2,4,6-tris(dimethylaminomethyl)phenol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-09-04 to 2017-10-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted January 22, 2001

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

May 30, 2008

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,4,6-tris(dimethylaminomethyl)phenol

EC Number:

202-013-9

EC Name:

2,4,6-tris(dimethylaminomethyl)phenol

Cas Number:

90-72-2

Molecular formula:

C15H27N3O

IUPAC Name:

2,4,6-tris[(dimethylamino)methyl]phenol

Test material form:

liquid

Details on test material:

2,4,6-tris(diemethylaminomethyl)phenol, Batch 2287872

Specific details on test material used for the study:

The test item was suspended in the vehicle to the appropriate concentrations and was administered orally at a constant volume once daily from the 6th to the 20th day of gestation

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ORGANISMS: 
- Species: Rat
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Strain: CD / Crl: CD(SD)
- Age: 56 days
- body weight: 200.2 g - 280.3 g
- Diet: ad libitum, Commercial diet ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water: ad libitum
- Acclimatisation period: 5 days
-Housing: Except during the mating period, the dams were kept singly in MAKROLON cages
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C +/- 3° C
- Humidity (%): 55% +/- 15 %
- Illumination: 12 hours artifical fluorescent light and 12 hours dark
- Ventilation rate: between fifteen to twenty air changes per hour.
ENVIROMENTAL ENRICHMENT
- The animals received one piece of wood (certified for animal use) to gnaw on once weekly at change of the cages.
- Octagon-shaped red-tinted huts (polycarbonate) were placed in the cages to offer the animals a resting and hiding place.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG400

Details on exposure:

ADMINISTRATION: 
- Frequency: once daily, day 6 to 20 of gestation
- Dose volume: 4 ml/kg b.w.
- Dose: 0, 15, 50, 150 mg/kg/bw
- Animals: 25 female rats/group
DOSAGE PREPARATION:
The test item formulations were freshly prepared every day.
The test item was suspended in the vehicle to the appropriate concentrations and was administered orally at a constant volume once daily from the 6th to the 20th day of gesta-tion.
The amount of the test item was daily adjusted to the current body weight of the animal. The control animals received the vehicle at the same administration volume daily in the same way.
The male rats for mating remained untreated.

Analytical verification of doses or concentrations:

yes

Remarks:

Concentration of the test item-vehicle mixtures

Details on analytical verification of doses or concentrations:

For the analysis of the test item-vehicle formulations, samples of approximately 4 mL were taken at the following times and stored at -20°C or colder until analysis at LPT:
At start of dosing
Analysis of concentration: During treatment always before administration to the last animal of the group (1 sample/test item group).
Number of samples: 1 x 3 = 3, (Sampling date: September 11, 2017)

At the end of the dosing period (at a time when the majority of animals was dosed):
Analysis of concentration: During treatment always before administration to the last animal of the group (1 sample/test item group).
Number of samples: 3 x 3 = 9, (Sampling date: September 25, 2017)

Sum of all samples: 12

Details on mating procedure:

- Sexually mature ('proved') male rats of the same breed served as partners.
- The female breeding partners were randomly chosen.
- Mating was monogamous: 1 male and 1 female animal were placed together in one cage during the dark period.
- Each morning a vaginal smear was taken to check for the pres-ence of sperm. If findings were negative, mating was repeated with the same partner.
- The day on which sperm was found was considered as the day of conception (day 0 of pregnancy).
- This procedure was repeated until 25 mated dams were available for all groups.
- The non-pregnant animals were excluded from the analysis of the results and replaced by other animals.
- A post-mortem negative staining according to SALEWSKI was carried out in the replaced animals in order to confirm the non-pregnancy status.

Duration of treatment / exposure:

From gestation day 6 until gestation day 20

Frequency of treatment:

Once daily

Duration of test:

On gestation day 21, the rats were laparotomised under CO2 narcosis.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Doses / concentrations 2

Dose / conc.:

15 mg/kg bw/day (nominal)

Remarks:

Low dose

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Intermediate dose

Doses / concentrations 4

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

HIgh dose

No. of animals per sex per dose:

25 female rats/dose , orally dosed with 0, 15, 50 or 150 mg test item/kg b.w.
Evaluated litters: 20 litters per group

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were selected in agreement with the Sponsor based on the results of a dose-range-finding study for a prenatal developmental toxicity (LPT study no. 34963).
In this dose-range finding study, test item was administered to pregnant female rats at dose levels of 15, 50 or 150 mg/kg b.w./day orally, by gavage, once daily from gestation day 6 to 20.
No premature deaths were noted.
Dams treated with 150 mg test item/kg b.w./day were noted with a reduced body weight and body weight gain.
No embryotoxic properties (no dead fetuses, no malformations and no test item-related variations) were noted at any of the tested dose levels.

Examinations

Maternal examinations:

Dated and signed records of all activities relating to the day to day running and maintenance of the study within the animal units, as well as to the group observations and examinations outlined in the Study Plan, were recorded in the appropriate documentation. In addition, observations relating to the individual animals made throughout the study were recorded.

The following observations were made during the course of the study:
Clinical signs
Individual animals were observed daily for any signs of behavioural changes, reaction to treatment, or illness.
Immediately after administration, any signs of illness or reaction to treatment were recorded. In case of changes, the animals were observed until the symptoms disappeared. In addition, animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m.
On Saturdays and Sundays, the animals were checked regularly starting from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately 3.30 p.m.
Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.


Viability
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This allowed post mortem examinations to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed except that the final check was carried out at approximately midday.
Animals showing signs of abortion or premature delivery would have been sacrificed on the same day. Fetuses obtained this way were examined for abnormal development, whenever possible. No abortion occurred in the study.


Body weight
The weight of each rat was recorded on day 0 of gestation (the day of detection of a positive mating sign), followed by daily weighing - always at the same time of the day.
The body weight gain was calculated in intervals (i.e. day 0-3, 3 6, 6-9, 9-12, 12-15, 15-18 and 18-21), for the whole study (gestation day 0 - 21) and for the period after the start of dosing (gestation day 6 to gestation day 21). Furthermore the carcass weight and the net weight gain from day 6 is given.
These values are stated in the report.
These measurements were also used for calculating the daily amount of test item to be administered.

Food and drinking water consumption
The quantity of food consumed by each rat was recorded daily. Food intake per rat (g/rat/day) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment day.

The relative food consumption (g/kg b.w./day) was calculated using the following formula:
Daily food consumption [g/kg b.w./day]= Total food intake in g / Body weight in g x 1000

Daily monitoring by visual appraisal of the drinking water bottles was maintained throughout the study. Dehydration of the dams was avoided.

EXAMINATIONS (NECROPSY), Examination of the dams
Dissection technique and evaluation of the animals:
On gestation day 21, the rats were laparotomised under CO2 narcosis. The ovaries and the uteri of the dams were removed; the gravid uteri (in toto) were weighed. In order to check for possible drug effects, a dissection with macroscopic examination of the internal organs and placentae of the dams was carried out on the day of sacrifice or on the day on which the animals were found dead. In case of macroscopical findings, the affected maternal tissues were preserved in 7% buffered formalin for possible future histopatho-logical examinations.

Ovaries and uterine content:

Corpora lutea
- number per dam
- absolute number per group
- mean per group

Implantations
- number per dam
- distributions in the uterine horns
- absolute number per group
- mean per group

Resorptions
- number per dam
- distributions in the uterine horns
- absolute number per group
- mean per group
- early resorptions < 2 mm
- Late resorptions > 2 mm

Weight of placentae
- individual data per fetus
- mean per litter
- mean per group
- mean per sex and group

Weight of fetuses
- individual data per fetus (alive and dead)
- mean per litter
- mean per group
- litter mean per sex and group

Fetuses
- number per dam (alive)
- number per dam (dead)
- number of fetuses (alive and dead) per sex and dam
- distribution in the uterine horns
- absolute number of fetuses alive per group
- mean number of fetuses alive per group
- mean % of fetuses alive per group
- male/female ratio (alive and dead)


Runts
- number per dam
- mean per group


Malformed fetuses
- individual data per fetus
- type of malformation
- number and incidence (%) per group and litter

Total malformation rate [%] = malformed fetuses per group / fetuses per group x 100

Fetuses with variations
- type of variation
- individual data per fetus
- number and incidence (%) per group and litter

Total variation rate [%] = fetuses per group with variations / fetuses per group x 100

