Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

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:
2008-09-25 to 2008-11-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
March 1996
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: Wistar Han (Crl:WI(Han))
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld. Germany
- Age at study initiation (F0-treatment): approximately 10 weeks
- Weight at study initiation: male:279 - 317 g, female: 180 - 215 g
- Housing:
Pre-mating: animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIII type, height 18 cm)
Mating: females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/sex/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
Lactation: Offspring was kept with the dam until termination in Macrolon cages (MIII type, height 18 cm)
- Diet: ad libitum, pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum, tap-water
- Acclimation period F0: at least 5 days prior to start of treatment


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3°C (actual range: 18.4 - 22.2° C)
- Humidity (%): 30 - 70% (actual range: 37 - 94%)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours artificial light and 12 hours darkness per day

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1 % aqueous carboxymethyl cellulose (Genfarma, Zaandam, The Netherlands).
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- Dose volume: 5 ml/kg bw. Actual dose volumes were calculated according to the latest body weight.

VEHICLE
- 1% aqueous carboxymethyl cellulose

TEST SUBSTANCE FORMULATION
- Stability of test substance in vehicle: stable in 1% aqueous carboxymethyl cellulose for at least 6 hours at room temperature over the concentration range 10 to 200 mg/mL (determined during this project).
- Method of formulation: formulations (w/w) were prepared daily, were homogenised to a visually acceptable level and dosed as soon as possible after preparation with a maximum of 2.5 hours after preparation. No adjustment was made for specific gravity of the test substance, vehicle, and/or
formulation. In order to obtain homogeneity, the test substance formulations were heated in a water bath with a maximum temperature of 45 °C for a maximum of 22 minutes. The test substance formulations were allowed to cool down to a temperature at a maximum of 40 °C prior to dosing.
Based on results of the thermal analysis performed by NOTOX (NOTOX project 488541), reaction and/or decomposition of the test substance were observed above approximately 175°C and, therefore, the test substance was considered to be stable at 45°C.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Analyses were performed on a single occasion after the treatment phase according to a validated method (NOTOX Project 488541).
- The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115%. Homogeneity was demonstrated if the coefficient of variation was <= 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
Males: exposed for 30 days, i.e, 2 weeks prior to mating, during mating, and up to termination
Females: exposed for 42-44 days, i.e, during 2 weeks prior to mating, during mating, post-coitum, and during at least 4 days of lactation
Offspring: not treated
Frequency of treatment:
- Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
Basis:
actual ingested
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
Basis:
actual ingested
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Basis:
actual ingested
No. of animals per sex per dose:
F0 males: 10, F0 females: 10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on results of the dose range finding study (NOTOX Project 488547, cited in this study under 138985-20-3_8.6.1_8.7.1_Evonik Goldschmidt_2009_OECD 422, Appendix 5)
- Rationale for animal assignment (if not random): This species and strain of rat has been recognized as appropriate for general and reproductive toxicity studies.
Positive control:
-

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily
- Once prior to start of treatment and at weekly intervals this was also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating, or housed individually.
- All symptoms were recorded and graded according to fixed scales:
Maximum grade 1: grade 0 = absent, grade 1 = present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe


BODY WEIGHT: Yes
- Time schedule for examinations: first day of exposure and weekly thereafter; mated females were weighed on days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on days 1 and 4


FOOD CONSUMPTION:
- Time schedule: weekly for males and females. It was not recorded during the breeding period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and during lactation on Days 1 and 4 post-partum

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes (iso-flurane (Abbott Laboratories Ud., Zwolle, The Netherlands) anaesthesia)
- Animals fasted: Yes
- How many animals: from the first five mated males and the first five females with live offspring from each group
- Parameters: WBC, differential leucocyte count, RBC, Reticulocytes, Red blood cell distribution width (RDW), Haemaglobin, Haematocrit, MCV, MCH, MCHC, platelets, PT, APTT

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
- Animals fasted: Yes
- How many animals: from the first five mated males and the first five females with live offspring from each group
- Parameters: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase,Total Protein, Albumin, Total Bilirubin, Urea, Creatinine,
Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The assigned males were tested during Week 4 of treatment and the assigned females were tested during lactation (all before blood sampling)
- Dose groups that were examined: In the first five mated males and the first five females with live offspring, from each group
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex and grip strength (Score 0 =normal/present, score 1 =abnormal/absent); motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system; Pearson Technical Services, Debenham, Stowmarket, England). During the motor activity test, males were caged individually and females were caged with their offspring

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Dose groups: the first 5 mated males per group and the first 5 females with live offspring per group
-- Macroscopic examination: Adrenal glands, Aorta, Brain (cerebellum, mid-brain, cortex), Caecum, Cervix, Clitoral gland, Colon, Coagulation gland, Duodenum, Epididymides,
(Eyes with optic nerve (if detectable) and Harderian gland), (Female mammary gland area), (Femur including joint), Heart, Ileum, Jejunum, Kidneys, (Larynx), (Lacrimal gland, exorbital), Liver, Lung, Ovaries, Pancreas, Peyer's patches (jejunum, ileum) if detectable, Pituitary gland, Preputial gland, Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, Seminal vesicles, (Skeletal muscle), (Skin), Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid includinq parathyroid (if detectable), (Tongue), Trachea, Urinary bladder, Uterus, Lymph nodes - mandibular, mesenteric, (Nasopharynx), Oesophagus, Vagina, all gross lesions

