Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction products of (1 mole) Pentaerythritol ethoxylated and propoxylated (4:1) with 2-propenoic acid (4 moles)
EC Number:
604-394-0
Cas Number:
144086-02-2
Molecular formula:
C17H28O12 (C3H6O.C2H4O)n
IUPAC Name:
Reaction products of (1 mole) Pentaerythritol ethoxylated and propoxylated (4:1) with 2-propenoic acid (4 moles)
Constituent 2
Reference substance name:
2,2-bis(hydroxymethyl)-1,3-propanediol, ethoxylated and propoxylated, esters with acrylic acid
IUPAC Name:
2,2-bis(hydroxymethyl)-1,3-propanediol, ethoxylated and propoxylated, esters with acrylic acid
Test material form:
liquid: viscous
Details on test material:
- Physical state: colourless liquid
- Lot/batch No.: 2A187468501VSP
- Expiry date: 31 December 2015
- Storage conditions: at room temperature and protected from light.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system 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.

Administration / exposure

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
Doses / concentrations
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)

Examinations

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)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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

Details on results (P0)

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.

Effect levels (P0)

open allclose all
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

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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).