Fetuses with retardations
- type of retardation
- individual data per fetus
- number and incidence (%) per group and litter

Total retardation rate [%] = fetuses per group with retardations / fetuses per group x 100


Indices of pre-implantation loss and post-implantation loss:

Calculation of group indices
Pre implantation
loss [%] = [Corpora lutea (per group) - Implantations (per group)] / Corpora lutea (per group) x 100


Post implantation
loss [%] = [Implantations (per group) - living fetuses (per group)] / Implantations (per group) x 100


Calculation of mean indices per litter
Pre implantation
loss [%] = sum of pre-implantation losses per litter in a group [%] / number of litters in a group

Post implantation
loss [%] = sum of post-implantation losses per litter in a group [%] / number of litters in a group

Fetal examinations:

The fetuses were removed and the following examinations performed:
(a) Macroscopic inspection (gross evaluation) of the placentae for example for focal indurations or abnormal appearance (e.g. size, colour, shape).
(b) The number of fetuses (alive and dead) and placentae (location in the uterus and the assignment of the fetuses) was determined.
(c) Sex and viability of fetuses were determined. Animals are said to be viable when they are found alive (spontaneous breathing, spontaneous movement).
(d) Number and size of resorptions were determined.
(e) Corpora lutea in the ovaries, implantations and location of fetuses in the uterus were determined.
(f) Weights of fetuses and weights of the placentae were determined (fetuses were considered as runts if their weight was less than 70% of the mean litter weight).
(g) All fetuses (dead and alive) were inspected externally for damages, especially for malformations .
(h) The fetuses were sacrificed by an ether atmosphere.
(i) Examination of fetuses and determination of number and kind of retardations, variations or malformations:
1) 50% of the number of fetuses in each litter were examined for skeletal anomalies. The thorax and peritoneal cavity (without damage to ribs and sternum) were opened and the location, size and condition of the internal organs were determined.
Then the skeleton was double-stained with Alcian blue for the examination of cartilage and with Alizarin red to reveal ossifications (according to DAWSON). The skeletal system was examined (determination of the number and type of retardations, variations as well as malformations).
2) The remaining 50% of the number of fetuses in each litter were examined for soft tissue anomalies. Body sections were made and examined according to WILSON.
The fetuses were allocated to the evaluation of DAWSON or WILSON on an alternating basis.

Statistics:

Parametrical data:
The statistical evaluation of the parametrical values was done by Provantis using the following settings:
Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILKS test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), inter-group comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

Non-parametrical data
The statistical evaluation of non-parametrical values was done using the FISHER or Chi2 test:
FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01)
or
Chi2 test, n ≤ 0.01 (p ≤ 0.05 and p ≤ 0.01)

The respective calculations for the FISHER and Chi2 test were performed using Provantis (maternal macroscopic findings at necropsy or findings during the external macro-scopic examination of the fetuses). or an internal computer program (e.g. findings during the fetal skeletal or soft tissue examination).
Note:
The statistical evaluation of the pre- and post-implantation index (per group) using the number of corpora lutea, implantation sites and/ or fetuses per group (see table 7-1 Re-production Data - Summary - Values per Group) was done using StatXact 4.0.1 software, as such a calculation is not possible in Provantis.

Significantly different data are indicated in the summary tables of the result sections of the report.

Indices:

Fertility Index, Viability Index, Resorption Index, Pre-Implantation Loss Index, Post-Implantation Loss Index, Runts Index, Variation Index,
Number of litters having abnormalities, Number of abnormalities per litter

Historical control data:

LPT Background Data, see Appendix 4
Summarized results of the 59 last embryotoxicity studies in Sprague-Dawley rats (Charles River Deutschland GmbH performed at LPT in the years 2004 to July 2017 .

