[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a subchronic repeated dose toxicity study (OECD TG 408) on rats with doses of up to and including the limit dose of 1000 mg/kg bw/day, no effects were observed on weight or histopathology of reproductive glands/tissues/organs. The oestrous cycle was also not affected by the treatment.

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A data waiver for reproductive toxicity studies according to OECD TG 421/422 and 443 is claimed.

In a subchronic repeated dose toxicity study (OECD TG 408) on rats with doses of up to and including the limit dose of 1000 mg/kg bw/day, no effects were observed on weight or histopathology of reproductive glands/tissues/organs. The oestrous cycle was also not affected by the treatment.

Effects on developmental toxicity

Description of key information

OECD TG 414 study on rats: no effects on fetal development; NOAEL 1000 mg/kg bw/day (highest dose tested)

OECD TG 414 study on rabbits: no effects on fetal development; NOAEL 600 mg/kg bw/day (highest dose tested)

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan 2009 - March 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

(2001)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Stability in the solvent was analytically proven.

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Hsd Cpb:WU (SPF-bred)
- Source: Harlan-Nederlands, 5960 AD Horst, The Netherlands
- Age at study initiation: 12-14 weeks
- Weight at study initiation: 204 - 261 g (females); 421 - 545 g (males)
- Housing: Starting from gestation day 0 individually in Type IIIh Makrolon cages on low-dust wood shavings.
- Diet and water: ad libitum
- Acclimation period: at least 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): approximately 55
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The formulations were prepared as needed taking into account the analytically determined stability.

VEHICLE
- Lutrol® (PEG 400).
- Justification for use and choice of vehicle (if other than water): The test item in the vehicle gave a solution which was analytically confirmed to be stable for at least 8 days.
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Content checks of the active ingredient in samples with concentrations of 20, 60, and 200 mg/mL were carried out twice during the study.
For this purpose representative samples were dissolved with a mixture of acetonitrile/ aqueous 0.05 % formic acid (4:1) and subsequently quantified by reversed phase (C18) HPLC with UV-detection (DAD, wavelength 200 nm). Standard solutions of the authentic test item were used for calibration. The results of the content checks in samples with concentrations of 20, 60, and 200 mg/mL during the study showed no meaningful deviation of the active ingredient content from the nominal value.

Details on mating procedure:

The animals were mated by placing two females overnight into a Type IIIh cage together with one male rat. If sperm was detected in the vaginal smear taken on the morning following mating, this day was regarded as day 0 of gestation.

Duration of treatment / exposure:

Days 6 - 20 p.c.

Frequency of treatment:

once daily (between 06:00 and 12:00 CET)

Duration of test:

From study initiation date to end of in-life-phase 35 days.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25 females

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were selected based on the results of a previous dose toleration study in rats with 1000 mg/kg, which revealed no treatment related findings.

Maternal examinations:

CLINICAL EXAMINATIONS: Yes
- Time schedule: The females were inspected from days 0 to 21 p.c. twice daily (once daily only on weekends, on public holidays, and on day 21 p.c.), and all findings were recorded. Attention was paid to disturbances in the general condition of the rats (appearance, behavior), and any alterations concerning their excretory products.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of the females was determined on day 0 p.c. and daily from day 6 to day 21 p.c. Corrected body weight gain was determined by subtracting the uterus weight on day 21 p.c. from the body weight gain from days 0 to 21 p.c.

FOOD CONSUMPTION: Yes
- The food intake of the animals was determined from the difference in weight between the food offered and the food not consumed for the following days of gestation: Days 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18, and 18 -21.

WATER CONSUMPTION: Yes
- Water intake was assessed daily by visual estimation of the quantities left over and reported together with clinical findings.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Cesarean sections were performed on day 21 p.c. without knowledge of treatment groups.
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of dead and live fetuses: Yes
- Other: individual weight and appearance of the placentas

Fetal examinations:

- Sex of live fetuses
- Individual weights of live fetuses
- External examinations: Yes: [findings in alive and dead fetuses are included]
- Soft tissue and head examinations : Yes: [evaluation of about half of alive fetuses per litter]
- Skeletal and cartilage abnormalities : Yes: [evaluation in about half of alive fetuses per litter]
Furthermore, the eyes of fetuses No. 533, 582, 1136, and 1175 exemplarily underwent histopathological examination after investigation according to the modified WILSON technique to clarify unusual findings.

Findings in abdominal, pelvic, and thoracic organs as well as skeletal and cartilage findings by the modified DA WSON technique with the addition of cartilage staining [method described by INOUYE, modified]: evisceration, cartilage staining with alcian blue GX, clearing of the fetuses with diluted potassium hydroxide solution, staining of the skeletal system with alizarin red S, and evaluation of the skeletal system including cartilaginous findings (processing and evaluation after cesarean section without knowledge of treatment group ). Every other fetus within a litter was prepared for either skeletal or visceral evaluation with generally the first fetus of each litter assigned to skeletal evaluation.

