25	24
Fetuses	N	119	132	122	130	136	134
Fetal incidence	N (%)	102 (86)	97 (73)	87 (71)	106 (82)	111 (82)	118 (88)
Litter incidence	N (%)	23 (100)	24 (96)	23 (92)	23 (92)	25 (100)	24 (100)
Affected fetuses/litter	Mean %	85.4	74.1	71.0	79.0	0.0	0.0

Tab. 12: Total fetal malformations

		group 0 - 0 mg/kg bw/d	group 1 -60 mg/kg bw/d	group 2 -200 mg/kg bw/d	group 3 -300 mg/kg bw/d	group 4 -0 mg/kg bw/d	group 5 -600 mg/kg bw/d
Litter	N	23	25	25	25	25	24
Fetuses	N	228	253	229	249	263	255
Fetal incidence	N (%)	1 (0.4)	0.0	2 (0.9)	2 (0.8)	0.0	0.0
Litter incidence	N (%)	1 (4.3)	0.0	1 (4.0)	2 (8.0)	0.0	0.0
Affected fetuses/litter	Mean %	0.4	0.0	0.7	0.8	0.0	0.0

Tab.13: Total fetal variations

		group 0 - 0 mg/kg bw/d	group 1 -60 mg/kg bw/d	group 2 -200 mg/kg bw/d	group 3 -300 mg/kg bw/d	group 4 -0 mg/kg bw/d	group 5 -600 mg/kg bw/d
Litter	N	23	25	25	25	25	24
Fetuses	N	228	253	229	249	263	255
Fetal incidence	N (%)	122 (54)	131 (52)	123 (54)	130 (52)	139 (53)	136 (53)
Litter incidence	N (%)	23 (100)	25 (100)	25 (100)	25 (100)	25 (100)	24 (100)
Affected fetuses/litter	Mean %	53.4	52.1	55.3	52.3	52.7	53.6

Applicant's summary and conclusion

Conclusions:

Under the conditions of this prenatal developmental toxicity study, the oral administration to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at a dose of 600 mg/kg bw/d caused evidence of maternal toxicity, such as reduced gross and corrected (net) body weight gain. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 300 mg/kg bw/d.
The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 600 mg/kg bw/d, the highest tested dose.

Executive summary:

In a prenatal developmental toxicity study the test substance was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential for maternal and prenatal developmental toxicity. Analyses confirmed the correctness of the prepared concentrations and the stability of the test substance in the vehicle. The test substance caused neither mortality nor clinical symptoms of systemic toxicity in any of the exposed groups. Two females of the high-dose group (600 mg/kg bw/d) showed transient (up to 10 minutes) salivation at two occasions during the treatment period (GD 14 and GD 17). Such transient salivation shortly after dosing most likely reflects a reaction of the animals to the taste and smell of the test substance. It is not considered to be a sign of systemic toxicity. The high-dose of the test substance (600 mg/kg bw/d) caused a decrease in body weight gain as well as a significant decrease in the corrected (net) body weight gain. These effects are considered minimal, but treatment-related and adverse. No toxicologically relevant clinical effect was noted for the animals exposed to 60, 200 or 300 mg/kg bw/d of the test substance. No differences of toxicological relevance between the control and the treated groups (60, 200, 300 or 600 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no influence of the test compound on fetal weight and sex distribution of the fetuses was noted at any dose. Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose. Thus, the test item is not teratogenic in rats.