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No test item-related changes in behaviour, the external appearance or the faeces that were considered to be of toxicological relevance were noted in the low and intermediate dose groups (15 or 50 mg test item/kg b.w./day).
The changes in behaviour and external appearance that were noted in the intermediate and high dose group (15 or 50 mg test item/kg b.w./day) are listed in the tables below.
At 15 mg test item/kg b.w./day, slight to moderate salivation was noted for 4 dams.
At 50 mg test item/kg b.w./day, slight to moderate salivation was noted for 6 dams.
Due to the low incidence of at maximum 2 days per dam for the low and intermediate dose group (15 or 50 mg test item/kg b.w./day), salivation was considered to be not adverse, especially as no signs of systemic toxicity were noted at these dose levels.
At 150 mg test item/kg b.w./day, slight to extreme salivation and piloerection were noted for 14 or 2 of 21 animals. The prematurely deceased dam no. 79 was noted with slightly to extremely reduced motility, breathing sounds lateral position and pultaceous faeces.
As reduced motility, breathing sounds, lateral position, pultaceous faeces and piloerec-tion were noted for dam no. 79 shortly before its premature death and therefore, were considered to be pre-mortal symptoms and not test item-related. Piloerection was additionally observed for 3 test days for dam no. 88. This observation was considered to be spontaneous, as none of the other surviving dams showed piloerection.
For salivation, an increase was noted for the severity, number of affected dams and the incidence compared to the low and intermediate dose groups. Therefore, at 150 mg test item/kg b.w./day, salivation was considered to be adverse.
 
Start and duration
Salivation
In the low and intermediate dose groups (15 or 50 mg test item/kg b.w./day), salivation started immediately to 5 minutes post administration and ended 0 to 20 minutes post administration.
At 150 mg test item/kg b.w./day, salivation started immediately to 5 minutes post administration and disappeared 5 to 20 or 20 to 60 minutes post administration.

Reduced Motility
Reduced Motility was noted for dam no. 79 of the high dose group (150 mg test item/kg b.w./day). It was noted consistently during GD 10 and 11. On GD 12 and 13, reduced motility started immediately to 5 minutes post administration and disappeared 20 to 60 minutes post administration.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

No premature deaths were noted in the control group and in the low and intermediate dose groups (15 or 50 mg test item/kg b.w./day).
Dam no. 79 treated with 150 mg test item/kg b.w./day was found dead during GD 14. Dam no. 79 was noted with pre-mortal symptoms in form of piloerection, reduced motility, breathing sounds lateral position and pultaceous faeces. The pre-mortal death was considered to be test item-related. Observations for dam no. 79 that were noted during necropsy

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight
No test item-related differences in body weight were noted between the dams of the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).

Body weight gain from GD 0 to 21
No test item-related difference for the body weight gain from GD 0 to 21 compared to the control group was noted for the animals treated with 15, 50 or 150 mg test item/kg b.w./day.
In the high dose group (150 mg test item/kg b.w./day), decreased values were noted for the body weight gain (33.1% below the value of the control group between GD 6 and 9, p < 0.01 and 12.5% below the value of the control group between GD 10 and 12, not significant) for the first 6 days after the start of the dosing between GD 6 and GD 12. As also a decreased body weight gain (8.3% below the value of the control group, not significant) was noted during the whole study period (see table), the reduced body weight gain was considered to be test item-related.

There were no test item-related influences on the gravid uterus weight. Therefore, the gravid uterus weight had the same influence on the body weight gain for all groups

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food and drinking water consumption
No test item-related differences were noted between the control group and the low and intermediate dose groups (15 or 50 mg test item/kg b.w./day).
At 150 mg test item/kg b.w./day, statistically significantly lower values were noted for the food consumption of the dams between GD 7 and 12 and between GD 20 and 21 in comparison to the control. This distinct decrease of food consumption was considered to be test item-related.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No test item-related changes in drinking water consumption were noted between the dams of the control group and the dams of the treatment groups by visual appraisal.

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:

effects observed, treatment-related

Description (incidence and severity):

Net body weight gain from GD 6 to 21
No test item-related differences were noted for the net body weight gain from GD 6 to 21 between the dams of the control group and the dams of the low and intermediate dose groups (15 or 50 mg test item/kg b.w./day).
A distinctly lower net body weight gain was noted for the dams treated with 150 mg test item/kg b.w./day (24.6% below the value of the control group). Although not statistically significant, the reduced net body weight gain was considered to be test item-related.