Statistics:

Differences between the control and test item treated groups were considered to be significant when p < 0.05. Significant differences from the control group are indicated with * for p < 0.05 and ** for p < 0.01. Statistical evaluation was performed on an Alpha 800 5/500 computer using the following methods:
Analysis of Variance (ANOVA); in case of significance Dunnett's test for feed intakes, body weights, body weight gains, and corrected body weight gains, uterine weights, number of corpora lutea per female, number of implantations per female, number of live fetuses per female and as percentage of implantations per female, placental weights per female, fetal weights per female.
2 by N CHI2 test; in case of significant differences Fisher's exact test with Bonferroni correction for fertility rate, gestation rate, number of implantations per group, number of preimplantation losses per group, number of postimplantation losses, early resorptions, late resorptions or dead fetuses per group, number of live fetuses per group as percentage of implantations per group, number of male or female fetuses or fetuses with indeterminable sex per group, number of placentas with findings or litters with placental findings per group, number of fetuses or litters with external, visceral or skeletal findings, with malformations or with external or visceral deviations per group.
Kruskal-Wallis test and in case of significant differences Dunn's test for number of preimplantation losses per female, number of postimplantation losses, early resorptions, late resorptions or dead fetuses per female, number of male or female fetuses or fetuses with indeterminable sex per female, number of placentas with findings per female, number of fetuses with external or visceral findings, with malformations or with external or visceral deviations per female.
CHI2 test (correction according to yates) for number of fetuses or litters with cartilaginous tissue observations.

Indices:

Body Weight Gains: Calculated against the body weight on Day 6 post-coitum.
Corrected Body Weight Gains: Body weight determined on Day 21 post-coitum minus the body weight on Day 0 to 21 post-coitum and the weight of gravid uterus on day 21 post-coitum.
Relative Food Consumption: Calculated against the body weight for scheduled intervals.
Fertility rate: percentage of inseminated females with implantations
Pre-implantation loss (%): number of corpora lutea minus number of implantation sites
Gestation rate: percentage of females with viable fetuses on day 21 pc of those with implantation sites
Post-implantation loss (%): ((number of implantation sites - number of live fetuses)/(number of implantation sites)) x 100

Historical control data:

historical control data

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One female of the 1000 mg/kg level revealed respiratory sounds and transient piloerection, and one further female ofthis dose group also revealed piloerection for one single day. A treatment related effect is not assumed due to the isolated occurrence, and since these fmdings
may occur spontaneously in the rat strain used (see Annex, Section 590). Furthermore, salivation after administration occurred in all dose groups, which is most likely related to the bad taste of the test item and considered as not adverse.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Absolute and corrected body weight gains were unaffected by treatment at dose levels up to and including 1000 mg/kg.

Food efficiency:

no effects observed

Description (incidence and severity):

The placental weights were unaffected by treatment, see table 1.

Description (incidence and severity):

One pale placenta occurred at the 1000 mg/kg bw level, for which a treatment related effect is not assumed due to its single occurrence, and as this finding is known as a spontaneous findin in the rat strain used. Thus, the appearance of placentas was unaffected by treatment.

Other effects:

no effects observed

Description (incidence and severity):

The fertility rate (percentage of inseminated females with implantations) was unaffected by treatment see table 1.

Number of abortions:

no effects observed

Description (incidence and severity):

see table 1

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

see table 1

Early or late resorptions:

no effects observed

Description (incidence and severity):

see table 1

Dead fetuses:

no effects observed

Description (incidence and severity):

see table 1

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

see table 1

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Appearance, behavior, and mortality were unaffected at dose levels up to and including 1000 mg/kg bw/day.
Absolute and corrected body weight gains were unaffected by treatment at dose levels up to and including 1000 mg/kg.
Food intake, water intake, and fecal and urinary excretions were unaffected by treatment at dose levels up to and including 1000 mg/kg.
No treatment related gross pathological findings occurred at dose levels up to and including 1000 mg/kg.

Dose descriptor:

NOAEL

Remarks:

Maternal toxicity

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: No adverse findings in maternal animals up to and incl. 1000 mg/kg

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

see table 1

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

see table 1

Changes in sex ratio:

no effects observed

Description (incidence and severity):

fetal sex distribution was unaffected by treatment.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

see table 1

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

see Attachments 1 (fetal malformations), 2 (historical control data for spontaneous malformations), 3 (external and visceral malformations) and 4 (historical control data for spontaneous external and visceral deviations):
The criteria for classifying the observed external or visceral findings as deviation or malformation are shown in the Attachment 6, Section 8.5. If fetuses revealed more than one external or visceral deviation, the number of deviations is higher than the number of affected fetuses in these groups.

The overall incidences of fetuses or litters with malformations lay within the range of historical control data, revealed no statistical significance, and were thus unaffected by treatment at dose levels up to and including 1000 mg/kg.
The types of malformations observed in this study are generally a representative sample of spontaneous malformations in the rat strain used (meningoencephalocele, abdominal hernia, microphthalmia/anophthalmia, malposition of forelimb, cardiovascular malformations, sternal malformations, vertebral malformations with pelvis shift, dysplastic forelimb bones or skull bones (see historical control data in Attachment 2).
Malformations of the eyes ( eye ball reduced in size) occurred in all dose groups and the control group in comparable incidences of affected fetuses and litters ( each two fetuses out of two litters) and were thus unaffected by treatment at dose levels up to and including l000mg/kg.
At the 1000 mg/kg level, fetal examinations for malformations revealed some further findings (malposition of forelimb, ventricular septal defect of the heart, atrial septal defect and ventricular septal defect of the heart with pulmonary trunk arisen from left ventricle,
dysplastic forelimb bones with malformation of sternum and ribs and one supernumerary lumbar vertebra, one supernumerary lumbar vertebra with misshapen 7th lumbar vertebral
arches and pelvis shift, and dysplastic humerus ), for which a treatment related effect is not assumed, as these findings also occurred in the current control group (ventricular septal defect of the heart as part of a multiple thoracic malformation) and/or historical control groups (see Attachment 2, Section 8.8.11: dysplasia of forelimb bones: 4.31 % affected fetuses and 20.00 % affected litters in study T5068551; atrial septal defects of the heart: 2.12 % affected fetuses and 18.18 % affected litters in study Tl067765; ventricular septal defects of the heart: 2.17 % affected fetuses and 26.32 % affected litters in study T0073280; malposition of limbs (malrotation of hind limb(s)): 0.5 % affected fetuses and 5.6 % affected litters in study T0076746, and remaining findings).
At the 300 mg/kg level, fetal examinations for malformations revealed two fetuses out of two litters with folded retina, each without a dose dependency, and two further isolated findings (multiple malformation including meningoencephalocele and abdominal hernia, ventricular septal defect of the heart), for which a treatment related effect is not assumed due to the single occurrences, and as these findings also occurred in the current control group (multiple malformation, folded retina, ventricular septal defect of the heart as part of a multiple thoracic malformation) and/or historical control groups (see Attachment 2, Section 8.8.11: multiple malformation, meningoencephalocele, abdominal hernia (umbilical hernia), folded retina, ventricular septal defect of the heart).
At the 100 mg/kg level, fetal examinations for malformations revealed some further isolated findings (folded retina, malformation of vertebrae, sternum, ribs, and skull bones), for which a treatment related effect is not assumed, as these findings also occurred in the current control group (folded retina) and/or historical control groups (see Attechment 2, Section 8.8.11: folded retina, malformation of vertebrae, sternum, ribs, and skull bones).
Thus, a treatment related effect on malformations (incidence or type) was not evident at dose levels up to and including 1000 mg/kg.