- From all remaining animals:
Cervix, Clitoral gland, Coagulation gland, Epididymides,Ovaries, Preputial gland, Prostate gland, Seminal vesicles, Testes, Uterus, Vagina, all gross lesions

- Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

-- Organ weights:
- From the first 5 mated males per group and the first 5 females with live offspring per group: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Prostate (when fixed for at least 24 hours), Seminal vesicles, Spleen, Testes, Thymus

- From all remaining males:
Epididymides, Testes, Prostate (when fixed for at least 24 hours), Seminal vesicles


HISTOPATHOLOGY: Yes
- Histopathologic examination was performed on an extensive list of organs and tissues from five males and five females of groups 1 and 4 as well as gross lesions from all rats. Sections of testes from five group 1 and 4 rats were assessed for spermatogenesis staging.
- Adrenal glands, aorta, bone - sternum [and femur including joint]; bone marrow - sternal, brain, clitoral glands, epididymides, esophagus, [eyes with optic nerve and Harderian glands); heart, [identification marks], kidneys, [Iacrimal glands - exorbital], large intestine cecum, colon and rectum; [larynx), liver, lungs, Iymph nodes - mandibular and mesenteric; [female mammary gland area], [nasopharynx], ovaries, pancreas, pituitary gland, preputial, glands, prostate gland, [salivary glands - mandibular and sublingual]; sciatic nerve, seminal vesicles with coagulation glands, [skeletal muscle], [skin], small intestine - duodenum, jejunum and ileum with Peyer's patches: spinal cord - cervical, midthoracic and lumbar; spleen, stomach, testes, thymus, thyroid glands with parathyroid glands, [tongue], trachea, urinary bladder, uterus with uterine cervix, vagina and all organs or tissues with macroscopic abnormalities.
Following fixation, organs (except those listed in brackets) from the selected animals of groups 1 and 4 along with all organs or tissues with macroscopic abnormalities from all rats, were trimmed , processed and embedded in paraffin wax, precision cut and stained with hematoxylin and eosin.

PATHOLOGY OFFSPRING: Yes
- Pups were killed by decapitation on Day 5 of lactation or shortly thereafter.
- All offspring was sexed and externally examined. The stomach was examined for the presence of milk. Descriptions of all external abnormalities were recorded. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin, for possible further examination.
Other examinations:
OFFSPRING
Mortality/Viability: The numbers of live and dead pups at the First Lilter Check (=check at Day 1 of lactation) and daily thereafter were determined. lf possible, defects or cause of death were evaluated.
Clinical signs: At least once daily, detailed clinical observations (including abnormal behaviour) were made in all animals.
Body weights: Live pups were weighed during lactation on Days 1 and 4.
Sex: was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).
Statistics:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher, 1950) was applied to frequency data.
- No statistical analysis was performed on histopathology findings
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
-- Mortality: no mortality occurred during the study period
-- Clinical signs: No toxicologically-relevant clinical signs were noted up to 1000 mg/kg.
- Slight salivation was noted among the dose groups and was considered to be a physiological response to the taste of the formulation rather than a sign of systemic toxicity, considering the nature, minor severity of the effect and its time of occurrence (i.e. after dosing). Therefore, this was considered not toxicologically relevant.
- Incidental findings that were noted included alopecia, scaling and scabbing of various body parts, piloerection and a broken tail apex. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.

BODY WEIGHT AND WEIGHT GAIN
- Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.

FOOD CONSUMPTION
- Food consumption before or after allowance for body weight was similar between treated and control animals.

HAEMATOLOGY
- At 1000 mg/kg, haemoglobin levels were slightly reduced for males (statistical significant at 5%) and white blood cell counts (WBC) were higher for females (statistical significant at 1%; only determined for two females). .
- A statistical significant reduction in prothrombin time (PT) was noted for males at 150 mg/kg, but in the absence of a dose-dependent trend it was considered to be of no toxicological relevance.

CLINICAL CHEMISTRY
- At 1000 mg/kg, males showed elevated alkaline phosphatase (ALP) levels (statistically significant at 1%), and significantly reduced levels of cholesterol and total protein compared to vehicle controls (both statistically significant at 5%).
- Aspartate aminotransferase (ASAT) levels showed a reduction for females at all treatment levels without a dose response relationship. However, for this parameter, the values for the concurrent control animals were much higher than data from historical controls (historical control mean = 66.6, Group 1 mean = 121.4), and as such, the reduced ASAT levels were not considered to be toxicologically relevant. The cause of these increased values for the concurrent control group was unclear. However, as no corroborative findings were noted, it was not considered toxicologically relevant.
- Chloride levels were slightly lower for females at 150 and 1000 mg/kg. However, as this change was very slight and within the normal range, it was not considered toxicologically relevant.
- All other statistically significant changes from controls (decreased glucose levels at 50 mg/kg and increased inorganic phosphate values at 150 mg/kg, both for males) were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution.