Gravid uterus weight and Carcass weight
No test item-related differences were noted between the gravid uterus weight and the carcass weight of the control dams and the dams of the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Slight reductions were noted for the gravid uterus weight for the dams treated with 50 or 150 mg test item/kg b.w./day (6.2% or 6.3% below the value of the control group, not significant). However, as these were only slight and not statistically significant reductions, the reduced gravid uterus weights were considered to be not test item-related.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related observations were noted for the dams of the treatment groups (15, 50 or 150 mg test item/kg b.w./day) during the macroscopic inspection of the organs and tissues.
However, macroscopic findings were noted for the kidneys (cystic or dilation of the renal pelvis) for the control and also for the treatment groups or to the uterus (hemorrhage of the left or right uterine horn) for the control and the high dose group. Additionally, the prematurely deceased dam no. 79 was noted with ulcera in the stomach and dark-brown contents in the intestines.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Details on results:

Dam no. 79 of the high dose group (150 mg test item/kg b.w./day) prematurely deceased during GD 14.
A high incidence of salivation, a slightly reduced body weight gain and a reduced food consumption were further noted at the high dose level.
No test item-related changes were noted in the macroscopic examination during laparotomy.

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation loss) were noted between the dams of the control group and the dams of the treatment groups (15, 50 or 150 mg test item/kg b.w./day). See table below "any other information on results"

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation loss) were noted between the dams of the control group and the dams of the treatment groups (15, 50 or 150 mg test item/kg b.w./day). See table below "any other information on results"
At 150 mg test item/kg b.w./day, a statistically significant increase was noted for the pre-implantation loss. As the implantation is completed before start of treatment, this increase was considered to be not test item-related but spontaneous.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation loss) were noted between the dams of the control group and the dams of the treatment groups (15, 50 or 150 mg test item/kg b.w./day). See table below "any other information on results"

Early or late resorptions:

no effects observed

Description (incidence and severity):

No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation loss) were noted between the dams of the control group and the dams of the treatment groups (15, 50 or 150 mg test item/kg b.w./day). See table below "any other information on results"

Dead fetuses:

no effects observed

Description (incidence and severity):

No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation loss) were noted between the dams of the control group and the dams of the treatment groups (15, 50 or 150 mg test item/kg b.w./day). See table below "any other information on results"

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation loss) were noted between the dams of the control group and the dams of the treatment groups (15, 50 or 150 mg test item/kg b.w./day). See table below "any other information on results"

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

2 runts were noted for a dam in the control group (nos. 10-08 and 10-10) and one runt (88-11) was noted in the high dose group (150 mg test item/kg b.w./day). The occurrence of one or two runts was within the normal range of variation and therefore not test item-related.

Details on maternal toxic effects:

The reproductive parameters (number of implantation sites, number of resorptions and number of fetuses) were not influenced by the test item.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The placental and fetal weights showed no test item-related differences between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): The placental and fetal weights showed no test item-related differences between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No dead fetus was noted in the control group and in the test item treated groups (15, 50 or 150 mg test item/kg b.w./day).

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No test item-related differences between the ratio of male and female fetuses (range: 0.83 - 1.03) were noted between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).

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 inspection at laparotomy
No macroscopically visible external alterations (malformations or variations) were noted for the fetuses of the control and the treatment groups (15, 50 or 150 mg test item/kg b.w./day) during the macroscopic inspection at laparotomy

Gross inspection of the organs and tissues at laparotomy
The macroscopic inspection of the organs and tissues for gross alterations at laparotomy revealed no malformations or variations for the fetuses of the control group and the fetuses of the treatment groups (15, 50 or 150 mg test item/kg b.w./day).

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations
No skeletal malformations were noted for the fetuses of the control group and the test item-treated groups (15, 50 or 150 mg test item/kg b.w./day) during the skeletal exami-nation according to DAWSON.

Skeletal variations
Skeletal variations were noted for the ribs (less than 13 ribs ossified, wavy or misshapen) and the sternum (bipartite, misshapen or misaligned to a slight degree).
No test item-related difference in the incidence of the observed skeletal variations in comparison to the control group was noted for the fetuses of the treatment groups (15, 50 or 150 mg test item/kg b.w./day).

