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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
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:
other: OECD TG 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

Constituent 1
Chemical structure
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:
[(uraniovanadio)methyl]borane
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
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
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.
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
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
Duration of test:
49 days
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

Examinations

Maternal 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

POST-MORTEM EXAMINATIONS: Yes
- 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. 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.
- Organs examined: 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.
Histopathological 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).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of implantations: Yes
Fetal examinations:
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.

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.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.
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.
Indices:
- Gestation index (%) = (number of females with live pups on the day of birth / number of females pregnant) * 100
- 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

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Clinically, distinct toxicity was noted in the F0 females at 500 mg/kg bw/d. Mortality in 3/10 high-dose females as well as clinical observations such as respiratory sounds, labored respiration and piloerection were observed during major parts of treatment. Detailed clinical examinations in an open field and in a functional observational battery (FOB) confirmed the findings made during cage-side observations, but measurements of motor activity did not reveal any indications of test substance-induced effects in low-, mid- and high-dose rats (50, 150 and 500 mg/kg bw/d).

Pathology (see below) confirmed that mortality and adverse clinical observations were subsequent to local irritant effects of the test substance in the fore- and glandular stomach. Also, reduced food consumption in females during premating is considered to be secondary to these local effects. Thus, these distinct adverse effects, in particular mortality, are not regarded to be due to systemic toxicity of Glycerin 3 EOTA.

All high-dose as well as most mid-dose animals of both sexes showed transient salivation for a few minutes immediately after each treatment. This was likely to be induced by the unpleasant taste of the test substance and/or by local irritation of the upper digestive tract. It is neither considered to be a sign of systemic toxicity nor as adverse.

Clinical pathology revealed a slight anemia in mid- and high-dose females (150 and 500 mg/kg bw/d) indicated by reduced RBC counts, haemoglobin and hematocrit values. Maybe, anemia was due to erosions and ulcers in the forestomach and glandular stomach of rats of both sexes in the mentioned dose groups. Females seemed to be more prone to anemia because of gestation and lactation. The increased synthesis of coagulation factors (i.e., reduced prothrombin time) in females of test group 3 (500 mg/kg bw/d) as well as the increased platelet counts in females of test groups 2 and 3 (150 and 500 mg/kg bw/d) happened consequently to the erosions in the stomach. Most probably, higher cholesterol levels in rats of both sexes of test group 3 (500 mg/kg bw/d) as well as higher triglyceride levels in females of this test group were due to an altered liver cell metabolism induced by the substance administration.

Pathology and histopathology revealed fore- and glandular stomach, liver lymph node, duodenum, and liver as target organs in this study.

In the forestomach, erosions or ulcers were noted in one female of test group 2 (150 mg/kg bw/day) as well as in all females of test group 3 (500 mg/kg bw/day). The erosions or ulcers were associated with inflammation. Their occurrence is regarded to be the primary response to an irritant effect of the test substance. As proliferative response secondary to ulceration, squamous cell hyperplasia was observed in one female of test groups 2 (150 mg/kg bw/day) as well as in 9 females of test group 3 (500 mg/kg bw/day). The severity of the squamous cell hyperplasia was dose-related increased. In test group 3 (500 mg/kg bw/day), all decedents showed erosions or ulcers and squamous cell hyperplasia in the forestomach. The occurrence of erosions or ulcers and squamous cell hyperplasia is considered to be treatment-related and adverse.

In the glandular stomach, the test substance led to erosions or ulcers that were observed in 2 females of test group 3 (500 mg/kg bw/day). In addition, focal hyperemia was noted in 2 females of test group 3 (500 mg/kg bw/day). Erosions or ulcers and the focal hyperemia are considered as consequence of irritant effects of the test substance and are regarded to be adverse.

In the liver lymph node, a lympho-reticulocellular hyperplasia was observed in 7 females of test group 3 (500 mg/kg bw/day). Furthermore, lymphoid cysts occurred in 5 females of test group 3 (500 mg/kg bw/day). These findings are considered to be secondary effects as consequence of the inflammatory processes in the forestomach and regarded to be adaptive and non-adverse.

In the duodenum, a diffuse thickening of duodenal mucosa was noted in 7 females of test group 3 (500 mg/kg bw/day). Because the histological structures of the duodenum in the affected animals were comparable to controls and because the severity was only minimal or slight, this finding is considered to be adaptive and non-adverse.

The increased liver weights in females of test group 2 (150 mg/kg bw/day) as well as in males and females of test group 3 (500 mg/kg bw/day) are related to treatment. They are regarded to be adaptive and non-adverse.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Remarks:
(local effects)
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Remarks:
(general, systemic toxicity)
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No signs of developmental toxicity were noted in any of the treated groups, numbers of liveborn pups, pup survival and growth were not influenced by the test compound. No malformations are reported.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion