Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 January 2015 - 15 March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Janvier, le Genest-Saint-Isle, France
- Age at study initiation: approximately 10 weeks old for males and approximately 9 weeks old for females
- Mean body weight at study initiation: a mean body weight of 396 g (range: 368 g to 423 g) for males and a mean body weight of 258 g (range: 243 g to 278 g) for females.
- Housing: the animals were individually housed in polycarbonate cages (Tecniplast 2154, 940 cm²)
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: a period of 7 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 13 January 2015 to 15 March 2015.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING FORMULATIONS:
Type of formulation (visual observation): solution in the vehicle.

Preparation procedure: according to a previous validation study, describing the preparation procedure for a range of concentrations covering the lowest and highest used in this study.

Frequency of preparation:
Test item dose formulations: daily.
Vehicle preparation: based on the CiToxLAB France in-house procedures.

Delivery conditions: at room temperature, protected from light, and in sealed vials.

VEHICLE
- Justification for use and choice of vehicle: homogenous formulation in this vehicle
- Concentration in vehicle: 5, 16 and 40 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.
Details on mating procedure:
- M/F ratio per cage: 1/1 during the night
- Length of cohabitation (mating period): until mating occurred
- Proof of pregnancy: vaginal plug or sperm in the morning vaginal lavage referred as day 0 post-coitum
- After successful mating each pregnant female was caged individually again
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: HPLC-UV
Test item concentrations: the test item concentrations in the administered dose formulations analyzed in Weeks 1, 3 and 6 were within an acceptable range of variations (-6.7% to +0.6%) when compared with the nominal acceptance values (± 10% of the target concentrations), except for the dose formulations prepared for group 2 in Week 1 found at -13.0% and for group 4 in Week 3 found at -73.8%. As the formulations were given only once in the study and as the formulation preparation process was validated before the study and confirmed at several occasions during this study, this was considered not to have a impact on the study conclusion.
Following the result obtained for test item concentration in group 4 formulation in Week 3, a new analysis was performed on groups 1 and 4 formulations prepared in Week 4. Group 4 formulation was found at 2.7% when compared to the nominal value and no test item was observed in the control dose formulation.
Homogeneity: homogenous formulation
Stability: not assessed, dose formulation prepared daily
Duration of treatment / exposure:
In the males:
− 2 weeks before mating,
− during the mating period,
− until sacrifice (at least 5 weeks of treatment in total for groups 1 to 3, 30 days for group 4),

In the females:
− 2 weeks before mating,
− during the mating period,
− during gestation,
− during lactation until Day 4 post-partum inclusive.
Frequency of treatment:
Daily
Details on study schedule:
- No F1 parents (only one generation mated)
- Age at mating of the mated animals in the study: 11-12 weeks
Remarks:
Doses / Concentrations:
25, 80 and 200 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, following the results of two previous studies performed in the same strain:
- in a previous preliminary repeated dose toxicity study by oral route (gavage) in rats: 4 groups of 10 male and 10 female rats received the test item, daily or bidaily by oral (gavage) administration in corn oil, at dose-levels of 0, 100 (2 weeks) then 200 (2 weeks), 300 or 1000 then 600 mg/kg/day.
All animals treated at 300 or 600/1000 mg/kg/day were found died or prematurely sacrificed within the first 9 days of treatment after showing numerous signs of poor clinical condition (mainly respiratory difficulties). The mortality was considered to be related to the irritant properties of the test item, with consecutive gastro-intestinal findings mainly. Mean body weight and mean food consumption were affected in males and/or females,
At 100/200 mg/kg/day, in-life effects were limited to hypersalivation. Necropsy revealed the presence of thickening, irregular surface and/or white deposits in the forestomach,
- in a previous 4-week toxicity study by the oral route (gavage) in rats: 5 male and 5 female rats were given 25, 80 or 240 mg/kg/day of the test item in corn oil by gavage daily for 4 weeks.
There were no deaths in the study. Ptyalism and loud breathing were the main clinical signs recorded at 80 and 240 mg/kg/day. There were no significant effects on mean body weight, mean food consumption and mean biochemistry parameters. Females had statistically significant prolonged mean prothrombin time at all dose-levels. There were no adverse effects at urinalysis. The test item administration did not induce any changes in mean organ weights. At necropsy, white discoloration was noted on the forestomach at 240 mg/kg/day in 4/5 males and 3/5 females. The main histopathological changes induced by the test item administration at 240 mg/kg/day were seen in the forestomach and were indicative of irritant properties of the test item. They consisted of squamous cell hyperplasia and hyperkeratosis (correlated with macroscopy), subacute inflammation and low grade erosions. Low grade inflammation and hyperplasia were additionally noted at this dose-level in a few animals in the stomach. Minimal epithelial degeneration/regeneration was noted in the trachea in two males at this dose-level and may have been related to irritation consecutive to aspiration of the test item during the gavage procedure. Increased myeloid cell numbers seen in the bone marrow of two males at 240 mg/kg/day were considered to be related to the inflammation noted in the forestomach. The increased incidence of minimal mucosal inflammation in the cecum in males at 240 mg/kg/day was of equivocal significance since there were no changes in the small intestine.

Based on both studies, the dose-levels from 300 mg/kg/day were considered to have exceeded the Maximun Tolerated Dose and the dose of 240 mg/kg/day was thought to be too high with increase of the treatment duration because of the adverse findings in the forestomach. Therefore, 200 mg/kg/day was selected as the high-dose level. The low-dose and mid dose were selected using a ratio representing approximately a 2.5-3-fold interval (i.e. 25 and 80 mg/kg/day).

- Rationale for animal assignment: stratified procedure.
Positive control:
no (not required)
Parental animals: Observations and examinations:
MORTALITY/MORBIDITY:
- Time schedule: once a day before the treatment period and at least twice a day during the treatment period, including weekends.

CLINICAL OBSERVATIONS:
- Time schedule: from arrival, each animal was observed once a day as part of routine examinations. From the start of the treatment period, each animal was observed once a day, at approximately the same time each day.

BODY WEIGHT:
- Time schedule: the body weight of each male was recorded on the first day of treatment (Day 1), then once a week until sacrifice.
The body weight of each female was recorded on the first day of treatment (Day 1), then once a week until mated and on Days 0/1, 7, 14 and 20 post-coitum (p.c.) and Days 1 and 5 p.p.

FOOD CONSUMPTION:
- Time schedule: Males: once a week. Females: once a week until mating, then on Days 0-7, 7-14 and 14-20 post-coitum and Days 1-5 post-partum.