Description (incidence and severity):

see Attachments 5 (historical control data for spontaneous skeletal findings (section 8.8.13).
The criteria for classifying the observed external or visceral findings as deviation or malformation are shown in Attachment 6, Section 8.5. If fetuses revealed more than one external or visceral deviation, the number of deviations is higher than the number of affected fetuses in these groups.

Fetal Skeletal Deviations Including Cartilaginous Deviations
At the 100 mg/kg and 300 mg/kg levels, fetal examinations for skeletal retardations and variations revealed statistically significantly retarded ossification of a few localizations (300 mg/kg: 1st distal phalanges of digits left; 100 mg/kg: 1st distal phalanges of digits left, 2nd proximal phalanges of digits left, 3rd proximal phalanges of digits left, 4th proximal phalanges of digits right, 5th proximal phalanges of digits right, 5th distal phalanges of toes right, parietal bones bilateral), when calculation was done on a fetal basis, for which a treatment related effect was not evident, because statistical significance was lacking, when calculation was done on a litter basis, and since a dose dependency was lacking.
At the 100 mg/kg and 300 mg/kg, and 1000 mg levels, statistically significantly progressed ossification occurred (1000 mg/kg and 300 mg/kg: calcaneus bilateral; 300 mg/kg: 8th caudal vertebral bodies; 100 mg/kg: 2nd sternebrae), when calculation was done on a fetal basis, for which a treatment related effect was not evident due to a lacking dose dependency except for calcaneus, because statistical significance was lacking, when calculation was done on a litter basis, and since the values lay inside the range of historical control data except for 8th caudal vertebral bodies (see Attachment 5, Section 8.8.13).
A statistically significantly increased incidence of wavy ribs (10th right, 11th right and sum) and a statistically significantly decreased incidence of punctiform 14th ribs right occurred at the 100 mg/kg level, when calculation was done on a fetal basis. A treatment related effect for these findings was not evident, because statistical significance is lacking, when calculation was done on a litter basis (except for wavy ribs sum), and due to a lacking dose dependency.
A statistically significantly decreased incidence of dumbbell shaped 11th thoracic vertebral bodies occurred at the 1000 mg/kg level, when calculation was done on a fetal basis. A treatment related effect for this finding was not evident, because statistical significance is lacking, when calculation was done on a litter basis.
Evaluation of fetal cartilaginous tissue revealed a statistically significantly increased incidence of unciformed cartilaginous ends of 8th ribs at the 300 mg/kg level, for which a treatment related effect was not evident due to a lacking dose dependency.

Thus, a treatment related effect on skeletal deviations (retardations, variations, including cartilaginous tissue findings) was not evident at dose levels up to and including 1000 mg/kg.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

see Attachments 1 (fetal malformations), 2 (historical control data for spontaneous malformations), 3 (external and visceral malformations) and 4 (historical control data for external and visceral deviations):
The criteria for classifying the observed external or visceral findings as deviation or malformation are shown in Attachment 6, Section 8.5. If fetuses revealed more than one external or visceral deviation, the number of deviations is higher than the number of affected fetuses in these groups.

