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Description of key information

- subacute (42 d study) combined 28 -day repeated dose toxicity study with reproduction/developmental toxicity screening test according to OECD guideline 422, GLP, RL1, NOAEL > 1000 mg/kg bw/day, read-across

- short-term repeated dose (28 d study) according to OECD guideline 422, GLP, RL2, dose selection for OECD guideline study 422: 50, 150 and 1000 mg/kg bw/day, read-across

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
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
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

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.
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:
-
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.
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.
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.
Key result
Critical effects observed:
not specified
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.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Reason / purpose:
reference to other study
Principles of method if other than guideline:
If not mentioned otherwise, test system and techniques were identical to those used during the main study (138985-20-3_8.6.1_8.7.1_Evonik Goldschmidt_2009_OECD 422).
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Wistar Han (Crl:WI(Han))
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld. Germany
- Age at study initiation: 10 - 12 weeks
- Housing: 3 animals per cage
- Diet: ad libitum, pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum, tap-water


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3°C (actual range: 19.8 -22.0°C),
- Humidity (%): 30 - 70% (actual range: 42 - 79%)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
other: 1 % aqueous carboxymethyl cellulose
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Chemical analysis not performed during the pilot study.
Duration of treatment / exposure:
5 days
Frequency of treatment:
daily
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
3
Control animals:
no
Details on study design:
- Dose selection rationale: The dose levels were based on an acute oral study (Acute toxicity: oral: 138985-20-3_8.5.1_Evonik Goldschmidt_2009_OECD 423) in which the oral LD50 was > 2000 mg/kg bw.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION:
- Time schedule: daily

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
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:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:

BODY WEIGHT AND WEIGHT GAIN
Slightly decreased body weight gain was noted at both dose levels (without a dose response).

FOOD CONSUMPTION
Slightly decreased food consumption was noted at both dose levels (without a dose response).

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Remarks on result:
other:
Remarks:
no treatment related effects at the highest dose tested
Critical effects observed:
not specified
Conclusions:
Based on these results, the dose levels for the main study (138985-20-3_8.6.1_Evonik Goldschmidt_2009_OECD 422) were selected to be 50, 150 and 1000 mg/kg body weight/day.
Executive summary:

The present study was conducted in order to set the dose levels for the main study (combined 28-day/reproductive/developmental screening study, OECD 422), a pilot study was

performed. Groups of 3 female Wistar rats (10-12 weeks old) were dosed at 500 mg/kg body weight/day (Group 1) and 1000 mg/kg body weight/day (Group 2) for 5 days by oral gavage. No mortality, clinical signs, macroscopic findings or organ weight effects were observed at 500 and 1000 mg/kg bw/day.

Slightly decreased body weight gain and food consumption was noted at both dose levels (without a dose response).

Based on these results, the dose levels for the main study (combined 28-day/reproductive/developmental screening study; OECD 422) were selected to be 50, 150 and 1000 mg/kg body weight/day.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source substance is constituted of the target substance (alpha methyl glucoside) esterified to Isostearic acid and therefore exhibit partially similar toxicological behaviour due to similarity in their structure. During synthesis of Isostearic acid esters with methyl-α-D-glucose the four OH-groups of the methyl glucose are partially esterified with isostearate groups. The molar ratio of methyl glucose and isostearic acid is chosen close to 1:2 which means that the products may be a mixture of non reacted methyl glucose and its 4 monostearates, 6 distearates, 4 tristearates and 1 tetrastearate isomers. These numbers will increase accordingly, if the cyclic form of glucose is in equilibrium with its linear form and the other anomer (hemiacetalic carbon). A possible hydrolysis results in alpha methyl glucoside and excess Isostearic acid.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance alpha methyl glucoside is a monoconstituent substance composed of a methyl-group which is o-glycosidic bound to the α-D-glucose core structure.

The source substance Isostearic acid esters with Methyl-α-D-glucose is an UVCB with a composition of 80% alpha methyl glucoside isostearate esters (mainly di-), 16% isostearic acid and 4% alpha methyl glucoside.

3. ANALOGUE APPROACH JUSTIFICATION
For detailed information please refer to the attached justification.

4. DATA MATRIX
For detailed information please refer to the attached justification.
Reason / purpose:
read-across source
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.
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.
Remarks on result:
other: Based on source substance used as test material
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 remarks
Key result
Critical effects observed:
not specified
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 for the source substance Isostearic acid, esters with methyl α-D-glucoside.
Executive summary:

In a Repeated Dose Toxicity Study according OECD 422 the source 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).

For the combined 28-days/reproductive/developmental screening Study a dose range finding study was conducted in order to set the dose levels for the main study.

Groups of 3 female Wistar rats (10-12 weeks old) were dosed at 500 mg/kg body weight/day (Group 1) and 1000 mg/kg body weight/day (Group 2) for 5 days by oral gavage. No mortality, clinical signs, macroscopic findings or organ weight effects were observed at 500 and 1000 mg/kg bw/day. Slightly decreased body weight gain and food consumption was noted at both dose levels (without a dose response).

Based on the available results the parental NOEL is 150 mg/kg bw/day, based on the findings noted at 1000 mg/kg bw/day in the study according to OECD 422.

The parental NOAEL for the source substance 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 during the main study and without any adverse effect during the dose range finding study.

The dose descriptor obtained from the existing combined repeated dose and reproduction/developmental screening study according to OECD 422 with the source substance is considered as an appropriate starting point for deriving a DNEL. In general, based on the diverging molecular weight of the target and the source substance this dose descriptor has to be adjusted. However, due to supporting information from varying sources adjustment of the dose descriptor is scientifically not justified. A justification is given below.

Although alpha methyl glucoside is the core structure of the source substance Isostearic acid, esters with α-methyl glucoside, it is also structurally almost identical to alpha D-glucopyranoside. Alpha D-glucopyranoside is the major carbon source and an indispensable part of the intermediary metabolism in many organisms including rodents and humans (common textbooks of Biochemistry). It was shown that both compounds are absorbed by the same transporter and both, alpha D-glucopyranoside and alpha methyl glucoside are known to be renally excreted. There are published information that indicate that higher doses (3000 mg/kg bw) of alpha methyl glucoside administered to rats via oral gavage do not result in adverse effects within 2h after administration (Demarest, K et al., 2012). Due to the structural similarity to a-D glucose, whose LD50 is > 20000 mg/kg bw in rabbits (Spector, 1955), the LD50 of alpha methyl glucoside, although non-metabolisable, is considered to be in the same order of magnitude. Data about the acute oral toxicity of α-D-glucopyranoside is mainly provided by studies conducted prior to implementation of GLP and/or OECD test-guidelines, i.e. collection of data/handbook data, thus, a detailed documentation of the occurring effects after administration of high doses of α-D-glucopyranoside is lacking. The exceptional high doses of glucose which are required to mediate a toxic effect (deterioration of physiological functions/mortality) suggest that toxicity results mainly from deterioration of cell functions mediated by dehydration. Based on the structural similarity it can be assumed that despite its non-metabolisable nature, alpha methyl glucoside causes the same dehydration symptoms at high doses. Furthermore, it is highly unlikely that other adverse effects occur at low doses of alpha methyl glucoside because it is considered to be excreted unchanged. Local effects were not detected during reliable and relevant irritation studies conducted according to OECD test guideline 439 and 437 with the target substance alpha methyl glucoside, thus, based on all available information an increased risk even during the intermittent workers exposure patterns or due to peak exposures during work is not expected.

Moreover, although alpha methyl glucoside is a non-metabolisable compound, information from other regulatory institutions like US EPA confirm a low toxicity. At the US EPA alpha methyl glucoside is listed on the ‘Safer Chemical Ingredients list’ as chemical that has been verified to be of low concern based on experimental and modeled data. Additionally, the FDA states that alpha methyl glucoside is considered to be safe in certain substances which are polyester resins and are safely used as articles which are intended for repeated use in contact with food.

In summary, the available information provides evidence that alpha methyl glucoside exhibits a low toxicity, thus, adjustment of the descriptor starting point is scientifically not justified.

 

 

References:

1)   Spector, W. S. (1955).HANDBOOK OF TOXICOLOGY. VOLUME 1. NATIONAL ACADEMY OF SCIENCES-NATIONAL RESEARCH COUNCIL WASHINGTON DC.

2)   Brot-Laroche, Edith, et al. "Characterization of the d-glucose/Na+ cotransport system in the intestinal brush-border membrane by using the specific substrate, methyl α-d-glucopyranoside."Biochimica et Biophysica Acta (BBA)-Biomembranes904.1 (1987): 71-80.

3)   Kimmich GA, Randles J: alpha-Methylglucoside satisfies only Na+-dependent transport system of intestinal epithelium. Am J Physiol 241:C227–C232, 1981

4)   Genel, M., London, D., Holtzapple, P. G., & Segal, S. (1971). Uptake of alpha-methylglucoside by normal and diabetic human jejunal mucosa.Translational Research,77(5), 743-750.

5)   Lostao, M. P., et al. "On the multiplicity of glucose analogues transport systems in rat intestine."Revista espanola de fisiologia47.4 (1991): 209-216.

6)   Segal, Stanton, et al. "Transport of alpha-methyl-D-glucoside by human kidney cortex."Metabolism22.1 (1973): 67-76.

7)   Demarest, Keith T., et al. "Methods of using alpha-methylglucoside (AMG) as an indicator for glucose absorption and excretion." U.S. Patent No. 8,147,801. 3 Apr. 2012.

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available study was conducted according to OECD guideline and is of high quality. Based on the different molecular weights of target and source substance the NOAEL was adjusted for alpha methyl glucoside.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

There are no data for alpha methyl glucoside regarding oral repeated dose toxicity, however, there are reliable and relevant data for the structurally similar Isostearic acid, esters with methylα-D-glucoside indicating a very low oral toxicity for the target substance.

 

In a dose range finding study 2 groups of 3 Wistar Han rats each were administered 500 mg/kg bw and 1000 mg/kg bw Isostearic acid, esters with methylα-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 for 5 days by oral gavage. Subsequently the animals were sacrificed and a necropsy was performed. Observation of clinical signs, body weights and food consumption were made daily, mortality was checked twice daily. No mortality, clinical signs, macroscopic findings or organ weight effects were observed at 500 and 1000 mg/kg bw/day. Slightly decreased body weight gain and food consumption was noted at both dose levels (without a dose response). Based on these results, the dose levels for a combined 28-day/reproduction/developmental screening study were selected to be 50, 150 and 1000 mg/kg body weight/day.

In the main combined 28-day/reproduction/developmental screening study according OECD 422 test substance Isostearic acid, esters with methylα-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.

Based on the available information, the subacute oral toxicity of alpha methyl glucoside is low. There are no data gaps in subacute toxicity. Even though there is no information on subacute toxicity in humans, there is no reason to believe that the low subacute toxicity observed in experimental animals would not be relevant for human health.

 

Justification for read-across

For details on substance identity, toxicokinetics and detailed toxicological profiles, please refer also to the general justification for read-across attached as pdf document to section 13 of the IUCLID file.

Analogue approach justification

The read-across hypothesis is based on similar break down products of target and source substance, i.e. the target substance (alpha methyl glucoside) is one of two break down products of the source substance (Isostearic acid, esters with Methyl-α-D-glucose). According to scenario 1 of the Read-across Assessment Framework (RAAF) this analogue approach is based on the (bio) transformation of the source substance to common compounds, i.e. the target substance alpha methyl glucoside and Isostearic acid. Based on the available experimental data, including genotoxicity studies, the read-across strategy is supported by a similar toxicological behaviour of the two substances and their break-down products, respectively. Although there is a difference in absorption, for both substances a very low acute toxicity and mutagenicity was shown. However, in accordance with ECHA guidance on information requirements chapter R 7.c absorption of Isostearic acid, esters with methyl-α-D-glucose in the GI-tract is considered unlikely due to its size. The main metabolism pathway of the unchanged source substance would then be biotransformation by xenobiotic metabolism, i.e. functionalisation, conjugation and subsequent biliary excretion. However, unspecific hydrolysis of the source substance (Isostearic acid, esters with methyl-α-D-glucose) during GI-tract passage is more likely to occur resulting in its breakdown products, namely alpha methyl glucoside and Isostearic acid.

Alpha methyl glucoside (target substance) is a non-metabolisable glucose analogue which is used in several published studies to investigate cellular glucose uptake (Segal et al., 1973; Lee et al., 2007; Prieto et al., 1996; Genel et al., 1970). Alpha methyl glucoside enters the cell via the ubiquitously occurring SGLT transporter. Recovery of nearly 100% of labelled alpha methyl glucoside revealed a full absorption in various tissues. Due to reabsorption by the kidney, its concentration is elevated in the renal brush border membrane (Lee et al., 2007). This increase of alpha methyl glucoside is only limited by the ion gradient resulting from sodium symport. Alpha methyl-glucoside is considered to be mainly renally excreted. The other remaining break down product Isotearic acid is metabolised via β-oxidation and is thereby eliminated by the intermediary metabolism and thus, indistinguishable from fatty acids from other sources including diet.

 

Structural similarity

a. Structural similarity and functional groups

The target substance, alpha methyl glucoside, consists of alpha-D-glucose which forms an o-glycosidic bond with a methyl-group at C1. Since several studies confirm that alpha methyl glucoside should be regarded to as a non-metabolizable sugar in mammalian cells, it can be concluded that the substance is renally excreted without transformation (Segal et al., 1973; Lee et al., 2007; Prieto et al., 1996; Genel et al., 1970). Although not metabolisable by mammalian cells it was shown that alpha methyl glucoside is metabolised by several bacteria except these ones occurring most frequently in the GI flora (Devriese et al., 1996, Tittsler & Sandholzer, 1935, Koser & Saunders, 1932).

The source substance, Isostearic acid esters with methyl-α-D-glucose, consists of one to four C18 aliphatic chains which are esterified to alpha methyl glucoside. Although this substance has a very lipophilic character (logKow > 6.5, water solubility < 5E-004 g/L and a high molecular weight (weighted mean) 690.31 g/mol) it cannot be excluded that it is absorbed to little extent. Subsequent to uptake unhydrolysed Isostearic acid, esters with methyl-α-D-glucose will to some extent undergo biotransformation by xenobiotic metabolism and subsequently biliary excreted, on the other hand unspecific hydrolysis is likely to occur and two fragments will emerge of which one is readily metabolised by fatty acid metabolism, i.e. β-oxidation of the aliphatic chain and the other one, alpha methyl glucoside (= target substance), is excreted unchanged. However, since Isostearic acid, esters with methyl-α-D-glucose is an UVCB consisting of an estimated amount of 4% of methyl-α-D-glucose, it must be assumed that studies conducted with the source substance are also performed with 4% of the target substance.

However, there are some differences between the target substance and the source substance regarding their physicochemical properties and consequently their toxicological effects.

 

b. Common breakdown products:

The metabolism expected to occur in the unhydrolysed source substance Isostearic acid, esters with methyl-α-D-glucose due to its size is functionalisation, conjugation and excretion. On the other hand hydrolysis of the isostearic acid moeity is likely to occur. The rate of hydrolysis is assumed to be enzyme-mediated and may thus be limited. Further degradation may be the metabolism of the fatty acid chain via β-oxidation. However, since the source substance is considered to be highly lipophilic, intestinal absorption may be limited for the unhydrolysed substance. However, the source substance is considered to be unspecifically hydrolysed in the GI-tract and subsequently absorbed and further metabolised. In case of alpha methyl glucoside (one of two breakdown products), a known non-metabolisable molecule, distribution preferentially to the kidney and in tissues exhibiting the SGLT1 and SGLT2 transporter occurs. This uptake and transient increase of concentration is also driven by renal reabsorption and slow excretion but this increase is limited by sodium symport. Finally, alpha methyl glucoside is considered to be renally excreted.