REPRODUCTION (apart from indices):
- Pre-coital time and duration of gestation were recorded.
Oestrous cyclicity (parental animals):
fresh vaginal lavage (stained with methylene blue), each morning during the pairing period, until females are mated.
Sperm parameters (parental animals):
Parameters examined in males of parental generation:
- testis weight (all groups) + microscopic evaluation (control and high-dose groups),
- epididymis weight (all groups) + microscopic evaluation (control and high-dose groups),
- special emphasis to stages of the spermatogenic cycle and testicular interstitial cells (control and high-dose groups) at microscopy.
Litter observations:
STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED:
- number and sex of pups,
- number of live, dead and cannibalized pups,
- presence of gross anomalies,
- weight gain,
- clinical signs.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all surviving animals after the end of the mating period
- Female animals: all surviving animals = Day 5 post-partum.

GROSS NECROPSY
Macroscopic post-mortem examination of principal thoracic and abdominal organs.

HISTOPATHOLOGY
- epididymides, ovaries, stomach + forestomach and testes from all animals of the control and high-dose groups (groups 1 and 4) sacrificed at the end of the treatment period (at the end of the mating period for males or on Day 5 p.p. for females),
- forestomach from all animals of the low- and intermediate-dose groups (groups 2 and 3) sacrificed at the end of the treatment period (at the end of the mating period for males or on Day 5 p.p. for females),
- all macroscopic lesions of all groups.

ORGAN WEIGHTS: see above sperm parameters
Postmortem examinations (offspring):
SACRIFICE: on Day 5 post-partum

GROSS NECROPSY: on all pups (surviving and found dead)

HISTOPATHOLOGY: No

ORGAN WEIGTHS: No
Statistics:
yes
Reproductive indices:
Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
Post-implantation loss = 100 * (Number of implantation sites - Number of live pups) / Number of implantations
Mating index = 100 * (Number of mated animals / Number of paired animals)
Fertility index = 100 * (Number of pregnant female partners / Number of mated pairs)
Gestation index = 100 * (Number of females with live born pups / Number of pregnant females)
Offspring viability indices:
Live birth index = 100 * (Number of live born pups / Number of delivered pups)
Viability index on day 4 p.p. = 100 * (Number of surviving pups on day 4 p.p. / Number of live born pups)
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined
Reproductive function: oestrous cycle:
not specified
Description (incidence and severity):
not enough data for interpretation
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
MORTALITY:
There were no premature deaths at 0, 25 and 80 mg/kg/day.

At 200 mg/kg/day there were 7 premature sacrifices: 4 males between Days 17 and 28 and 3 females during the mating period on Day 17 (one female) or during gestation between on Days 6 and or 23 p.c. (two females). The sacrifice decisions were taken following poor health condition and body weight losses (down to -26%).
These deaths and their poor health condition were considered to be test item treatment-related.
Following these premature deaths in the high-dose group and as the clinical condition of the two other high-dose males was degrading, it was decided in agreement with the Sponsor to move forward of a few days the terminal sacrifice of the high-dose males.

At histopathology, there were test item-related white discoloration and/or thickening in the forestomach in most of the unscheduled dead animals which correlated with hyperplasia of squamous cells and hyperkeratosis often associated with ulcer/erosion and inflammation of the submucosa at 200 mg/kg/day. These findings contributed to the death/moribundity and thus were considered to be adverse.

One female at 200 mg/kg/day was found dead on Day 11 p.c. and had before death similar clinical signs (emaciated appearance, loud and abdominal breathing, hypoactivity and ptyalism) and body weight loss as the prematurely sacrificed females described above. The cause of death was a stomach perforation, with unclear relationship to test item administration.
One another female at 200 mg/kg/day was sacrificed on Day 24 p.c. The female was trying to deliver (dystocia) for 2 days and on Day 24 p.c. presented clinical signs of poor health condition (piloerection, pallor of extremities, ptyalism and loud breathing) with 31% of body weight loss from Day 20 p.c. A relationship of this dystocia with test item treatment at the high-dose cannot be excluded. There were test item-related marked squamous cell hyperplasia and hyperkeratosis in the forestomach, correlated with gross thickening and white discoloration in this female.

CLINICAL SIGNS:
At 200 mg/kg/day, ptyalism, respiratory difficulties (abdominal and loud breathing) and round back noted in surviving animals were also observed in the prematurely dead animals and were considered to be test item treatment-related and adverse.

At 80 mg/kg/day, loud breathing noted for 5 days in one male was considered of limited toxicologically significance in view of the isolated occurrence.
There was one female with piloerection, pallor of extremities and eyes and reddish vaginal discharge from Day 2 p.p. until sacrifice on Day 5 p.p. (or until Day 3 p.p. for vaginal discharge). As this concerned only one female in the mid-dose group, a relationship with the test item treatment was considered unlikely.

At 25 mg/kg/day, ptyalism noted in 7/20 animals was considered to be of non toxicological significance in the absence of any dose-relationship (clinical sign not observed at 80 mg/kg/day). Reddish vaginal discharge in two females on Day 2 p.p. was not attributed to the test item treatment in the absence of dose-relationship.

BODY WEIGHT:
At 200 mg/kg/day, about half the animals in each sex lost weight ones after the others, most of them were prematurely sacrificed.
- in males, more than half of the males started to lose weight in Weeks 2 (one male prematurely sacrificed on Day 17) or 4 (one male sacrificed on Day 28) but mainly 3 (two males sacrificed on Day 26, one male sacrificed on Day 22 and one surviving male). Three of them lost between 19 and 26% of body weight. Thus mean body weight changes (decrease vs. controls or body weight loss) and slightly lower mean body weights (up to -8% vs. controls) were observed during these weeks,
- in females, no statistically significant lower mean body weight or mean body weight change were noted. However, one female lost 26% of body weight from the pre-mating period and was sacrificed on Day 17. During gestation period, three other females lost weight (one female sacrificed on Day 6 p.c., another one found dead on Day 11 p.c. and another one sacrificed on Day 23 p.c.: between 13 and 21%) from the start of the mating period or from Day 20 p.c.
Female in dystocia lost 31% of body weight from Day 20 p.c..

There were no effects on mean body weights and mean body weight changes at 25 and 80 mg/kg/day.

FOOD CONSUMPTION:
At 200 mg/kg/day and when compared with controls, statistically significant lower mean food consumption was observed in Week 2 in both sexes and generally concerned the animals that were prematurely sacrificed or found dead thereafter.
There were no effects on mean food consumption at 25 and 80 mg/kg/day.

REPRODUCTIVE PERFORMANCE:
Pairing, mating and fertility data
There were no effects on mean pairing, mating and fertility data.
At 200 mg/kg/day, the four surviving pregnant females gave birth to live pups.
Four mated females were not pregnant (3 from the control group and one female from the high-dose group) and were sacrificed on Day 26 p.c.