Skeletal retardations
Retardations (delayed ossifications) were related to the skull (incomplete ossification of nasal, frontal, parietal and/or interparietal areas), the hyoid (unossified), the sternum (sternebra(e) incompletely ossified, reduced in size or unossified), the thoracic vertebral bodies (bipartite or dumbbell-shaped), the caudal vertebral bodies (only one body ossi-fied), the os pubis and the os ischii (incompletely ossified) and the metacarpalia and metatarsalia (absence of ossification in metacarpalia 2 to 5 and metatarsalia 2 to 5).
No test item-related increase in the incidence of skeletal retardations at 15, 50 or 150 mg test item/kg b.w./day was noted during skeletal examination according to DAWSON.
See table below "overall remarks"

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Malformations
No malformations were noted for the fetuses of the control group and the fetuses of the treatment groups (15, 50 or 150 mg test item/kg b.w./day) during the soft tissue examination according to WILSON.

Variations
During the examination of the organs and tissues according to WILSON, variations were noted for the brain (dilatation of the 4th cerebral ventricle or subdural hemorrhage in the cerebellum), the kidneys (uni- or bilateral dilatation of the renal pelvis, formation of a hydro ureter or malpositioned) and the liver (haemorrhagic focus/foci).
No test item-related differences and no statistically significant differences in the incidences of the observed variations were noted between the control group and the low and intermediate dose groups (15 or 50 mg test item/kg b.w./day).
At 150 mg test item/kg b.w./day, a statistically significantly increased incidence was noted for the incidence of the dilatation of the renal pelvis and for the total incidence of variations. As both incidences were within the range of the LPT background data (see table on the following page), these increased incidences were considered to be not test item-related.

Other effects:

no effects observed

Description (incidence and severity):

Unclassified observations
No unclassified observation was noted for any fetus of the control group and the treat-ment groups (15, 50 or 150 mg test item/kg b.w./day).

Details on embryotoxic / teratogenic effects:

No dead fetuses, no malformations and no test item-related variations or retardations were noted.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: prenatal developmental toxicity; highest dose tested

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Examination of the dams

test item	Group 1 Control	Group 2 15 mg/kg	Group 3 50 mg/kg	Group 4 150 mg/kg
Treated dams	25	25	25	25
Not pregnant dams	1	1	0	0
Dams without viable fetuses	0	0	0	0
Dams with early delivery	0	0	0	0
Prematurely deceased animals	0	0	0	1
Not examined dams (spare animals)	4	4	5	4
Evaluated litters	20	20	20	20

test item	Group 1 Control	Group 2 15 mg/kg	Group 3 50 mg/kg	Group 4 150 mg/kg
Animal nos. of mated rats	1 - 25	26 - 50	51 - 75	76 - 100
Animal nos. with evaluable litters at laparotomy	1, 3-21	26-28, 30‑46	51‑70	76-78, 80‑96
Dams not pregnant (animal nos.)	2	29	none	none
Dams with total implantation loss (animal nos.)	none	none	none	none
Prematurely deceased animals (animal nos.)	none	none	none	79
Reserve animals (animal nos.)	22-25	47-50	71-75	97-100

Reproduction data of the dams

Parameter				Group 1 Control (n=20)	Group 2 15 mg/kg (n=20)	Group 3 50 mg/kg (n=20)	Group 4 150 mg/kg (n=20)
Corpora lutea			total mean per dam	304 15.2	319 16.0	291 14.6	311 15.6
Implantation sites			total mean per dam	300 15.0	315 15.8	283 14.2	298 14.9
Resorptions			total mean per dam	8 0.4	8 0.4	10 0.5	14 0.7
Early resorptions			total mean per dam	7 0.4	5 0.3	8 0.4	12 0.6
Late resorptions			total mean per dam	1 0.1	3 0.2	2 0.1	2 0.1
Live fetuses			total mean per dam	292 14.6	307 15.4	273 13.7	284 14.2
Dead fetuses			total	0	0	0	0
Pre-implantation loss [%]			per group #^1 mean per dam	1.3 1.2	1.3 1.2	2.7 3.3	4.2 * 4.8
Post-implantation loss [%]			per group #^2 mean per dam	2.7 2.8	2.5 2.5	3.5 3.5	4.7 5.1
		Statistical analyses were performed for the mean values per dam using an ANOVA/DUNNETT test.
	#^1	The statistical comparison of the pre-implantation loss per group was done by comparing the values of implantation sites/corpora lutea of the test group with the ratio of implantation sites/corpora lutea of the control group using the Chi^2test (*/**: p ≤ 0.05/p ≤ 0.01).
	#^2	The statistical comparison of the post-implantation loss per group was done by comparing the values of live fetuses/implantation sites of the test group with the ratio of fetuses/implantation sites of the control group using the Chi^2test (*/**: p ≤ 0.05/p ≤ 0.01).

