Phenol, 2-[4,6-bis([1,1'-biphenyl]-4-yl)-1,3,5-triazin-2-yl]-5-[(2-ethylhexyl)oxy]-

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014/2015

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)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test animals

Species:

rat

Strain:

Wistar

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: 10-15 weeks- Weight at study initiation:- Housing: single caging- Diet (e.g. ad libitum): ad libitum- Water (e.g. ad libitum): ad libitum- Acclimation period: six daysENVIRONMENTAL CONDITIONS- Temperature (°C): 20-24°C- Humidity (%): 30-70%- Air changes (per hr): 15- Photoperiod (hrs dark / hrs light): 12/12IN-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 bwThe 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance preparations was demonstrated over a period of 8 days at room temperature.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant- Proof of pregnancy: referred to as day 0 of pregnancy

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.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

Doses / concentrations 5

Dose / conc.:

600 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: range finding studyAs 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: Mortality/Morbidity, pertinent behavioral changes and/or signs of overt toxicity. 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: No - Sacrifice on gestation day: GD 20.On GD 20, the dams were sacrificed under isoflurane anesthesia by decapitation, in randomizedorder.After the dams had been sacrificed, they were necropsied and assessed for gross pathology, in randomized order. The uteri and the ovaries were removed and the following data were recorded:- Weight of the unopened uterus- Number of corpora lutea- Number and distribution of implantation sites classified as:• Live fetuses• Dead implantations:a) Early resorptions (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single horn pregnancy)b) Late resorptions (embryonic or fetal tissue in addition to placental tissue visible)c) Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)After the weight of the uterus had been determined, all subsequent evaluations of the dams and the gestational parameters were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose animal numbers were encoded.These data were used to calculate conception rate and pre- and postimplantation losses.The conception rate (in %) was calculated according to the following formula:(number of pregnant animals / number of fertilized animals) x 100The preimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:((number of corpora lutea – number of implantations) / number of corpora lutea) x 100The postimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:((number of implantations – number of live fetuses) number of implantations) x 100

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: YesExaminations 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):MalformationA permanent structural change that is likely to adversely affect the survival or health. VariationA 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, DUNNETT's testcorrected body weight gain, carcass weight, weight ofthe unopened uterus, weight of the placentas andfetuses, corpora lutea, implantations, pre- andpostimplantation losses, resorptions and live fetusesFISHER's exact testNumber of pregnant animals at the end of the study, FISHER's exact test mortality rate (of the dams) and number of litters with fetal findingsWILCOXON testProportion of fetuses with findings per litter

Indices:

sex ratio

Historical control data:

yes, period over 4 years

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

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). No further clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female at dose levels of 60, 200, 300 or 600 mg/kg bw/d during the entire study period.

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 mean body weights (BW) of the high-dose dams (600 mg/kg bw/d) were comparable to the concurrent control group. The mean body weight gain (BWC) of this group was slightly decreased during the treatment period (GD 6-19), the dams gained 9% less weight during this time. However, a lower weight gain of the animals was also noted before the beginning of treatment (GD 0-6, minus 10%), both effects together resulted in a lower gross weight gain (minus 8%) for the entire study. The mean body weights and the average body weight gain of the dams in test groups 1-3 (60, 200 and 300 mg/kg bw/d) were in general comparable to the concurrent controls throughout the entire study.

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.Corrected (net) body weight gain:Mean carcass weights were unaffected by the treatment (test groups 1, 2, 3 and 5). 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.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Uterus weightThe 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.

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.

Maternal developmental toxicity

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 implantation sites.

Total litter losses by resorption:

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 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

Details on maternal toxic effects:

Reproduction data:The conception rate was 96% in test groups 0 and 5 (0 and 600 mg/kg bw/d) and 100% in test groups 1, 2, 3 and 4 (60, 200, 300 and 0 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of the study. 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, in the mean number of corpora lutea and implantation sites or in the value calculated for the postimplantation loss, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

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

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

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1, 2, 3 and 5 (60, 200, 300 and 600 mg/kg bw/d) was not influenced by the test substance. 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).

External malformations:

no effects observed

Description (incidence and severity):

Fetal external malformationsExternal 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.Fetal external variationsOne 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.Fetal external unclassified observationsTwo 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.Fetal soft tissue unclassified observationsNo unclassified soft tissue observations were recorded.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Fetal skeletal malformationsSome 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.Fetal skeletal variationsFor all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeleton and appeared without a relation to dosing. The overall incidences of skeletal variations were comparable to the historical control data.Fetal skeletal unclassified cartilage observationsAdditionally, 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

Description (incidence and severity):

Fetal soft tissue malformationsNo soft tissue malformations were recorded.Fetal soft tissue variationsThree 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 relevantFetal soft tissue unclassified observationsNo unclassified soft tissue observations were recorded.

Other effects:

no effects observed

Description (incidence and severity):

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.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed at the highest dose tested

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

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

Total indcidence of 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

                                                                             

Total 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

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

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

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

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.