Delivery data
There were no effects on mean delivery data.
At 200 mg/kg/day, the four surviving females had delivery data comparable with the other groups. However, among the dead females, one female had dystocia and a relationship with the test item treatment cannot be ruled out.

The control group had a mean number of implantations and a mean pre-implantation loss higher than in the other groups. This was due to one female which had a high post-implantation loss and thus a low number of implantations. The mean number of pups delivered was thus lower in the control group [also because of a second female with a low number of pups delivered].

ORGAN WEIGHTS:
There were no test item-related mean organ weight differences.
The non-statistically minimal testes absolute and relative-to-body weight changes were not considered to be test item related because they were of insufficient magnitude, not dose-related and uncorrelated with microscopic findings.

GROSS PATHOLOGY:
Unscheduled deaths
In most prematurely sacrificed animals, there were test item-related gross white discoloration and/or thickening in the forestomach. These findings correlated with hyperplasia of squamous cells and hyperkeratosis. These findings contributed to the death/moribundity and thus were considered to be adverse.
In addition, distension of stomach and intestines was observed in some of these animals, and were considered to be probably agonal changes.
The small thymus and spleen noted in one prematurely sacrificed male and in two prematurely sacrificed females correlated with lymphoid atrophy and were considered to be secondary to stress.

The stomach perforation recorded in one female and associated with thinned forestomach and yellow content in the abdominal cavity. The stomach findings were considered to be probably the cause of death but the relationship to test item administration could not be determined in view of the isolated occurrence of these findings.

Terminal sacrifice
Test item-related gross white discoloration and/or thickening in the forestomach in all males and most females. These findings correlated with hyperplasia of squamous cells and hyperkeratosis.

The small thymus noted in one female treated at 80 mg/kg/day and in one female treated at 200 mg/kg/day was considered to be secondary to stress.

The few other gross observations were considered to be consistent with spontaneous findings encountered in the rats of these strain and age because they were non dose related, isolated and/or correlated with common background lesions, including the unilateral cryptorchidism/small testis and small epididymis seen in one male treated at 200 mg/kg/day.

MICROSCOPIC EXAMINATION
Unscheduled deaths
Test item-related microscopic minimal to marked hyperplasia of squamous cells and hyperkeratosis were noted in most decedents and correlated with gross white discoloration and/or thickening in the forestomach. There was also erosion/ulcer in these animals. These findings contributed to the death/moribundity and thus were considered to be adverse.

The slight atrophy/fusion of villi in ileum, the moderate regeneration of glands in cecum and the minimal degeneration/necrosis in the stomach from one female were possibly related to test item administration, although seen with minimal incidence.

Terminal sacrifice
Test item-related microscopic slight to marked hyperplasia of squamous cells and hyperkeratosis were noted in most surviving animals treated at 200 mg/kg/day and correlated with gross white discoloration and/or thickening in the forestomach. These lesions were often associated with ulcer/erosion and inflammation of the submucosa and/congestion. These lesions were considered to be adverse since they were associated with mortality and were related to the irritant properties of the test item. At 25 or 80 mg/kg/day, there were minimal or slight hyperplasia of squamous cells and hyperkeratosis in occasional females and thus these findings were considered to be non adverse at these dose-levels.

There were no test item-related findings in the testes, epididymides or ovaries at microscopic examination.

There were a few other microscopic observations seen only in males treated with test item. These findings were seen at low incidence, at minimal severity, were not dose-related and correlated with background lesions seen in the rats of these strain and age, including the unilateral severe tubular atrophy/degeneration in testis and severe reduced epididymis sperm content seen in one male treated at 200 mg/kg/day. They were considered to be consistent with spontaneous findings and unrelated to test item.

Dose descriptor:
NOAEL
Remarks:
(Parental systemic toxicity)
Effect level:
80 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Toxicity at 200 mg/kg/d (deaths, clinical signs, body weight loss, reduced food consumption, hyperplasia of squamous cells and hyperkeratosis in the forestomach)
Dose descriptor:
NOAEL
Remarks:
(Parental reproductive performance)
Effect level:
> 80 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
MORTALITY/VIABILITY/CLINICAL SIGNS:
There were no clinical signs in pups in any groups.
There were no effects on Day 4 p.p. viability index.
At 200 mg/kg/day, there were no significant pup deaths in the four litters.
At 25 and 80 mg/kg/day, there were no test item treatment-related effects on pup viability. The slightly lower viability index at 25 mg/kg/day was mainly due to one litter which lost 10 pups out of 14 by Day 1 p.p.

BODY WEIGHT:
There were no effects on mean pup body weight or body weight changes.
At 200 mg/kg/day, the four litters had comparable data with the other groups.

The lower mean body weight gain at 80 mg/kg/day was particularly due to one litter in this group. Moreover, two controls litters had a high mean body weight gain related to their low number of pups. It was thus not considered to be test item treatment related.

SEX RATIO:
At 25 and 200 mg/kg/day, there was no indication of a test item treatment effect.
At 80 mg/kg/day, there was a trend towards a sex ratio in favour of females with half of the litters having more than twice females than males. A relationship to the test item treatment cannot be totally excluded.

GROSS PATHOLOGY:
At 200 mg/kg/day, surviving pups of the four litters had no necropsy findings.

At 25 and 80 mg/kg/day, there were two and one litters, respectively, with one or two fetuses scheduled sacrificed having marked dilated renal pelvis and ureter or having dilated ureter. These findings were likely not test item treatment-related as dilated renal pelvis and dilated ureter can be found spontaneously in Sprague-Dawley fetuses and as there was no dose-relationship.

In prematurely dead pups, autolysis and absence of milk in the stomach (also noted in one surviving control pup) were observed at necropsy in comparable litter and/or pup incidence in the control group and at 25 and 200 mg/kg/day. Therefore these findings, which often occur in prematurely dead rat pups in this kind of study, were not considered to be test item treatment-related.
Dose descriptor:
NOAEL
Remarks:
(Toxic effects on progeny)
Generation:
F1
Effect level:
> 80 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed
Reproductive effects observed:
not specified

Mortality

 

Sex

Male

Female

Dose-level (mg/kg/day)

200

200

Number of sacrificed animals

4

3

Ptyalism

4

3

Loud breathing

4

3

Abdominal breathing

2

3

Dyspnea

2

3

Piloerection

2

1

Pallor (extremities)

3

1

Cold to the touch

2

1

Eyes half-closed

2

2

Hypoactivity

 

1

Emaciated appearance

 

2

Round back

1

 

Loss of balance

1

 

Dorsal decubitus

1

 

Swollen abdomen

1

 

Exteriorized penis

1

 

Chromodaccryorrhea

 

1

 Clinical signs

 

Sex

Male

Female

Dose-level (mg/kg/day)

0

25

80

200

0

25

80

200

Number of surviving animals

10

10

10

6

7

10

10

4

Pre-mating (females) or whole study (males)

 

 

 

 

 

Ptyalism

 

3

 

6

 

 

 

4

Loud breathing

 

 

1

4

 

 

 

 

Abdominal breathing

 

 

 

1

 

 

 

 

Round back

 

 

 

1

 

 

 

 

Gestation

 

 

 

 

 

 

 

 

Ptyalism

/

/

/

/

 

2

 

4

Loud breathing

/

/

/

/

 

 

 

1

Lactation

 

 

 

 

 

 

 

 

Ptyalism

/

/

/

/

 

2

 

4

Loud breathing

/

/

/

/

 

 

 

1

Reddish vaginal discharge

/

/

/

/

 

2

1

 

Piloerection

/

/

/

/

 

 

1

 

Pallor (extremities, eyes)

/

/

/

/

 

 

1

 

/: not applicable.