Attachment 3 (Table 5-7) gives an overview on external and visceral deviations (findings other than malformations) in live fetuses.
The overall incidences of fetuses or litters with external and visceral deviations were
unaffected at dose levels up to and including 1000 mg/kg and revealed the highest value on a fetal basis in the 100 mg/kg group.
The external and visceral deviations observed in this study were of a common type and
comparable with spontaneous findings in the current control group and/or historical control groups (see Attachment 4, 8.8.12) and represented the normal range of scattering in the rat strain used.
Exemplarily histopathological evaluation of fetuses no. 533, 582, 1136, and 1175 (whitish
mass between lens and cornea) revealed lens material in the anterior eye chamber, which is regarded as artifacts, so that remaining fetuses with this finding were judged equally.
Histopathological evaluation of additional circumscribed hard whitish tissue in the nasopharynx was performed in a recently conducted prenatal developmental toxicity study
with the same rat strain (T0076746) and revealed calcium concrements without connection to the underlying tissue in the affected localizations. Calcium might have been dissolved from the fetal bones by the Wilson fixative and precipitated in the nasopharyngeal duct so that these findings were regarded as artifacts.
The finding of a small hollow structure in different locations of the head is also considered as a fixation artifact, which also occurred in all dose groups of a recent prenatal developmental toxicity study (T7078264; control group: two fetuses out of two litters; low dose group: one fetus; medium dose group: two fetuses out of two litters; high dose group: one fetus) with the same rat strain used, without showing a dose dependency.
A statistically significantly increased incidence of slight dilation of renal pelvis occurred at the 100 mg/kg level, for which a treatment related effect is not assumed due to a lacking dose dependency, and as the incidence lay inside the range of historical control data (see Attachment 4, Section 8.8.12).
Slight dilation of lateral brain ventricle(s) occurred in all dose groups, for which a treatment related e:ffect is not assumed due to a lacking dose dependency, and as the incidences lay inside the range of historical control data (see Attachment 4, Section 8.8.12: 5.16 % affected fetuses and 47.37 % affected litters in study T6062954).
The remaining findings of the dose groups showed no dose dependency or were isolated findings.
Thus, a treatment related effect on external and visceral deviations was not evident at dose levels up to and including 1000 mg/kg.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The gestation rate was unaffected by treatment at dose levels up to and including 1000 mg/kg.
Appearance and weights of placentas were unaffected by treatment at dose levels up to and including 1000 mg/kg.
Postimplantation loss and correspondingly the number of fetuses were unaffected by treatment at dose levels up to and including 1000 mg/kg.
Fetal sex distribution was unaffected by treatment at dose levels up to and including 1000 mg/kg.
The fetal weights were unaffected by treatment at dose levels up to and including 1000 mg/kg.
A treatment related effect on malformations was not evident at dose levels up to and including 1000 mg/kg.
A treatment related effect on external and visceral deviations was not evident at dose levels up to and including 1000 mg/kg.
A treatment related effect on skeletal deviations (retardations, variations, including cartilaginous tissue findings) was not evident at dose levels up to and including 1000 mg/kg.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment related effects at any dose

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 1: general reproduction data

dose (mg/kg bw/day)	0	100	300	1000
inseminated females	25	25	25	25
inseminated females evaluated	25	25	25	25
females with implantations	23	25	22	24
in % of those inseminated	92.0	100	88.0	96.0
mean values per female with implantation sites
corpora lutea	14.6	15.6	15.4	14.8
preimplantation loss	1.3	1.5	1.4	1.9
implantations	13.3	14.1	14.0	12.9
gestation rate
females with viable fetuses on day 21 pc	23	25	22	24
in % of meles with implantations	100	100	100	100
total resorptions	0	0	0	0
intrauterine development; mean value per female
placental with in g	0.59	0.60	0.60	0.60
number of fetuses	12.8	13.7	13.7	12.6
postimplantation loss	0.5, 0.5	0.4, 0.4	0.2, 0.2	0.3, 0.3
male fetuses in %	48.5	49.7	51.9	53.5
fetal weight in g	4.98	5.00	5.03	5.02

Executive summary:

A developmental toxicity study with the substance was performed according to OECD TG 414. Twenty-five inseminated female Wistar rats per dose group were administered (gavage) daily from day 6 to 20 p.c. the test substance at doses of 0, 100, 300, and 1000 mg/kg bw in Lutrol^®(PEG 400). The fetuses were delivered by cesarean section on day 21 of gestation. Investigation was performed on general tolerance of the test substance by the females as well as on its effect on intrauterine development.

Appearance, behavior, mortality, absolute and corrected body weight gains, food intake, water intake, and fecal and urinary excretions were unaffected at dose levels up to and including 1000 mg/kg bw.

No treatment related gross pathological findings occurred at dose levels up to and including 1000 mg/kg bw.

The gestation rate, appearance and weights of placentas, postimplantation loss and correspondingly the number of fetuses, fetal sex distribution, and fetal weights were unaffected by the treatment at dose levels up to and including 1000 mg/kg bw.

A treatment related effect on malformations, external, visceral, and skeletal (including cartilage) deviations was not evident at dose levels up to and including 1000 mg/kg bw.

Summarizing and evaluating all data investigated revealed a NOAEL of 1000 mg/kg bw/day for each maternal toxicity and developmental toxicity.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Feb 2020 - 26 Jun 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

d.d. 24 February 2020

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Age at study initiation: 18-20 weeks
- Weight at study initiation: 3076 - 4188 g
- Fasting period before study: No
- Housing: Females were housed individually in cages with perforated floors
- Diet: Pelleted diet for rabbits (KLIBA NAFAG Rabbit Diet 3409 maintenance and breeding, from Kliba NAFAG Granovit AG, Kaiseraugst, Swizerland) was provided ad libitum throughout the study, except during designated procedures. In addition, pressed hay (Tecnilab-BMI BV, Someren, The Netherlands) was provided during the study period.
- Water: Municipal tap water was freely available to each animal via water bottles/containers.
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

The feed was analyzed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water was performed. It was considered that there were no known contaminants in the feed or water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-19
- Humidity (%): 51-58
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES main study: From: 20 Apr 2020 To: 15 May 2020
IN-LIFE DATES pilot study: March 2020

Route of administration:

oral: gavage

Vehicle:

other: 1% Aqueous Hydroxypropyl Methylcellulose

Details on exposure:

VEHICLE
- Justification for use and choice of vehicle: Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure.

Elix water (approximately half of the end volume prepared) was brought into a suitable beaker. Water was stirred while heated to approximately 75 ºC. The heat was turned off once the temperature was within the appropriate range (actual range 70-76ºC). During stirring, the necessary amount of Methocel F4M, Premium was added into the water. The Methocel solution was stirred until completely dissolved and the mixture had reached room temperature (actual range 22-25ºC) spontaneously. The water was added to reach the end volume. The solution was stirred until, on visual inspection, a homogeneous solution was obtained. The vehicle was stable for 1 month in a refrigerator set to maintain 4°C. The prepared vehicle was removed from the refrigerator and stirred for at least 30 minutes before dosing.

