Glycerol, ethoxylated, esters with acrylic acid

Toxicity to reproduction

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:

26 Jan 2010 - 12 Oct 2010 (experimental)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted in compliance with GLP regulations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Glycerol, ethoxylated, esters with acrylic acid

EC Number:

500-322-6

EC Name:

Glycerol, ethoxylated, esters with acrylic acid

Cas Number:

144086-03-3

Molecular formula:

UVCB substance

IUPAC Name:

Glycerol, ethoxylated, esters with acrylic acid

Details on test material:

- Name of test material (as cited in study report): Glycerin 3 EOTA
- Physical state: liquid / colorless, clear
- Analytical purity: 98.9 g/100 g
- Lot/batch No.: GK2656/104
- Test substance No. (testing lab.): 04/0114-3
- Expiration date of the lot/batch: 31 Jul 2010

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:WI(Han)
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 11-13 weeks old
- Weight at study initiation: 350.0 g – 377.5 g (males); 186.5 g – 213.5 g (females)
- Fasting period before study: no
- Housing: During overnight matings, male and female mating partners were housed together in Makrolon type M III cages. Pregnant animals and their litters were housed together until PND 4 (end of lactation). For motor activity (MA) measurements the animals were housed individually in polycarbonate cages.
- Diet (ad libitum): ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water (ad libitum): drinking water
- Acclimation period: about 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% in drinking water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the test substance preparation, the specific amount of test substance was weighed, topped up with 1% Carboxymethylcellulose suspension in drinking water in a calibrated beaker and intensely mixed with a magnetic stirrer.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw/day

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: overnight for a maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as [day 0] of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in 1% Carboxymethylcellulose suspension in drinking water at room temperature for a period of 24 hours was given (analytical report: Project No.: 09L00308).

Concentration control analyses were carried out in samples taken at the beginning and towards the end of the administration period while homogeneity of the preparations was verified only in samples from the beginning of the administration period. For homogeneity analyses three samples (one from the top, middle and bottom in each case) of the low and high concentrations (50 and 500 mg/kg bw/d) were withdrawn, while preparations were agitated by means of a magnetic stirrer.

The homogeneous distribution of the test substance in the vehicle (1% Carboxy-methylcellulose in drinking water) was demonstrated.
All measured mean values for Glycerin 3 EOTA were in the expected range of the target concentrations (94 – 107%), demonstrating the correctness of the aqueous preparations.

Duration of treatment / exposure:

The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females (females: 49 days; males: 30 days).

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 150, and 500 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations checked: any signs of morbidity, pertinent behavioral changes and signs of overt toxicity, parturition and lactation behavior of the dams

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the first administration and thereafter at weekly intervals during the administration period
- Observations: abnormal behavior during “handling”, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmos, pupil size, feces (appearance/consistency), urine, other findings

BODY WEIGHT: Yes
- Time schedule for examinations: once a week with the following exceptions:
- During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
- Females with litter were weighed on the day of parturition (PND 0) and on PND 4.

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes
- Time schedule for examinations: once a week with the following exceptions:
- Food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined for gestation days (GD) 0-7, 7-14, 14-20.
- Food consumption of F0 females, which gave birth, was determined for prenatal days (PND) 1-4.

WATER CONSUMPTION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Haematological examinations were carried out on study days 30 (males) and 49 (females).
- Anaesthetic used for blood collection: Yes: isoflurane (Isoba®, Essex GmbH Munich, Germany)
- Animals fasted: Yes
- How many animals: 5 animals per test group and sex
- Parameters checked: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes, Prothrombin time (Hepato Quick’s test) (HQT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Clinicochemical examinations were carried out on study days 30 (males) and 49 (females).
- Anaesthetic used for blood collection: Yes: isoflurane (Isoba®, Essex GmbH Munich, Germany)
- Animals fasted: Yes
- How many animals: 5 animals per test group and sex
- Parameters checked: Alanine aminotransferase (ALT) (L-alanine: 2-oxoglutarate aminotransferase; EC 2.6.1.2.), Aspartate aminotransferase (AST) (L-aspartate: 2-oxoglutarate aminotransferase; EC 2.6.1.1.), Alkaline phosphatase (ALP) (orthophosphoric acid monoester phosphohydrolase; EC 3.1.3.1.), Gamma-Glutamyltransferase (GGT) (Gamma -glutamyl) peptide: aminoacid-gamma-glutamyl-transferase; EC 2.3.2.2.), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Magnesium (MG).

URINALYSIS: Yes
- Time schedule for collection of urine: From 5 male and 5 female animals (with litter) urinalysis were carried out on study days 29 (males) and 46 (females).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color, Turbidity, Volume.