NEUROBEHAVIOUR
- Hearing ability, pupillary reflex, static righting reflex, grip strength and motor activity were normal in the selected animals.

ORGAN WEIGHTS
- At 1000 mg/kg, higher absolute Iiver weights and liver to body weight ratios were observed for both sexes (not statistically significant for absolute liver weights of the males).
- At 150 mg/kg, increased testes weights (absolute and body weight ratio) were noted and an increase in seminal vesicle weight (body weight ratio) was seen at 50 mg/kg. These findings in both treatment groups were considered not to be a sign of toxicity as no dose response relationship was noted.

GROSS PATHOLOGY
- Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
- Incidental findings included foci on the stomach glandular mucosa, pelvic dilation of the kidneys,reduced size of the seminal vesicles, ectopic splenic tissue, a bent tail apex, and alopecia of several body parts. These findings are occasionally seen among rats used in these types of studies. As they remained within the range of biological variation for rats of this age and strain, these findings were considered to be changes of no toxicological significance.

HISTOPATHOLOGY: NON-NEOPLASTIC
- All recorded microscopic findings were within the range of background pathology encountered in Wistar Han rats of this age in this type of study and occurred at similar incidences and severity in both control and treated rats.
- The spermatogenic staging profiles were normal for all Group 1 and Group 4 males evaluated.


HISTORICAL CONTROL DATA (if applicable)
- Results were compared wit historical control data.
- No detailed data available in study

OTHER FINDINGS:
REPRODUCTION
- Reproduction parameters were unaffected by treatment up to 1000 mg/kg body weight/day.
- All pairs mated within four days and all females were pregnant.
- No treatment related findings were observed for mating performance, fertility parameters, gestation duration, number of dead and living pups at first Iitter check, number of implantation sites and number of corpora lutea.

BREEDING DATA
- Breeding parameters were unaffected by treatment up to 1000 mg/kg body weight/day.
- Postnatal loss and viability index were similar for the control and treated groups.

PUP DEVELOPMENT
- Development of pups was unaffected by treatment up to 1000 mg/kg body weight/day.
- Pup (mean) body weights were in the same range for the control and treated groups.
- Incidentai clinical symptoms consisted of small size, bluish colour, blue spot on the neck and eye, scabbing of the right cheek, pale appearance and insufficient milk in the stomach.
- Incidental macroscopic findings consisted of autolysis of pups found dead at the first Iitter check, scabbing of the right cheek, and insufficient milk in the stomach. No relationship with treatment was established for these observations and they were considered to be of no toxicoiogical significance.

Effect levels

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: - Effects see NOEL - Establishment of >=1000 mg/kg bw NOAEL because the described findings at 1000 mg/kg bw were not considered adverse and were without corroborative findings.

Target system / organ toxicity

Key result
Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, treatment with Isostearic acid, esters with methyl α-D-glucoside by oral gavage in male and female Wistar Han rats at dose levels of 50, 150 and 1000 mg/kg body weight/day revealed parental toxicity at 1000 mg/kg body weight/day. No reproduction, breeding and developmental toxicity was observed for treatment up to 1000 mg/kg body weight/day.
Based on these findings, the parental No Observed Effect Level (NOEL) was established at 150 mg/kg body weight/day. As these findings noted at 1000 mg/kg were not considered adverse and were without corroborative findings, the parental No Observed Adverse Effect Level (NOAEL) was established to be at least 1000 mg/kg body weight/day.
Executive summary:

In a Repeated Dose Toxicity Study according OECD 422 test substance Isostearic acid, esters with methyl α-D-glucoside (100% UVCB-Substance (80% Methyl Glucoside Isostearate Esters (mainly Di-), 16% Isostearic Acid, 4% Methyl Glucoside (not soluble in Olive Oil))

in 1 % aqueous carboxymethyl cellulose was administered to 10 male and 10 female Wistar Han rats/dose group by daily oral gavage at dose levels of 0, 50, 150, and 1000 mg/kg bw/day. The males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 30 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 42 to 44 days).

At 1000 mg/kg bw/day statistically significantly reduced haemoglobin, cholesterol and total protein levels (males), and elevated white blood cell counts (determined for only two females) plus alkaline phosphatase levels (males) were found. Increased liver weights (absolute and relative) were noted for high dose males and females.

No treatment-related changes were noted in any of the remaining parameters investigated in this study (i.e. mortality, clinical appearance, functional observations, body weight, food consumption, macroscopic and microscopic examination, reproduction, breeding and pup development).

The parental NOEL is 150 mg/kg bw/day, based on the findings noted at 1000 mg/kg bw/day.

The parental NOAEL is >= 1000 mg/kg bw/day, based on the findings noted at 1000 mg/kg bw/day which were not considered adverse and were without any corroborative findings like histopathological changes.

This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirement for a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422) in rat.