- Concentration in vehicle: 0, 12, 40, 120 mg/mL
- Amount of vehicle: 5 mL/kg

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared at least weekly as a suspension, filled out in daily portions and stored in the refrigerator until use. If not used on the same day of preparation, the dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
Adjustment was made for specific gravity of the test item. No correction was made for the purity/composition of the test item.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis during week 1. Analyses were performed by using a validated analytical procedure (Test Facility Study No. 20215812).
Duplicate sets of samples were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10%.

Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20215812) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study (at least 24 hours at room temperature under normal laboratory light conditions and for at least 8 days in the refrigerator).

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant

Duration of treatment / exposure:

from Day 7 to Day 28 post-coitum, inclusive

Frequency of treatment:

7 days a week

Duration of test:

23 days

Dose / conc.:

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

Remarks:

Group 2

Dose / conc.:

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

Remarks:

Group 3

Dose / conc.:

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

Remarks:

Group 4

No. of animals per sex per dose:

22 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of the dose range finder, and in an attempt to produce graded responses to the test item.

Dose range finder:
No guidelines were applicable as this study was intended for dose level selection purposes only.
If not mentioned otherwise, test system, procedures and techniques were identical to those used during the main study. No formulation analyses were performed during the dose range finder study.
Time-mated female New Zealand White rabbits were received from Charles River (Chatillon sur Chalaronne, France). The females arrived on Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating). They were 18-20 weeks old and weighed between 3070 and 4100 g at the initiation of dosing. The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 6 days prior to the commencement of dosing.
The actual daily mean temperature during the study period was 19°C with an actual daily mean relative humidity of 54 to 60%.

Dose levels: 0 (control), 300, 600, 1000 mg/kg bw/day

The test item and vehicle were administered to the appropriate animals by once daily oral gavage from Day 7 to Day 28 post-coitum, inclusive.
The dose levels were selected based on the results of a tolerability study with Polyether P 293 in non-pregnant rabbits dosed by oral gavage (Test Facility Study No. 20215810). Treatment with doses of either 500 or 1000 mg/kg bw/day for 7 days was well tolerated.

Terminal Procedures – F0-Generation
If necessary for humane reasons, animals were euthanized by intravenous injection of sodium pentobarbital. Surviving animals were sacrificed on Day 29 post-coitum.

Terminal Procedures – F1-Generation
Scheduled necropsies
Each viable fetus of animals surviving to planned necropsy was externally examined in detail and weighed. All live fetuses were euthanized by administration of sodium pentobarbital into the oral cavity using a small metal feeding tube. For late resorptions a gross external examination was performed (if possible). Sex was not determined. No visceral (internal) or skeletal examination was performed. Fetuses and late resorptions without malformations were discarded.

Unscheduled necropsies
For expelled uterine contents, late resorptions and recognizable fetuses in development of females sacrificed before planned necropsy, no external examinations were performed. The stage of development was assessed, and fetuses were counted and euthanized by decapitation or by administration of sodium pentobarbital into the oral cavity using a small metal feeding tube. No tissues were preserved.

RESULTS
Maternal Findings
There were 4 preterm decedents during the study, 2 at 600 mg/kg bw/day and 2 at 1000 mg/kg bw/day.
Two high dose females were sacrificed on Days 22 and 21 post-coitum, respectively. Both females lost 8-10% body weight on Day 21 post-coitum when compared with start of treatment and food consumption was severely reduced from Day 12 of 15 postcoitum onwards. Clinical sings observed prior to sacrifice included hunched posture, pale appearance, piloerection, lean posture, reduced faeces production (severe) and half closed eyes (ptosis). Similar findings were observed for one female at 600 mg/kg bw/day that was sacrificed on Day 24 post-coitum. This female lost 6% body weight on Day 24 post coitum when compared with start of treatment and food consumption was severely reduced during Days 15-24 post-coitum. Apart from some of the earlier mentioned clinical signs, this female also presented with pale faeces and dark urine prior to sacrifice. These 3 mortalities in the mid and high dose groups were considered test item-related, based on the similar timing and pattern regarding clinical signs and effects on body weight and food consumption. Additionally, one female at 600 mg/kg bw/day was sacrificed on Day 15 post-coitum. This female already lost 7% body weight between Day 0 and Day 7 post-coitum prior to start treatment and lost additional weight combined with a severely reduced food consumption from start treatment onwards. Given that the effect on body weight commenced prior to start treatment for this female, this mortality was considered incidental.
Reduced faeces production was noted in treated animals at a higher incidence than observed in concurrent controls in a dose-related trend. Other clinical signs were either observed in animals that were sacrificed prior to scheduled necropsy or occurred within the range of background findings.
Mean body weight gain of females at 1000 mg/kg bw/day was lower than concurrent control from Day 15 post coitum onwards and mean body weight of these females was 7% lower than concurrent control animals on Day 21 post coitum. After the two females of this high dose group were sacrificed in extremis, mean body weight gain was comparable to concurrent controls. Body weight development of females at 300 and 600 mg/kg bw/day were considered to be unaffected by the test item. There were no clear effects of the test item on the weight gain corrected for the gravid uterus weight up to 1000 mg/kg bw/day.
Mean (relative) food consumption of females at 600 and 1000 mg/kg bw/day was lower than concurrent control mean over Days 12-24. After the two females in each group were sacrificed in extremis, food consumption was similar to concurrent control. Food consumption of females at 300 mg/kg bw/day was considered unaffected by the test item.
Macroscopic observations at necropsy did not reveal any alterations that were considered to be toxicologically relevant.
All females (including the preterm decedents) were pregnant. The mean numbers of corpora lutea and implantation sites, and mean litter incidence of early and late resorptions and pre- and post-implantation loss in the control and test groups were considered unaffected by treatment with the test item up to 1000 mg/kg bw/day.