Food consumption

Sex

Male

Female

Dose-level (mg/kg/day)

0

25

80

200

0

25

80

200

Pre-mating

 

 

 

 

 

 

 

 

Days 1 - 8

27

28

28

27

20

20

20

18

Days 8 -15

27

26

26

21**

18

19

19

15**

Gestation

 

 

 

 

 

 

 

 

Days 0 - 7p.c.

/

/

/

/

23

22

23

21

Days 7 - 14p.c.

/

/

/

/

25

26

27

25

Days 14 - 20p.c.

/

/

/

/

29

31

31

29

Lactation

 

 

 

 

 

 

 

 

Days 1- 5p.p.

/

/

/

/

39

39

38

42a

/: not applicable; **: p<0.01; a: only four animals left in the group.

Conclusions:
Under the experimental conditions of this study:
- the No Observed Adverse Effect Level (NOAEL) for parental systemic toxicity was considered to be 80 mg/kg/day, based on adverse effects at 200 mg/kg/day (deaths, clinical signs, body weight loss, reduced food consumption, hyperplasia of squamous cells and hyperkeratosis in the forestomach),
- the NOAEL for parental reproductive performance was considered to be higher than 80 mg/kg/day (no adverse effects on reproductive performance were observed in this study, but there were only four available litters at 200 mg/kg/day excluding a definitive conclusion at this dose),
- the NOAEL for toxic effects on progeny was considered to be higher than 80 mg/kg/day (no adverse effects on progeny were observed in this study, but there were only four available litters at 200 mg/kg/day excluding a definitive conclusion at this dose).
Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item following daily oral administration (gavage) to male and female rats from before mating, through mating and, for females, through gestation until Day 4 post-partum (p.p.).

This study provided initial information on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

 

Methods

Three groups of ten male and ten female Sprague-Dawley rats received the test item, 2,2-bis(hydroxymethyl)-1,3-propanediol, ethoxylated and propoxylated, esters with acrylic acid, daily by oral (gavage) administration before mating, through mating and, for the females, through gestation until Day 4 p.p. The test item was administered as a solution in the vehicle, corn oil, at dose-levels of 20, 80 and 200 mg/kg/day.

Another group of ten males and ten females received the vehicle alone, under the same experimental conditions and acted as a control group. A constant dose-volume of 5 mL/kg/day was used.

The concentrations of the dose formulations were checked in study Weeks 1, 3, 4 (control and high-dose formulations only) and 6.

The animals were checked twice daily for mortality and morbidity and once daily for clinical signs during the treatment period. Body weight and food consumption were recordedonce a week during pre-mating and mating periods (food consumption not during mating), and during gestation on Days 0, 7, 14 and 20 post‑coitum (p.c.) and lactation on Days 1 and 5 p.p.

The animals were paired for mating after 2 weeks of treatment and the females were allowed to litter and rear their progeny until Day 5 p.p. The total litter sizes and numbers of pups of each sex were recorded after birth. Pups were observed daily for clinical signs, abnormal behavior and external abnormalities and weighed on Days 1 and 5 p.p.

Males were sacrificed after at least 5 weeks of treatment and females on Day 5 p.p. Final body weights and selected organs weights (epididymides, testes) were recorded and a macroscopicpost-mortemexamination of the principal thoracic and abdominal organs was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on ovaries (with oviducts), uterus, stomach/forestomach, epididymides and/or testes from all males sacrificed at the end of the treatment period and all females sacrificed on Day 5 p.p. in the control and high-dose groups, on stomach/forestomach of animals in the low- and mid-dose groups and on all macroscopic lesions.

Pups were sacrificed on Day 5 p.p.and submitted for a macroscopic post-mortem examination of the principal thoracic and abdominal organs.


Results

Chemical analyses

No test item was observed in the control dose formulation and the test item concentrations in the analyzed dose formulations were within an acceptable range, except for the dose formulations at 25 mg/kg/day given once in Week 1 (-13.0% of nominal concentration) and for 200 mg/kg/day formulation given once in Week 3 (‑73.8%). However, formulation concentrations were correct when checked again in the weeks thereafter. The deviations were considered not to have an impact on the study conclusion.

 

Parents

At 200 mg/kg/day, four males and three females were prematurely sacrificed at different occasions during the study, following poor health condition (starting with respiratory problems) and body weight loss, which besides lead to the decision tomove forward of a few days the terminal sacrifice of the high-dose males. At pathology, they had test item treatment-related white discoloration and/or thickening in the forestomach correlated with adverse hyperplasia of squamous cells and hyperkeratosis often associated with ulcer/erosion and inflammation of the submucosa.

One female was sacrificed on Day 24 p.c.for dystocia; a relationship with test item treatment cannot be excluded. At pathology, it had comparable pathology findings as the animals above.

In surviving animals given 200 mg/kg/day, ptyalism, respiratory difficulties (abdominal and loud breathing) and round back were observed too.

About half the animals in each sex (mainly those prematurely dead) lost weight (between 13 and 26%) ones after the others throughout the study. When compared with controls, lower mean food consumption was observed in Week 2 (-22% and -17% in males and females, respectively, p<0.01) and generally concerned the animals that were prematurely sacrificed or found dead thereafter. The effects on survival, clinical condition, body weight and food consumption at 200 mg/kg/day were considered to be adverse.

At pathology in survival animals treated at 200 mg/kg/day, there were no test item treatment-related organ weight differences. Test item treatment-related gross white discoloration and/or thickening in the forestomach were seen in all males and most females and correlated microscopically with adverse hyperplasia of squamous cells and hyperkeratosis noted in most survival animals. These lesions were sometimes associated with erosion/ulcer and were related to the irritant properties of the test item.

There were no effects on mean pairing and mating data and the four surviving pregnant females gave birth to live pups and had comparable delivery data with the other groups.