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

not examined

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities.

In general, a check was made for any dead or moribund pups twice daily on workdays or as a rule, only in the morning on Saturdays, Sundays or public holidays. The live pups were examined daily for clinical symptoms (including gross morphological findings) during the clinical inspection of the dams. The pups were weighed on PND 1 and PND 4. Pups' body weight change was calculated from these results.


GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
Females were allowed to litter and rear their pups until day 4 after parturition. Female animals were sacrificed 49 days after the beginning of the administration, and examined.
Male animals were sacrificed 30 days after the beginning of the administration, and examined.
All parental animals were sacrificed by decapitation using isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention was given to the reproductive organs.
Animals which died intercurrently or were sacrificed in a moribund state were necropsied as soon as possible after their death and assessed by gross pathology.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

- Organ weights: Adrenal glands, Brain, Cauda epididymis, Epididymides, Heart, Kidneys, Liver, Ovaries, Pituitary gland, Prostate, Testes, Seminal vesicles including coagulation glands, Spleen, Thymus, Thyroid glands (with parathyroid glands), Uterus.

- The following organs or tissues of parental animals were fixed in 4% buffered formaldehyde solution or modified Davidson’s solution:
adrenal glands, all gross lesions, aorta, brain, bone marrow (femur), cecum, cervix, coagulation glands, colon, duodenum, eyes with optic nerve, esophagus, epididymides (modified Davidson’s solution), female mammary gland, femur with knee joint, heart, ileum, jejunum (with Peyer’s patches), kidneys, larynx, liver, lungs, lymph nodes (axillary and mesenteric), nose (nasal cavity), ovaries (modified Davidson’s solution), oviducts, pancreas, parathyroid glands, pharynx, pituitary gland, prostate gland, rectum, salivary glands (glandula mandibularis and glandula sublingualis), sciatic nerve, seminal vesicles, skeletal muscle, spinal cord (cervical, thoracic and lumbar cord), spleen, sternum with marrow, stomach (forestomach and glandular stomach), testes (modified Davidson’s solution), trachea, thymus, thyroid glands, urinary bladder, uterus, vagina.

From the liver each slice of the Lobus dexter medialis and the Lobus sinister lateralis was fixed in Carnoy’s solution and embedded in paraplast.

Fixation was followed by histotechnical processing and examination by light microscopy and assessment of findings: from the control and high dose animals all organs and tissues were assessed histopathologically. From the animals of the low and mid dose only selected organs and tissues were assessed: all gross lesions, liver and stomach (forestomach and glandular stomach).

Postmortem examinations (offspring):

SACRIFICE
-On PND 4, all pups were sacrificed and gross necropsied.

GROSS NECROPSY
- All pups were examined externally and eviscerated; their organs were assessed macroscopically.

Statistics:

- DUNNETT-test (two-sided) for food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), number of mating days, duration of gestation, number of pups delivered per litter, implantation sites, post implantation loss.
- FISHER'S EXACT test for male and female mating index, male and female fertility index, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, number of litters with affected pups at necropsy.
- WILCOXON-test (one-sided) for proportions of affected pups per litter with necropsy observations.

Reproductive indices:

- Male mating index (%) = (number of males with confirmed mating / number of males placed with females) * 100
- Male fertility index (%) = (number of males proving their fertility / number of males placed with females) * 100
- Female mating index (%) = (number of females mated / number of females placed with males) * 100
- Female fertility index (%) = (number of females pregnant /number of females mated) * 100
- Gestation index (%) = (number of females with live pups on the day of birth / number of females pregnant) * 100

Offspring viability indices:

- Live birth index (%) = number of liveborn pups at birth / number of females pregnant) * 100
- Post implantation loss (%) = (number of implantations - number of pups delivered / number of implantations) * 100
- Viability index (%) = (number of live pups on day 4 after birth / number of liveborn pups on day of birth) * 100
- Sex ratio = (number of live male or female pups on day 0/4 / number of live male and female pups on day 0/4) * 100

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- 500 mg/kg bw:
- Mortality in 4/10 males and 3/10 females of the high dose group.
- Adverse clinical observations such as respiratory sounds, labored respiration and piloerection during major parts of treatment; reduced activity, cyanosis, hypothermia, soft feces, reduced nutritional state and abdominal position in individual animals on several occasions.
- 150 mg/kg bw: No mortality; adverse clinical observations such as respiratory sounds, labored respiration and piloerection in individual animals during major parts of treatment.
- 50 mg/kg bw: No test substance-related adverse findings.