Fetal Findings
There were no test item-related effects on litter size of any group. There were no dead fetuses in any of the test groups. Mean fetal weight at 1000 mg/kg bw/day appeared to be lower than concurrent control mean, however when compared to historical control data the mean fetal weight of the control animals was relatively high. This difference was therefore considered not to be test item related. External examination of the fetuses did not show any abnormalities.

CONCLUSION
The dose level of 1000 mg/kg bw/day was considered inappropriate based on the test item-related mortality of 2/6 females, as the observed incidence of mortality could have potentially resulted in an insufficient number of available pregnant females at necropsy for a thorough toxicological evaluation in the subsequent main prenatal developmental toxicity study.
One of the mortalities observed at 600 mg/kg bw/day was regarded as test item-related. In absence of other signs of toxicity and recognizing the requirements for signs of toxicity and the minimum number of pregnant females at the highest dose level, the dose level of 600 mg/kg bw/day was selected as high dose for the Main study. Low and mid dose levels were selected to be 60 and 200 mg/kg bw/day, respectively.

- Rationale for animal assignment: random

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily, beginning on Day 7 post-coitum and lasting up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were individually weighed on Days 7, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was quantitatively measured for Days 7-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 post-coitum.

WATER CONSUMPTION: Yes
- Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
One animals with early delivery: Within 24 hours of delivery
- Organs examined: All animals (including animals sacrificed before planned necropsy and the female with early delivery) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No tissues, except the uterus, were weighed.

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

Blood sampling:

None

Fetal examinations:

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

The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination of all groups using the Wilson sectioning technique. After examination, the tissues without variation or malformations were discarded. Tissues with variations or malformations were stored in 10% formalin. The heads from the remaining one-half of the fetuses in each litter of all groups were examined by a mid-coronal slice.
While selected for soft-tissue examinations, heads of two fetuses were inadvertently examined by a mid-coronal slice and therefore not placed in Bouin's solution. Additionally, one fetus was not placed in Bouin's solution due to a malformation of the head.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

Non-Parametric
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution were compared using the Mann Whitney test.
Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences.

Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level.
The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Indices:

Body Weight Gains: Calculated against the body weight on Day 7 post-coitum.
Corrected Body Weight Gains: Body weight determined on Day 29 post-coitum minus the body weight on Day 7 post-coitum and the weight of gravid uterus.
Relative Food Consumption: Calculated against the body weight for scheduled intervals.

Pre-implantation loss (%): ((number of corpora lutea - number of implantation sites)/(number of corpora lutea)) x 100
Post-implantation loss (%): ((number of implantation sites - number of live fetuses)/(number of implantation sites)) x 100
Viable fetuses affected/litter (%): ((number of viable fetuses affected/litter)/(number of viable fetuses/litter)) x 100

Historical control data:

See attached background material.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Observed clinical signs either occurred in individual animals only or occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

see also Table 1: summary of results
There were 4 preterm decedents during the study, 2 in the control group, 1 at 200 mg/kg bw/day and 1 female at 600 mg/kg bw/day.
One female at 600 mg/kg bw/day was sacrificed in extremis on Day 24 post-coitum based on observed clinical signs. This female appeared moderately lethargic, lean and pale and showed piloerection. Faeces production was also reduced from Day 18 post-coitum onwards. Additionally, a body weight loss of 7% and reduced food consumption were observed between Days 21 and 24 post-coitum. At necropsy, dark red foci and a watery-clear gelatinous fluid were observed in the stomach. This female was gravid with 7 early resorptions and 4 dead fetuses. The mortality of this female was considered to be incidental, given the single occurrence in a high-dose female in absence of any maternal toxicity in the remaining females of this dose group.
One female at 200 mg/kg bw/day was sacrificed in extremis on Day 23 post-coitum due to an early delivery. The following clinical signs were observed on the day of sacrifice: red fluid on the manure tray, red staining of the vagina, severe lethargy, pale appearance, quick breathing. Additionally, the animal had a hunched and flat posture while laying on its side (ventro-lateral recumbency). Hemorrhagic fluid was found in the uterus and the vagina was dark red discolored at necropsy. This female was gravid with 10 dead fetuses of which 8 were within the uterus. Given the single occurrence in a mid-dose female, this early delivery was considered to be unrelated to treatment with the test item.
Two control females were sacrificed on Day 20 and 15 post-coitum, respectively. Both animals were gasping for air after dosing was completed and blood was noted in the dosing tube for one female. Findings at necropsy were considered confirmatory of an oral gavage event and consisted of the following: foam in the trachea, lungs did not collapse and/or watery-cloudy fluid in the thoracic cavity. One female was non-gravid and one female was gravid with 11 normal implantations in development.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

see also Table 1: summary of results
Mean body weights, body weight gain and weight gain corrected for gravid uterus of treated animals remained in the same range as controls over the treatment period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after correction for body weight was similar to the control level over the study period.

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

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment with the test item.
Any findings that were noted among control and/or treated animals were considered to be of no toxicological significance, since they remained within the range of biological variation for rabbits of this age and strain.