 

At 25 and 80 mg/kg/day, there were no premature deaths and no effects on mean body weights, mean body weight changes and mean food consumption. There were also no effects on mean pairing, mating, fertility and delivery data.

At 80 mg/kg/day, loud breathing noted for 5 days in one male was considered of limited toxicologically significance. At 25 mg/kg/day, there were no toxicologically significant clinical signs.

There was non-adverse minimal to slighthyperplasia of squamous cells and hyperkeratosis in the forestomach from occasional males and females at 25 and 80 mg/kg/day.

 

Pups

There were no clinical signs in pups in any groups. At 25 and 80 mg/kg/day, there were no test item treatment-related effects on pup viability, mean pup body weight and mean body weight change, and at 200 mg/kg/day the four surviving litters had data comparable with the other groups.

At 80 mg/kg/day, there was a trend towards a sex ratio in favour of females (36.7% of males vs. 47.6% in controls). A relationship to the test item treatment cannot be totally excluded. At 25 and 200 mg/kg/day, there was no indication of a test item treatment effect on the percentage of male pups.

At necropsy, the findings at 25 and 80 mg/kg/day were not considered to be test item-related, and at 200 mg/kg/day the four litters had no necropsy findings.


Conclusion

Under the experimental conditions of this study:

.         the No Observed Adverse Effect Level (NOAEL) for parental systemic toxicity was considered to be 80 mg/kg/day, based on adverse effects at 200 mg/kg/day (deaths, clinical signs, body weight loss, reduced food consumption, hyperplasia of squamous cells and hyperkeratosis in the forestomach),

.         the NOAEL for parental reproductive performance was considered to be higher than 80 mg/kg/day (no adverse effects on reproductive performance were observed in this study, but there were only four available litters at 200 mg/kg/day excluding a definitive conclusion at this dose),

.         the NOAEL for toxic effects on progeny was considered to be higher than 80 mg/kg/day (no adverse effects on progeny were observed in this study, but there were only four available litters at 200 mg/kg/day excluding a definitive conclusion at this dose).  

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
80 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The OECD 421 study is considered to be reliable.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Screening on reproduction toxicity (OECD 421)

The objective of this study was to evaluate the potential toxic effects of the test item following daily oral administration (gavage) to male and female rats from before mating, through mating and, for females, through gestation until Day 4 post-partum (p.p.). This study provided initial information on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

 

Three groups of ten male and ten female Sprague-Dawley rats received the test item, 2,2-bis(hydroxymethyl)-1,3-propanediol, ethoxylated and propoxylated, esters with acrylic acid, daily by oral (gavage) administration before mating, through mating and, for the females, through gestation until Day 4p.p.The test item was administered as a solution in the vehicle, corn oil, at dose-levels of 20, 80 and 200 mg/kg/day.

Another group of ten males and ten females received the vehicle alone, under the same experimental conditions and acted as a control group. A constant dose-volume of 5 mL/kg/day was used.

The animals were checked twice daily for mortality and morbidity and once daily for clinical signs during the treatment period. Body weight and food consumption were recordedonce a week during pre-mating and mating periods (food consumption not during mating), and during gestation on Days 0, 7, 14 and 20 post‑coitum (p.c.)and lactation on Days 1 and 5 p.p.

The animals were paired for mating after 2 weeks of treatment and the females were allowed to litter and rear their progeny until Day 5 p.p. The total litter sizes and numbers of pups of each sex were recorded after birth. Pups were observed daily for clinical signs, abnormal behavior and external abnormalities and weighed on Days 1 and 5p.p.

Males were sacrificed after at least 5 weeks of treatment and females on Day 5 p.p. Final body weights and selected organs weights (epididymides, testes) were recorded and a macroscopicpost-mortemexamination of the principal thoracic and abdominal organs was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on ovaries (with oviducts), uterus, stomach/forestomach, epididymides and/or testes from all males sacrificed at the end of the treatment period and all females sacrificed on Day 5 p.p.in the control and high-dose groups, on stomach/forestomach of animals in the low- and mid-dose groups and on all macroscopic lesions.

Pups were sacrificed on Day 5 p.p.and submitted for a macroscopicpost-mortem examination of the principal thoracic and abdominal organs.

 

Parents

At 200 mg/kg/day, four males and three females were prematurely sacrificed at different occasions during the study, following poor health condition (starting with respiratory problems) and body weight loss, which besides lead to the decision tomove forward of a few days the terminal sacrifice of the high-dose males. At pathology, they had test item treatment-related white discoloration and/or thickening in the forestomach correlated with adverse hyperplasia of squamous cells and hyperkeratosis often associated with ulcer/erosion and inflammation of the submucosa.

One female was sacrificed on Day 24 p.c.for dystocia; a relationship with test item treatment cannot be excluded. At pathology, it had comparable pathology findings as the animals above.

In surviving animals given 200 mg/kg/day, ptyalism, respiratory difficulties (abdominal and loud breathing) and round back were observed too.

About half the animals in each sex (mainly those prematurely dead) lost weight (between 13 and 26%) ones after the others throughout the study. When compared with controls, lower mean food consumption was observed in Week 2 (-22% and -17% in males and females, respectively, p<0.01) and generally concerned the animals that were prematurely sacrificed or found dead thereafter. The effects on survival, clinical condition, body weight and food consumption at 200 mg/kg/day were considered to be adverse.

At pathology in survival animals treated at 200 mg/kg/day, there were no test item treatment-related organ weight differences. Test item treatment-related gross white discoloration and/or thickening in the forestomach were seen in all males and most females and correlated microscopically with adverse hyperplasia of squamous cells and hyperkeratosis noted in most survival animals. These lesions were sometimes associated with erosion/ulcer and were related to the irritant properties of the test item.

There were no effects on mean pairing and mating data and the four surviving pregnant females gave birth to live pups and had comparable delivery data with the other groups.

 

At 25 and 80 mg/kg/day, there were no premature deaths and no effects on mean body weights, mean body weight changes and mean food consumption. There were also no effects on mean pairing, mating, fertility and delivery data.

At 80 mg/kg/day, loud breathing noted for 5 days in one male was considered of limited toxicologically significance. At 25 mg/kg/day, there were no toxicologically significant clinical signs.

There was non-adverse minimal to slighthyperplasia of squamous cells and hyperkeratosis in the forestomach from occasional males and females at 25 and 80 mg/kg/day.

 

Pups

There were no clinical signs in pups in any groups. At 25 and 80 mg/kg/day, there were no test item treatment-related effects on pup viability, mean pup body weight and mean body weight change, and at 200 mg/kg/day the four surviving litters had data comparable with the other groups.

