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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
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

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:
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 oral 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.
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:
none

Examinations

Observations and examinations performed and frequency:
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

OPHTHALMOSCOPIC EXAMINATION: 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.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: on study day 28 (males) and study day 42 (females)
- Dose groups that were examined: five male and female animals per group, respectively
- Battery of functions tested: sensory activity / grip strength / motor activity:
- Home cage observations: posture, tremors, convulsions, abnormal movements, impairment of gait, other findings
- Open field observations: behavior when removed from cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait, activity/arousal level, feces (number of fecal pellets/appearance/consistency) within two minutes, urine (appearance/quantity) within two minutes, number of rearings within two minutes, other findings
- Sensory motor tests/Reflex tests: approach response, touch response, vision (“visual placing response”), pupillary reflex, pinna reflex, audition (“auditory startle response”), coordination of movements (“righting response”), behavior during “handling”, vocalization, pain perception (“tail pinch”), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings
- Motor activity: The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in cages for the time of measurement. Eighteen beams were allocated per cage. The number of beam interrupts was counted over 12 intervals for 5 minutes in each case.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

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.

- 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).

Statistics:
- DUNNETT-test (two-sided) for food consumption (parental animals), body weight and body weight change (parental animals).
- KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians. For feces, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity.
- KRUSKAL-WALLIS test (two-sided) or WILCOXON-test (two-sided) for clinical pathology parameters, urine volume, urine specific gravity.
- FISHER's exact test for urinalysis, except color, turbidity, volume and specific gravity.
- KRUSKAL-WALLIS test (two-sided) or WILCOXON-test (two-sided) for weight parameters.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
- 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
- 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
- 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
- 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
- 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
No treatment-related changes among urinalyses parameters were measured.

NEUROBEHAVIOUR
- Home cage observations:
No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.
- Open field observations:
The open field observations revealed some findings in the male animals of test group 3 (500 mg/kg bw/d), such as piloerection and respiratory sounds in one male, respectively, and a reduced exploration of area in 4 high-dose males. Furthermore, one mid-dose male (150 mg/kg bw/d) showed piloerection, a labored respiration, respiratory sounds and a reduced exploration of area.
The open field observations did not reveal any test substance-related findings in female animals of all test groups.
- Sensorimotor tests/reflexes:
There were no test substance-related findings in male and female animals of all test groups.
- Motor activity measurement:
Significant changes of motor activity were observed in the high-dose and in the low-dose F0 females during interval 7. These sporadical changes were not considered to be related to treatment. There were no other significant deviations of motor activity in the male and female animals of all test groups in comparison to the concurrent control group.

ORGAN WEIGHTS
For details see tables 1 and 2 (any other information on results incl. tables).

The significantly 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, 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 in the prostate (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
- 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
- Forestomach:
Most of the macroscopically diagnosed erosions or ulcers in the forestomach could be confirmed histopathologically. In most of affected males and females the erosions or ulcers occurred multifocal. They were noted in 7 males and one female of test group 2 (150 mg/kg bw/day) as well as in all males and females of test group 3 (500 mg/kg bw/day). In addition, squamous cell hyperplasia was observed in treated males and females of test groups 2 (150 mg/kg bw/day) and 3 (500 mg/kg bw/day). The squamous cell hyperplasia was characterized by an increased number of epithelial cells showing papillary structures into the lumen and small endophytic finger-like projections. The hyperplasia was accompanied by hyperkeratosis and chronic inflammation and resulted in a thickening of the epithelium. The hyperplasia occurred diffuse or multifocal and was correlated with the grossly thickened margo plicatus. Incidence and gradings are given in the table 3 (any other information on results incl. tables).
The occurrence of erosions/ ulcers and of squamous cell hyperplasia is considered to be treatment-related.

- Glandular stomach:
Most of the macroscopically diagnosed erosions/ ulcers or hyperemia in the glandular stomach could be confirmed histopathologically. In test group 3 (500 mg/kg bw/day) erosions or ulcers of the glandular stomach occurred in 5 males and 2 females, a minimal to severe focal hyperemia was observed in 4 males and 2 females. The occurrence of erosions/ ulcers or hyperemia in the glandular stomach is considered to be treatment-related.

- Intestine:
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.
In test group 3 (500 mg/kg bw/day), the macroscopically dilated jejunum and cecum could be confirmed in 2 males and in one female. In the third male with a grossly dilation of the jejunum, the organ was autolytic. The hyperemia that was macroscopically observed in one male (animal no. 36) could be confirmed in the jejunum, ileum, and cecum. All these findings occurred in animas that died or were sacrificed in a moribund state. They are considered to be incidental.

- Liver lymph node:
Most of macroscopically enlarged liver lymph nodes corresponded with lympho-reticulocellular hyperplasia, histopathologically. Lympho-reticulocellular hyperplasia was observed in 3 males of test group 2 (150 mg/kg bw/day) as well as in 7 males and 7 females of test group 3 (500 mg/kg bw/day). In addition, lymphoid cysts occurred in 5 males and 5 females of test group 3 (500 mg/kg bw/day). These findings were associated with the findings in the forestomach. For the enlargement of the liver lymph nodes in the 2 males of test group 1 (150 mg/kg bw/day) there were no histopathological correlates.

- Liver:
5 males of test group 3 (500 mg/kg bw/day) showed in the liver a minimal central hepatocellular hypertrophy. The hepatocellular hypertrophy is assessed as treatment-related and adaptive.

All other findings occurred either individually or were biologically equally distributed over the control group and the treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
(local)
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 clinical observations as well as hematological changes at 150 and 500 mg/kg bw/d.
Dose descriptor:
NOAEL
Remarks:
(systemic)
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.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Absolute organ weights (in % of control)

 

Male animals

Female animals

Test group

(mg/kg bw/day)

1

(50)

2

(150)

3

(500)

1

(50)

2

(150)

3

(500)

Terminal body weight

99%

95%

94%*

 

 

 

Brain

97%*

96%*

96%*

 

 

 

Cauda epididymis

91%*

86%**

90%*

 

 

 

Liver

 

 

 

105%

110%*

114%**

Prostate

103%

94%

73%**

 

 

 

Seminal vesicle

100%

93%

83%*

 

 

 

Thymus

 

 

 

90%

98%

82%**

* : p <= 0.05; **: p <= 0.01

Table 2: Relative organ weights (in % of control)

 

Male animals

Female animals

Test group

(mg/kg bw/day)

1

(50)

2

(150)

3

(500)

1

(50)

2

(150)

3

(500)

Kidneys

 

 

 

102%

106%

109%*

Liver

101%

102%

118%**

104%

108%**

115%**

Prostate

104%

99%

78%**

 

 

 

* : p <= 0.05; **: p <= 0.01

Table 3:  Incidence and grading of forestomach hyperplasia

Forestomach

Male animals

Female animals

Test group

(mg/kg bw/day)

0

1

(50)

2

(150)

3

(500

0

1

(50)

2

(150)

3

(500)

Number of animals

10

10

10

10

10

10

10

10

Hyperplasia, squamous, diffuse

 

 

8

10

 

 

1

9

·       Grade 2

 

 

5

1

 

 

 

1

·       Grade 3

 

 

2

3

 

 

1

3

·       Grade 4

 

 

1

6

 

 

 

5

Applicant's summary and conclusion