BODY WEIGHT AND WEIGHT GAIN (PARENTAL ANIMALS)
- 500 mg/kg bw: Reduced body weights/body weight gain in males.
- 150 and 50 mg/kg bw: Mean body weights and mean body weight gain of the F0 parental males in the low- and mid-dose groups were comparable to the concurrent control during the study period.

FOOD CONSUMPTION (PARENTAL ANIMALS)
- 500 mg/kg bw: Reduced food consumption in males (-13%) and females (during premating, -7%).
- 150 and 50 mg/kg bw: Food consumption of the low- and mid-dose male and female F0 rats was comparable to the respective control animals throughout the entire study.

HAEMATOLOGY (PARENTAL ANIMALS)
- 500 mg/kg bw:
- Reduced prothrombin time in both sexes.
- Decreased RBC counts, haemoglobin and hematocrit values in females.
- Increased platelet counts in females.
- 150 mg/kg bw:
- Decreased RBC counts, haemoglobin and hematocrit values in females.
- Increased platelet counts in females.
- 50 mg/kg bw: No test substance-related adverse findings.

CLINICAL CHEMISTRY (PARENTAL ANIMALS)
- 500 mg/kg bw:
- Increased cholesterol levels in both sexes.
- Decreased alkaline phophatase activities in males.
- Increased triglyceride levels in females.
- 150 mg/kg bw:
- Increased triglyceride levels in females. Although this alteration might be treatment-related, it was regarded as non adverse
- 50 mg/kg bw: No test substance-related adverse findings.

URINALYSIS (PARENTAL ANIMALS)
No treatment-related changes among urinalyses parameters were measured.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Male reproduction data:
For most F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index varied between 89% (test group 3 – 500 mg/kg bw/d), 90% (control group) and 100% (test groups 1 and 2 – 50 and 150 mg/kg bw/d).
Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter.
One control male, one low-dose male and one high-dose male did not generate F1 pups. No histomorphological correlate was found to explain these apparent infertilities.
Thus, the male fertility index was 89% in test group 3, 90% in test groups 0 and 1 and 100% in test group 2. This reflects the normal range of biological variation inherent in the strain of rats used for this study. All respective values are within the range of the historical control data of the test facility.
- Female reproduction and delivery data:
The female mating index varied between 89% (test group 3 – 500 mg/kg bw/d), 90% (control group) and 100% (test groups 1 and 2 – 50 and 150 mg/kg bw/d). One control female and one high-dose female had no sperm in vaginal smear (no GD 0) and therefore had no implants or pups.
The mean duration until sperm was detected (GD 0) was 3.1, 3.8, 3.7 and 3.6 days (0, 50, 150 and 500 mg/kg bw/d, respectively).
All sperm positive rats delivered pups or had implants in utero with the exception of one low-dose female.
The female fertility index varied between 90% (test group 1) and 100% (test groups 0, 2 and 3). These values reflect the normal range of biological variation inherent in the strain of rats used for this study. All respective values are within the range of the historical control data of the test facility and do not show any relation to dosing.
There were no corroborative histopathological findings in the sexual organs of the non-pregnant females.
The mean duration of gestation values varied between 21.9 and 22.1 days without any relation to dosing.
Two high-dose F0 parental females, which were sperm positive, were found dead or were sacrificed moribund during gestation. Therefore, the gestation index reached 75% in the high-dose group, but was 100% in all remaining groups including the control.
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account (12.7 / 14.1 / 13.9 and 14.5 implants/dam in test groups 0-3).
Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since postimplantation loss did not show any statistically significant differences between the groups, and the mean number of F1 pups delivered per dam remained unaffected (12.3 / 13.3 / 13.4 / 13.8 pups/dam at 0, 50, 150 and 500 mg/kg bw/d).
The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 97% (test group 2), 98% (test group 1) and 99% (test groups 0, 3). The number of stillborn pups was comparable between the groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
For details see IUCLID Chapter 7.5.1, Key.BASF SE 85R0114/04R001.Repeated dose toxicity: oral.OECD422.rat