At 80 mg/kg/day, there was a trend towards a sex ratio in favour of females (36.7% of malesvs.47.6% in controls). A relationship to the test item treatment cannot be totally excluded. At 25 and 200 mg/kg/day, there was no indication of a test item treatment effect on the percentage of male pups.

At necropsy, the findings at 25 and 80 mg/kg/day were not considered to be test item-related, and at 200 mg/kg/day the four litters had no necropsy findings.

Under the experimental conditions of this study:

.         the No Observed Adverse Effect Level (NOAEL) for parental systemic toxicity was considered to be 80 mg/kg/day, based on adverse effects at 200 mg/kg/day (deaths, clinical signs, body weight loss, reduced food consumption, hyperplasia of squamous cells and hyperkeratosis in the forestomach),

.         the NOAEL for parental reproductive performance was considered to be higher than 80 mg/kg/day (no adverse effects on reproductive performance were observed in this study, but there were only four available litters at 200 mg/kg/day excluding a definitive conclusion at this dose),

.         the NOAEL for toxic effects on progeny was considered to be higher than 80 mg/kg/day (no adverse effects on progeny were observed in this study, but there were only four available litters at 200 mg/kg/day excluding a definitive conclusion at this dose). 


Short description of key information:
The available guideline compliant developmental toxicity and teratology screening studies for PETIA (analoguous substance) in rabbits and rats support the conclusion that 2,2-bis(hydroxymethyl)-1,3-propanediol, ethoxylated and propoxylated, esters with acrylic acid does not represent a developmental toxicity hazard in humans.
Particularly, the recent OECD Guideline 414 developmental toxicity study in rabbits on PETIA as well as the OECD Guideline 421 reproductive/developmental screening study in rats on 2,2-bis(hydroxymethyl)-1,3-propanediol, ethoxylated and propoxylated, esters with acrylic acid did not reveal any evidence of developmental toxicity. These findings are supported by absence of developmental effects of PETIA at maternally non-toxic doses in a series of teratology studies in rats (Serota, 1982; Serota, 1983; Serota, 1984).

Justification for selection of Effect on fertility via oral route:
ONly one study is available to evaluation the effects of 2,2-bis(hydroxymethyl)-1,3-propanediol, ethoxylated and propoxylated, esters with acrylic acid on fertility.

Effects on developmental toxicity

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From May 20, 2014 to September 02, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to standard guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France.
- Age at study initiation: 17-19 weeks
- Housing: individually housed in labelled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) and shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days prior to pairing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12-h light/12-h dark cycle
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on trial formulations performed at WIL Research Europe
- Amount of vehicle (if gavage): 1 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (10 July 2014). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 h at room temperature under protection from light was also determined (highest and lowest concentration).

Duplicate samples (approximately 500 mg), which were taken from the formulations using a pipette, were accurately weighed into volumetric flasks of 20 mL. For determination of accuracy, samples were taken at middle position (50% height) or at top, middle and bottom position (90%, 50% and 10% height). The samples taken at 90%, 50% and 10% height were also used for the determination of the homogeneity of the formulations. For determination of stability, additional samples were taken at 50% height and stored at room temperature protected from light for 6 h. The volumetric flasks were filled up to the mark with acetonitrile. The solutions were further diluted to obtain an end solution of 25/75 (v/v) acetonitrile/water and concentrations within the calibration range. All solutions containing the test substance were protected from light.

Analytical conditions:
- Instrument: Acquity UPLC system (Waters, Milford, MA, USA)
- Detector: Acquity UPLC TUV detector (Waters)
- Column: Acquity UPLC BEH Shield RP-18, 100 mm × 2.1 mm i.d., dp = 1.7 μm (Waters)
- Column temperature: 40°C±1°C
- Injection volume: 10 μL
- Mobile phase: 30/70 (v/v) acetonitrile/water
- Flow 0.45 mL/min
- UV detection: 225 nm


The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Details on mating procedure:
One female was placed on a one-to-one-basis in the cage of a male rabbit. The time of mating was established by visual observation of mating. This day was designated Day 0 post-coitum.
Duration of treatment / exposure:
From Day 6 to Day 28 post-coitum, inclusive.
As a misgavage was suspected, female no. 45 (Group 3) was not dosed on Days 13 and 14 post-coitum. This animal died on Day 16 post-coitum.
Frequency of treatment:
Once daily for 7 days/week, approximately the same time each day
dose.
Duration of test:
From Day 6 to Day 28 post-coitum, inclusive
Remarks:
Doses / Concentrations:
0, 25, 50 and 75 mg/kg bw/day
Basis:

No. of animals per sex per dose:
24, 22, 26 and 24 females in groups 1, 2, 3 and 4, respectively; group 1 being control
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were selected based on the results of a dose range finding study. In this study, six mated rabbits per groups were dosed at 0, 25, 50 or 75 mg/kg bw/day for Days 6 to 28 postcoitum,inclusive, by oral gavage. At 75 mg/kg bw/day, toxicity consisted of reduced faeces production, reduced body weight gain (with a body weight loss on Days 6 to 9 post-coitum) and reduced food consumption on Days 6 to 9 post-coitum. At 75 mg/kg bw/day, one female had dark red fluid in the uterus. This animal had 5 live fetuses and 6 early resorptions, which resulted in a slightly increased post-implantation loss at this dose. At all dose levels, fetal body weights were slightly lower compared to controls, but this was not statistically significant and did not show a dose relationship.
Maternal examinations:
MORTALITY/VIABILITY:
At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ (2000)7). The circumstance of any death was recorded in detail.

CLINICAL SIGNS:
At least once daily from Day 0 post-coitum onwards up to the day prior to necropsy. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) were scored. Cage debris was examined in case of abortions.

BODY WEIGHTS:
Days 0, 3, 6, 9, 13, 16, 20, 23, 26, 29 post-coitum.

FOOD CONSUMPTION:
Days 0-3, 3-6, 6-9, 9-13, 13-16, 16-20, 20-23, 23-26 and 26-29 post-coitum.

WATER CONSUMPTION:
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

POST-MORTEM EXAMINATIONS:
All animals surviving to the end of the observation period, all moribund animals and all animals showing abortion were euthanized and 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). Females which had abortion or spontaneous death, were necropsied within 24 h of abortion.
Ovaries and uterine content:
Each ovary and uterine horn of animals surviving to planned necropsy was dissected and examined as quickly as possible to determine:

- The number of corpora lutea.
- The weight of the (gravid) uterus.
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths.
- The weight of each fetus.
- The sex of each fetus.
- Externally visible macroscopic fetal abnormalities.

Animals found dead or sacrificed before planned necropsy, were subjected to relevant examinations of the ovaries and uterine horns.
Fetal examinations:
External, visceral, and skeletal findings were recorded as developmental variations or malformations.