Analysis of test item formulations

The results of the test item-formulation analyses for the investigated parameters are listed in the table below:

Parameter	Sampling / dealing	Range of % nominal concentration
Concentration	before administration to the last animal of the group on test day 7	95.7% - 100.4%
	before administration to the last animal of the group on test day 21	98.7% - 101.2%

The measured actual concentrations of the test item in the test item vehicle-mixtures were between 95.7% and 101.2% of the nominal concentrations, indicating correctly prepared formulations.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, test item did not show any teratogenic potential.

Executive summary:

The aim of this prenatal developmental toxicity study (OECD 414, oral) was the examination of the influence of the test item administered orally during the critical period of organogenesis and the fetal development (6th to 20th day of gestation) on the pregnant rat and the fetus.

Findings:

Examination of the dams:
Mortality	No test item-related premature deaths were noted for the low and intermediate dose groups (15 or 50 mg test item/kg b.w./day). At 150 mg test item/kg b.w./day, dam no. 79 prematurely deceased during GD 14.
Clinical signs	No test item related influence on the behaviour, external appearance or faeces were noted for the low and intermediate dose groups (15 or 50 mg test item/kg b.w./day). At 150 mg test item/kg b.w./day, salivation was noted in 14 of 21 evaluated dams.
Body weight and body weight gain	No test item-related difference between the control group and the treatment groups was noted for the body weight. Additionally, no test item-related differences were noted for the body weight gain and the net weight gain at the low and intermediate dose levels (15 or 50 mg test item/kg b.w./day). At 150 mg test item/kg b.w./day, a reduction was noted for the body weight gain (8.3% below the value of the control group, not significant) and for the net body weight gain (24.6% below the value of the control group, not significant).
Food consumption	No test item-related difference was noted between the control group and the dams treated with15 or 50 mg test item/kg b.w./day. In the high dose group (150 mg test item/kg b.w./day), a reduced food consumption (at maximum 17.1% below the value of the control group between GD 11 to 12, p < 0.01) was noted between GD 7 and 12 and between GD 20 and 21.
Drinking water consumption	No differences were noted.
Necropsy findings	No test item-related changes were noted during the macroscopic inspection of the dams at necropsy.
Uterus and carcass weights	No test item-related differences were noted.
Reproduction data	No test item-related influence was noted on the reproductive parameter (number of implantation sites, resorptions and fetuses).

Examination of the fetus:
Mortality	No dead fetuses were noted in any of the test groups.
Body weight of the fetuses and the placentae	No test item-related differences were noted between the control group and the treatment groups.
Fetal alterations
Malformations	No malformations were noted during the macroscopic examinations at laparotomy (external inspection and inspection of the organs and tissues for gross lesions), the skeletal examination according to DAWSON and the soft tissue examination according to WILSON.
Variations	The macroscopic examinations at laparotomy, the skeletal examination according to DAWSON and the soft tissue examination according to WILSON revealed no test item-related variations.
Retardations	No test item-related retardations (delays in ossification) were noted.
Analysis of test item formulations	The measured actual concentrations of the test item in the test item vehicle mixtures were between 95.7% and 101.2% of the nominal concentrations, indicating correctly prepared formulations.

Conclusion

In this prenatal developmental toxicity study, the test item was administered orally to female rats at dose levels of 15, 50 or 150 mg/kg b.w./day from the 6th to 20th day of pregnancy.

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 50 mg test item/kg b.w./day for the dams.

Dam no. 79 of the high dose group (150 mg test item/kg b.w./day) prematurely deceased during GD 14.

A high incidence of salivation, a slightly reduced body weight gain and a reduced food consumption were further noted at the high dose level.

No test item-related changes were noted in the macroscopic examination during laparotomy.

The no-observed-adverse-effect level (NOAEL) for the fetal organism was above150 mg test item/kg b.w./day.

The reproductive parameters (number of implantation sites, number of resorptions and number of fetuses) were not influenced by the test item.

No dead fetuses, no malformations and no test item-related variations or retardations were noted.

Under the conditions of the study, test item did not show any teratogenic potential.