The significantly decreased terminal body weight in males of test group 3 (500 mg/kg bw/day) as well as the increased liver weights in females of test group 2 (150 mg/kg bw/day) and in males and females of test group 3 (500 mg/kg bw/day) are regarded to be treatment-related. The liver weight changes are adaptive and non-adverse.
The absolute and relative prostate weights were decreased in males of test group 3 (500 mg/kg bw/day). Because there were no histopatholocical correlates (the histological architecture was comparable to controls), a treatment-related effect seems rather unlikely.
The mean absolute weights of cauda epididymis were decreased in males of all treatment groups. There was no dose-response relationship and there were no histopathological correlates. Furthermore, the relative weights of cauda epididymis as well as the absolute and relative weights of the complete epididymides did not show significant weight changes. Therefore, the decreased weights of the cauda epididymis are considered to be incidental.
Since there were no histopathological correlates for the decreased mean absolute weight of seminal vesicles in males of test group 3 (500 mg/kg bw/day) and because the relative weight did not show significant weight changes, the decreased absolute weight of the seminal vesicles is related to the reduced terminal body weight in these animals.
The mean absolute brain weight was slightly decreased (p <= 0.05) in males of all treatment groups. Since the relative brain weights did not show significant weight changes and because there were no histopathological correlates, the decrease of the absolute brain weights is considered to be incidental.
Since there were no histopathological correlates, the decrease of the absolute thymus weight and the increase of the relative kidney weight in females of test group 3 (500 mg/kg bw/day) are considered to be incidental.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- 500 mg/kg bw:
- Erosion/ ulcer in the forestomach of all males and females.
- Thickened margo plicatus in the forestomach of 6/10 males and of 5/10 females.
- Erosions or ulcers in the glandular stomach of 1/10 males.
- Hyperemia in the glandular stomach of 3/10 males and 1/10 females.
- Thickened duodenal wall occurred in 7/10 males and 6/10 females.
- Hyperemia of duodenum, jejunum, ileum, and cecum in 1/10 males.
- Enlargement of liver lymph nodes in 7/10 males and 7/10 females.
- 150 mg/kg bw:
- Erosion/ ulcer in the forestomach of 8/10 males and of 1/10 females.
- Thickened margo plicatus in the forestomach of 9/10 males and of 3/10 females.
- Erosions or ulcers in the glandular stomach of 1/10 females.
- Enlargement of liver lymph nodes in 6/10 males.
- 50 mg/kg bw:
- Enlargement of liver lymph nodes in 2/10 males.
Since this finding is considered adaptive (see Discussion), no test substance-related adverse findings were made at this dose-level.

HISTOPATHOLOGY: NON-NEOPLASTIC (PARENTAL ANIMALS)
For details see IUCLID Chapter 7.5.1, Key.BASF SE 85R0114/04R001.Repeated dose toxicity: oral.OECD422.rat

Most of the macroscopically diagnosed erosions or ulcers in the forestomach could be confirmed histopathologically.
Most of the macroscopically diagnosed erosions/ ulcers or hyperemia in the glandular stomach could be confirmed histopathologically.
Most of the macroscopically diagnosed thickening of wall in the duodenum corresponded histopathologically with a minimal or slight diffuse thickening of duodenal mucosa, whereas the histological structures of the duodenum in the affected animals were comparable to the controls. The thickening of duodenal mucosa was observed in 9 males and 7 females of test group 3 (500 mg/kg bw/day) and is regarded treatment-related.
5 males of test group 3 (500 mg/kg bw/day) showed in the liver a minimal central hepatocellular hypertrophy that is regarded treatment-related and adaptive.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

(local effects)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Local irritation in fore- and glandular stomach led to mortality at 500 mg/kg bw/d and triggered adverse clincial observations as well as hematological changes at 150 and 500 mg/kg bw/d.

2

Dose descriptor:

NOAEL

Remarks:

(general, systemic toxicity)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Clinical chemistry changes at 500 mg/kg bw.

3

Dose descriptor:

NOAEL

Remarks:

(reproduction)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The NOAEL for fertility and reproductive performance was 500 mg/kg bw/d for the parental rats, the highest tested dose.

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
The mean number of delivered pups per dam and the rate of liveborn and stillborn pups were evenly distributed among test groups 0 (control) and 3 (500 mg/kg bw/d). The respective values reflect the normal range of biological variation inherent in the strain used in this study.
The viability index as indicator for pup mortality between PND 0-4 was 96% (test group 3), 97% (test group 1) and 98% (test groups 0 and 2).
The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 4 did not show biologically relevant differences between test groups 0 (control) and 3 (500 mg/kg bw/d).

CLINICAL SIGNS (OFFSPRING)
The F1 pups did not show any test substance-induced or spontaneous clinical signs up to scheduled sacrifice on PND 4.

BODY WEIGHT (OFFSPRING)
Mean pup body weights and pup body weight gain of all test substance-treated groups were comparable to the concurrent control group throughout the lactation period.
Two female runts were seen in test groups 0, 1 and 2 (0, 50 and 150 mg/kg bw/d) and one female runt was seen in test group 3 (500 mg/kg bw/d). No relationship to treatment is assumed.

GROSS PATHOLOGY (OFFSPRING)
There were no test substance-related findings in any of the F1 pups at necropsy. One control and one low-dose F1 pup had an empty stomach which was not considered to be associated to the test substance.

Overall reproductive toxicity

Reproductive effects observed:

not specified