External:
Each viable fetus was examined in detail and weighed. All live fetuses were euthanized. The nonviable fetus (the degree of autolysis was minimal or absent) was examined, crown-rump length measured and weighed. The crown-rump length of late resorptions was measured and a gross external examination performed. Late resorptions with malformations were fixed in 10% buffered formalin.

Visceral (Internal):
All fetuses were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development. The sex of all fetuses was determined by internal examination. The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution. Tissues were then transferred to a 70% aqueous ethanol for subsequent processing and soft-tissue examination of all groups using the Wilson sectioning technique. After examination, the tissues were stored in 10% formalin. All carcasses, including the carcasses without heads, were eviscerated, skinned and fixed in identified containers containing 96% aqueous ethanol for subsequent examination of skeletons.

Skeletal:
The eviscerated fetuses from Groups 1 and 4, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S. Subsequently, the skeletal examination was done on all fetuses from Groups 1 and 4. Since no treatment related effects in the high dose group were seen, skeletal examination was not extended to the fetuses from the low and mid dose group. All specimens were archived in glycerin with bronopol as preservative. A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. Evaluation by the fetal pathologist and study director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
MORTALITY:
No toxicologically relevant mortalities occurred up to 75 mg/kg bw/day.
Ten animals died preterm
-Five animals showed an abortion and had to be terminated early. Macroscopic examination showed abnormalities of the heart (enlarged, pale or soft), stomach (many black foci on glandular mucosa, many black-brown or dark red foci, irregular surface of the forestomach or gelatinous contents), gallbladder (enlarged or many greenish foci), oviducts (several watery-clear cysts), pancreatic lymph nodes (enlarged and dark red), alopecia, thoracic cavity (containing watery-clear fluid), lungs (many, black foci), liver (pale), general pale discolouration, or watery-clear contents in the caecum.
- Five animals died due to complications of the gavage procedure. Macroscopic examination of these animals revealed abnormalities in the trachea (contents: reddish/dark red foamy or hemorrhagic/clotted blood), lungs (reddish foamy contents, many dark red foci, dark red discolouration or isolated tan focus), or advanced autolysis.

CLINICAL SIGNS
No toxicologically relevant clinical signs were noted up to 75 mg/kg bw/day. Incidental findings that were noted included alopecia, scars, scales, scabs, lean appearance, hunched posture, laboured respiration, rales, ulcer, diarrhoea, a wound, and salivation. These findings 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. At the incidence observed, these were considered signs of no toxicological relevance.

BODY WEIGHTS
At 50 and 75 mg/kg bw/day, overall a body weight loss was noted on Day 9 post-coitum and reduced body weight gain was noted on Day 13 post-coitum. These changes were statistically significant but did not show a dose response. During the remainder of treatment, mean body weights recovered to normal. Corrected (for uterus) body weight gain was unaffected at these dose levels. At 25 mg/kg bw/day, body weights and (corrected) body weight gain remained in the same range as controls over the treatment period.


FOOD CONSUMPTION
Absolute and relative food consumption were statistically significantly lower on Day 6-13 post-coitum at 75 mg/kg bw/day and on Day 6-9 post-coitum at 50 mg/kg bw/day. Food consumption was slightly lower on Day 13-16 post coitum (not statistically significant). Subsequently, mean food consumption recovered to normal. Food consumption before or after allowance for body weight of treated animals at 25 mg/kg bw/day remained in the same range as controls.

WATER CONSUMPTION
During the observation period several animals in all groups (vehicle control and test item treated) showed reduced water intake on one or more days. In the absence of a clear treatment-related effect, no quantitative measurement of water consumption was introduced during this study.

MACROSCOPIC EXAMINATION
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment. The incidence of incidental findings among control and treated animals was within the background range of findings that are encountered among rabbits of this age and strain, and did not show a dose-related trend. These necropsy findings were therefore considered to be of no toxicological relevance.

MATERNAL PREGNANCY DATA
For the control, low, mid and high-dose groups, there were respectively 22, 21, 21 and 19 litters with viable fetuses available on Day 29 post-coitum. In the control group, out of 24 mated animals two died preterm; all rabbits were pregnant. At 25 mg/kg bw/day, out of 22 mated animals one had an abortion. At 50 mg/kg bw/day, out of 26 mated animals two aborted, two died preterm (both pregnant) and one was not pregnant. At 75 mg/kg bw/day, out of 24 mated animals two aborted, one died preterm (pregnant) and two were not pregnant.
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
LITTER SIZE
Litter sizes were not affected by treatment up to 75 mg/kg bw/day. Mean litter sizes were 9.5, 8.6, 9.2 and 8.4 viable fetuses per group for the control, 25, 50 and 75 mg/kg bw/day group, respectively. There was no effect on fetal viability up to 75 mg/kg bw/day. Litter proportions of viable or dead fetuses and early resorptions were all within the normal range of biological variation. The percentage of late resorptions at 25 mg/kg bw/day was slightly outside the historical control data (4.0% versus a maximum of 3.5% in the historical control data). As no dose response was noted, this slight increase was not considered toxicologically relevant.

SEX RATIO
Sex ratio was unaffected by treatment up to 75 mg/kg bw/day.

FETAL BODY WEIGHT
Fetal body weight was unaffected by treatment up to 75 mg/kg bw/day. Mean fetal body weights (both sexes combined) were 36.3, 37.8, 35.5 and 37.5 grams in the control, 25, 50 and 75 mg/kg bw/day group, respectively.

FETAL MORPHOLOGICAL EXAMINATIONS
The numbers of fetuses (litters) available for external and visceral morphological evaluation were 208 (22), 180 (21), 194 (21) and 160 (19) in control, 25, 50 and 75 mg/kg bw/day groups respectively. Soft tissue cephalic examination was done for approximately half of the fetuses of all groups. Skeletal examinations were performed for all fetuses of control and 75 mg/kg bw/day groups.

EXTERNAL MALFORMATIONS AND VARIATIONS
There were no external developmental malformations or variations up to 75 mg/kg bw/day for fetuses at planned necropsy. Two fetuses of one litter from 50 mg/kg bw/day group showed hyperextension of the right hind limb. As this only affected one litter and this malformation was noted in the historical control data, it was not considered toxicologically relevant.

VISCERAL MALFORMATIONS AND VARIATIONS
There were no treatment related effects on visceral morphology following treatment up to 75 mg/kg bw/day. Retrocaval ureter was noted at 0.8% in the control group, 1.8% at 25 mg/kg bw/day, 1.1% at 50 mg/kg bw/day and 2.6% at 75 mg/kg bw/day. At 25 and 75 mg/kg bw/day, this was just outside the historical control maximum of 1.4%. Without a clear dose response relationship, these slight increases were not considered toxicologically significant. Any remaining visceral malformations and variations were not considered treatment related as they occurred infrequently, did not follow a dose-related trend, or occurred at frequencies that were within the range of available historical control data.