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

One female at 200 mg/kg bw/day was sacrificed in extremis on Day 23 post-coitum due to an early delivery. The following clinical signs were observed on the day of sacrifice: red fluid on the manure tray, red staining of the vagina, severe lethargy, pale appearance, quick breathing. Additionally, the animal had a hunched and flat posture while laying on its side (ventro-lateral recumbency). Hemorrhagic fluid was found in the uterus and the vagina was dark red discolored at necropsy. This female was gravid with 10 dead fetuses of which 8 were within the uterus. Given the single occurrence in a mid-dose female, this early delivery was considered to be unrelated to treatment with the test item.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

see also Table 1: summary of results
Mean litter incidence of pre- and post-implantation loss of females at 200 mg/kg bw/day was higher when compared with concurrent controls. This was mainly attributed to one female, that had 11 corpora lutea of which only 2 were implantation sites (i.e. pre-implantation loss incidence of 81.8%). Additionally, one of the 2 implantation sites was an early resorption (i.e. post-implantation loss incidence of 50%). In absence of a dose-related response and as treatment with the test item was initiated on Day 7, after completion of implantation, the higher incidences noted for this female were considered to be unrelated to treatment with the test item.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

see also Table 1: summary of results

Early or late resorptions:

no effects observed

Description (incidence and severity):

see also Table 1: summary of results

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

see also Table 1: summary of results and attachments
Except for 3 control females (including one preterm decedent), 1 female at 60 mg/kg bw/day, 2 females at 200 mg/kg bw/day and 3 females at 600 mg/kg bw/day, all females were pregnant with viable fetuses. Based on the occurrence in the concurrent control group, these cases of non-pregnancy were considered unrelated to treatment with the test item.

Other effects:

no effects observed

Description (incidence and severity):

see also Table 1: summary of results and attachments
The mean number of corpora lutea were similar in the control and test groups.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: Absence of adverse effects up to and including the highest dose level tested.

Remarks on result:

other: A higher dose level than 600 mg/kg bw/day was considered unfeasible due to mortality (2/6 pregnant females) observed at 1000 mg/kg bw/day in the dose range finder

Fetal body weight changes:

no effects observed

Description (incidence and severity):

see also Table 1: summary of results and attachments
Mean combined (male and female) fetal body weights were 41.1, 41.1, 42.0 and 42.2 gram for the control, 60, 200 and 600 mg/kg bw/day groups, respectively.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

see also Table 1: summary of results
Mean litter sizes were 9.4, 9.1, 9.0 and 8.7 live fetuses/litter for the control, 60, 200 and 600 mg/kg bw/day groups, respectively. There were no dead fetuses in any of the test groups.
The lower mean of 8.7 live fetuses/litter noted for females at 600 mg/kg bw/day was considered incidental as no statistical significance was achieved and the mean value remained well within the historical control range.

Changes in sex ratio:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

see also Table 1: summary of results and attachments
There were no treatment related effects on external morphology following treatment with the test item up to 600 mg/kg bw/day. External malformations occurred in 4 fetuses from 2 litters at 600 mg/kg bw/day and in 1 fetus each in the control and 60 mg/kg bw/day groups.
At 600 mg/kg bw/day, three littermates of one dam (01, 02 and 04) had a cleft palate leading to a group mean litter incidence of 4.2% per litter. This was above the historical control maximum value for this finding (1.1% per litter). However, the presence of a cleft palate in three littermates suggests a familiar cause. The maximum historical control value was also caused by three affected fetuses in one litter. One of the fetuses with a cleft palate also had an omphalocele that occurred in another fetus as well, resulting in an incidence of 1.9% per litter at 600 mg/kg bw/day. This is below the historical control maximum value and even below the p95-percentile (3.0% and 2.0% per litter, respectively). As such, the observed incidences of cleft palate and omphalocele were considered unrelated to the test item. Additionally, a small incisor was noted in one fetus which was possibly
related to the cleft palate that occurred in this fetus as well. As such, this finding was considered unrelated to treatment with the test item.
At 60 mg/kg bw/day, one fetus had a spina bifida which was considered a chance finding due to single occurrence and in absence of a dose-related response.
One control fetus had multiple malformations which were considered to be spontaneous in origin given the occurrence in the control group. Observed malformations were thoracogastroschisis, scoliosis, absent forelimb and hyperextended hindlimbs.
External variations were not observed in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

see also Table 1: summary of results and attachments
There were no treatment related effects on skeletal morphology following treatment with the test item up to 600 mg/kg bw/day. Skeletal malformations occurred in 2 fetuses from 2 litters at 600 mg/kg bw/day, 1 fetus at 60 mg/kg bw/day and 4 fetuses from 3 litters in the control group.
A skull anomaly was observed in a total of 3 fetuses (i.e one fetus at 600 mg/kg bw/day; one fetus at 60 mg/kg bw/day and one control fetus). Additionally, a rib anomaly was observed at 600 mg/kg bw/day in one fetus. Due to absence of a dose relationship, the low incidence and/or occurrence in historical control fetuses, both malformations were considered unrelated to the test item.
The two remaining skeletal malformations were observed in control fetuses only. These were a vertebral and sternal anomaly, that both also occurred in one fetus that had multiple external and visceral malformations. In addition, three other control fetuses had a vertebral anomaly. These malformations were considered spontaneous in origin given the occurrence in the control group.
All skeletal variations observed in this study occurred at low incidences, in the absence of a dose-related incidence trend, in control fetuses only and/or at frequencies that were within the range of available

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

see also Table 1: summary of results and attachments
There were no treatment related effects on visceral morphology following treatment with the test item up to 600 mg/kg bw/day. Visceral malformations occurred in 3 fetuses of the control and high dose group each.
At 600 mg/kg bw/day, two fetuses in the litter of dam A087 (03 and 04) had a lung cyst and another fetus of the same litter showed abnormal lung lobation. Both these findings occurred previously at higher incidences in historical control fetuses and the occurrence within a single litter does not indicate a test item relationship. Therefore, these lung findings were considered to be chance findings.
Malformations noted in control fetuses were a diverticulum of the intestine (two littermates) and internal hydrocephaly and diaphragmatic hernia (one fetus with multiple external malformations). These malformations were considered spontaneous in origin given the occurrence in the control group.
All variations noted were considered unrelated to treatment as they occurred in the absence of a dose-related trend, infrequently, in control fetuses only and/or at frequencies that were within the range of available historical control data.