SKELETAL MALFORMATIONS AND VARIATIONS
At 75 mg/kg bw/day, there was a slight increase in skeletal developmental variations. This included three parameters indicating a developmental delay: unossified metacarpals and/or metatarsals (11.7% at 75 mg/kg bw/day versus 7.7% in the control group), slightly or moderately mal-aligned sternebrae (6.0% at 75 mg/kg bw/day versus 1.8% in the control group) and unossified tarsals (2.9% at 75 mg/kg bw/day versus 1.1% in the control group). As these increases were only slight and not statistically significant, these were not considered toxicologically significant. Additionally, the number of fetuses with caudal shift of the pelvic girdle was increased at 75 mg/kg bw/day (31.7% per litter; 52 fetuses in 11 litters) compared to the control group (17.1% per litter; 36 fetuses in 13 litters). Without any corroborative findings and as the number of affected litters was even less than controls, this finding was not considered toxicologically relevant. All malformations and remaining skeletal variations recorded in this study were not considered to be treatment related as they occurred infrequently, at lower levels in the high dose group, occurred at frequencies that were within the range of available historical control data, or were noted in control fetuses only.
Abnormalities:
not specified
Developmental effects observed:
not specified

None

Conclusions:
Under the conditions of the study, the No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity for the test substance was established as being at least 75 mg/kg bw/day, since no adverse effect was observed.
Executive summary:

A prenatal developmental toxicity study was conducted with PETIA in rabbits after oral exposure according to OECD Guideline 414, EU method B.31 and EPA OPPTS 870.3700 in compliance with GLP.

Ninety six mated female rabbits were assigned to four dose groups. The number of animals was 24, 22, 26 and 24 for Groups 1, 2, 3 and 4, respectively. The test substance was administered once daily by oral gavage from Days 6 to 28 post-coitum, inclusive, at doses of 25, 50 and 75 mg/kg bw/day (Groups 2, 3 and 4, respectively). The rabbits of the control group received the vehicle, Arachid oil, alone. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. All animals surviving to Day 29 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations.

No maternal or developmental toxicity was observed in any of the groups. Under the conditions of the study, the No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity for the test substance was established as being at least 75 mg/kg bw/day, since no adverse effect was observed.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
75 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
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

Studies on foetal development with PETIA (read-across)

A prenatal developmental toxicity study was conducted with PETIA (analoguous substance of 2,2-bis(hydroxymethyl)-1,3-propanediol, ethoxylated and propoxylated, esters with acrylic acid ) in rabbits after oral exposure according to OECD Guideline 414, EU method B.31 and EPA OPPTS 870.3700, in compliance with GLP. Ninety six mated female rabbits were assigned to four dose groups. The number of animals was 24, 22, 26 and 24 for Groups 1, 2, 3 and 4, respectively. The test substance was administered once daily by oral gavage from Days 6 to 28 post-coitum, inclusive, at doses of 25, 50 and 75 mg/kg bw/day (Groups 2, 3 and 4, respectively). The rabbits of the control group received the vehicle, arachid oil, alone. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. All animals surviving to Day 29post-coitumwere subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations andcorpora luteawere recorded. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. Neither maternal nor developmental toxicity was observed in any of the groups. Under the conditions of the study, the NOAEL for maternal and developmental toxicity for the test substance was established as being at least 75 mg/kg bw/day (Beekhuijzen, 2014).

 

A study was conducted to evaluate the embryo/fetal toxicity and teratogenic effects of PETIA when administered to a group of 22 pregnant female rats. The test substance was administered at a single dose level of 100 mg/kg bw/day to pregnant rats from Gestation Day (GD) 6-15. All animals were observed once daily and records made of mortality, moribundity and clinical signs. Body weights were recorded. On Day 20 of gestation, females were sacrificed by carbon dioxide asphyxiation and the fetuses were taken by caesarean section. Maternal, ovarian, uterine, litter and fetal data were evaluated. Test substance related maternal toxicity consisting of decreased survival, decreased body weight gains, or clinical signs (wheezing, dyspnea, rough haircoat, hunched posture, bloody crust around eyes, nose, mouth, and forepaws, salivation, and alopecia) were observed. Fetotoxic effects (probably related to maternal toxicity) were observed and consisted of an increase in resorptions and decreases in percent fetal viability, mean fetal weight, and mean gravid uterine weight. Under the conditions of the study, maternal toxicity and fetotoxicity was observed for test substance, when administered to pregnant rats at a level of 100 mg/kg bw/day. The fetotoxic effect was probably resulted from maternal toxicity. Treatment was terminated at the request of the sponsor due to excessive mortality at the dose administered (Serota, 1982; Serota, 1983).

A study was conducted to evaluate the embryo/fetal toxicity and teratogenic effects of PETIAwhen administered to a group of 25 pregnant rats. The test substance was administered at a single dose level of 10 mg/kg bw/day to pregnant rats from Gestation Day (GD) 6-15. All animals were observed once daily and records made of mortality, moribundity, and clinical signs. Body weights were recorded. On Day 20 of gestation, females were sacrificed by carbon dioxide asphyxiation and the fetuses were taken by caesarean section. Maternal, ovarian, uterine, litter and fetal data were evaluated. Suggestive signs of maternal toxicity were noted with the death of one female and the observation of wheezing in several animals. No effects, however, were noted in body weights, food and water consumption values, or gross pathology. An increased incidence of skeletal variants above the normal expected incidence was noted in the fetuses examined and consisted primarily of lagging ossification of various skeletal elements. This effect was considered treatment related but whether it resulted from maternal toxicity or from a direct fetotoxic effect could not be established. Under the conditions of the study, probable maternal toxicity and/or fetotoxicity was observed for test substance, when administered to pregnant rats at a level of 10 mg/kg bw/day. The fetotoxic effect was considered treatment related but whether it resulted from maternal toxicity or from a direct fetotoxic effect could not be established.No evidence of a teratogenic effect was noted (Serota, 1984).


Justification for selection of Effect on developmental toxicity: via oral route:
The most recent and the most reliable study was chosen.

Justification for classification or non-classification

A reproductive/developmental screening study was conducted in rats to investigate potential adverse effect of 2,2-bis(hydroxymethyl)-1,3-propanediol ethoxylated and propoxylated, esters with acrylic acid on reproduction, including embryo/foetal development. No adverse effects on reproductive/developmental parameters were noted at doses up to 80 mg/kg bw/d.

Thus, no classification is repuired for the reproduction endpoint.