Key result

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: Absence of adverse effects up to and including the highest dose level tested.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Dose Formulation Analyses

Accuracy
The concentrations analysed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%, actual values 97-99%).
No test item was detected in the Group 1 formulation.

Homogeneity
The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%, actual values 0.62-2.1%).

 

Table 1: Summary of results

Dose level (mg/kg bw/day)	0	60	200	600
Pregnant/total dams	19/22	21/22	20/22	19/22
Females that aborted or delivered	0	0	1	0
Females that died	0	0	0	0
Females that were euthanized	2	0	0	1
Females examined at scheduled necropsy	20	22	21	21
Pregnant	18	21	19	18
non-pregnant	2	1	2	3
Mean number of corpora lutea	11.1	10.7	11.6	10.5
Mean number of implantation sites	9.7	9.8	10.0	9.3
-early resorptions -late resorptions (total number and % per litter)	5 (3.5%) 1 (0.5%)	9 (4.1%) 5 (2.6%)	11 (7.6%) 8 (4.3%)	6 (3.0%) 5 (2.4%)
Dams with stillbirths, resorptions only and/or dead fetuses	-	-	-	-
Pre-implantation loss (total number and % per litter)	24 (12.1%)	18 (7.7%)	30 (13.8%)	21 (12.4%)
Post-implantation loss (total number and % per litter)	6 (4.0%)	14 (6.7%)	19 (11.9%)	11 (5.3%)
Mean body weight on day 29 pc (g)	4026	3950	4101	4041
Mean corrected body weight gain day 7-29 pc (%)	-2.1%	-3.0%	-3.9%	-2.0%
Gravid uterine weight (g)	531.9	516.8	530.9	522.4
Mean live offspring (number and percent)	9.4 (96.0%)	9.1 (93.3%)	9.0 (88.1%)	8.7 (94.7%)
Mean fetal body weight (males; g)	41.3	41.7	42.0	43.6
Mean fetal body weight (females; g)	40.9	40.3	41.1	41.1
Mean fetal body weight (sexes combined; g)	41.1	41.1	42.0	42.2
Malformations (including runts) (number and % per litter) -external -soft tissue -skeletal	1 (1.9%) 3 (2.7%) 4 (3.4%)	1 (0.5%) 0 (0%) 1 (0.5%)	0 (0%) 0 (0%) 0 (0%)	4 (4.7%) 3 (1.5%) 2 (1.8%)
Variations (% per litter) -external -soft tissue -skeletal	0% 8.0% 76.4%	0% 11.7% 69.1%	0% 13.5% 78.1%	0% 15.1% 79.5%

 

Conclusions:

In a prenatal developmental toxicity study in female New Zealand White rabbits according to OECD TG 414 the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Polyether P 293 was established as the highest dose tested of 600 mg/kg bw/day. A higher dose level than 600 mg/kg bw/day was considered unfeasible due to mortality (2/6 pregnant females) observed at 1000 mg/kg bw/day in the dose range finder.

Executive summary:

In a prenatal developmental toxicity study (performed according to OECD TG 414) 22 pregnant female New Zealand White rabbits per group were treated by oral gavage with Polyether P 293 in doses of 60, 200 and 600 mg/kg bw from GD 7 to 28. A higher dose level than 600 mg/kg bw/day was considered unfeasible due to mortality (2/6 pregnant females) observed at 1000 mg/kg bw/day in the dose range finder.

No mortality occurred during the study period that was considered to be related to treatment with the test item. No maternal or developmental toxicity was observed in the 60, 200 and 600 mg/kg bw/day groups.

In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Polyether P 293 was established as being at least 600 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

in a study on rabbits the NOAEL was 600 mg/kg bw/day (highest dose tested)

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

A developmental toxicity study (performed according to OECD TG 414) revealed no treatment related effects for appearance, behaviour, mortality, absolute and corrected body weight gains, food intake, water intake, and fecal and urinary excretions up to and including 1000 mg/kg bw. No treatment related gross pathological findings occurred at dose levels up to and including 1000 mg/kg. The gestation rate, appearance and weights of placentas, postimplantation loss and correspondingly the number of fetuses, fetal sex distribution and fetal weights were unaffected by treatment at dose levels up to and including 1000 mg/kg. A treatment related effect on malformations, external, visceral and skeletal (including cartilage) deviations was not evident at dose levels up to and including 1000 mg/kg.

Summarizing and evaluating all data investigated revealed a NOAEL of 1000 mg/kg bw for each maternal and developmental toxicity.

In a prenatal developmental toxicity study (performed according to OECD TG 414) 22 pregnant female New Zealand White rabbits per group were treated by oral gavage with Polyether P 293 in doses of 60, 200 and 600 mg/kg bw from GD 7 to 28. A higher dose level than 600 mg/kg bw/day was considered unfeasible due to mortality (2/6 pregnant females) observed at 1000 mg/kg bw/day in the dose range finder.

No mortality occurred during the study period that was considered to be related to treatment with the test item. No maternal or developmental toxicity was observed in the 60, 200 and 600 mg/kg bw/day groups.

In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Polyether P 293 was established as being at least 600 mg/kg bw/day.

Justification for classification or non-classification

No classification required for toxicity to reproduction according to Regulation (EC) No 1272/2008, Annex I.