c. Differences

As described above alpha methyl glucoside and Isostearic acid, esters with methyl-α-D-glucose are distinguished by the presence or absence of the C18 alkyl-chains.Associated with the presence of these alkyl chains also differences in the physicochemical properties of both substances occur, i.e. differences in water solubility, partition coefficient, molecular weight etc. These physicochemical characteristics are also decisive for absorption of the source substance either oral (via GI-tract), dermal or by inhalation. Exposure to the source substance via inhalation route can be excluded due to its waxy appearance and its low vapour pressure. Due to its high molecular weight and log Kow the absorption of the unhydrolysed source substance is also limited via the dermal and the oral route. However, as explained above the composition of the source substance substantiates an exposure to the target substance as well. In contrast, dermal absorption of alpha methyl glucoside is likely due to its small molecular weight. Respiratory absorption of alpha methyl glucoside cannot be fully excluded due to its granular form although its median particle size is 100 µm, thus, it is considered that the substance is not inhalable according to ECHA guidance on information requirements Chapter R.7.c.

Since alpha methyl glucoside is produced and handled only as an intermediate consumer exposure is assumed to be marginal, thus, exposure via the oral route is not expected.

Dermal absorption of alpha methyl glucoside is also considered to be low due to its low log Kow (-2.5) it is presumably to hydrophilic to cross the stratum corneum.

 

Comparison of subacute oral toxicity data

Endpoint

Target substance

Source substance

 

Alpha methyl glucoside

 

Isostearic acid, esters with methyl-α-D-glucoside

 

Acute oral toxicity

 

No data, read-across

 

OECD 422, rat, RL 1, GLP

NOEL > 150 mg/kg bw females

NOAEL > 1000 mg/kg bw females

 

Specific target organ toxicity after repeated exposure - no classification according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).

No experimental data are available for the target substance.

The oral NOAEL of the closely related source substance Isostearic acid, esters with methyl-α-D-glucoside in female rats was > 1000 mg/kg bw. The oral NOEC was > 150 mg/kg bw.

Quality of the experimental data of the analogues:

The source substance Isostearic acid, esters with methyl-α-D-glucoside has been tested in a reliable study according to OECD TG 422. The tests have been conducted according to GLP criteria. Therefore, these data have no uncertainties and can be used in an analogue approach. The available data from the source chemical is sufficiently reliable to justify the read-across approach.

 

Conclusion for read-across

The physico-chemical and structural similarities between the breakdown products of the source substance and the target substance as presented above support the read-across hypothesis. Adequate and reliable scientific information indicates that the source and target substance and the degradation products of the source substance have similar toxicity profiles under the experimental conditions in the considered studies for the endpoint oral repeated dose toxicity.

Thus, the results obtained with the source substance Isostearic acid, esters with methyl-α-D-glucoside are considered to be also relevant for the target substance alpha methyl glucoside.

 

References

Lee, Y. J., & Han, H. J. (2007). Regulatory mechanisms of Na+/glucose cotransporters in renal proximal tubule cells.Kidney International,72, S27-S35.

Segal, S., Genel, M., Holtzapple, P., & Rea, C. (1973). Transport of alpha-methyl-D-glucoside by human kidney cortex.Metabolism,22(1), 67-76.

Prieto, R. M., Ferrer, M., & Tur, J. A. (1996). Changes in intestinal alpha-methyl-D-glucoside uptake due to pregnancy and lactation in rats.Digestion,57(1), 16-21.

Genel, M., London, D., Holtzapple, P. G., & Segal, S. (1971). Uptake of alpha-methylglucoside by normal and diabetic human jejunal mucosa.Translational Research,77(5), 743-750.

Enoch, S. J., Madden, J. C., & Cronin, M. T. D. (2008). Identification of mechanisms of toxic action for skin sensitisation using a SMARTS pattern based approach.SAR and QSAR in Environmental Research,19(5-6), 555-578.

Koser, S. A., & Saunders, F. (1932). The Fermentation of Alpha-Methyl-D-Glucoside by Members of the Coli-areogenes Group.Journal of bacteriology,24(4), 267.

Tittsler, R. P., & Sandholzer, L. A. (1935). The fermentation of alpha-methylglucoside by bacteria.Journal of bacteriology,29(4), 363.

Devriese, L. A., Pot, B., & Collins, M. D. (1993). Phenotypic identification of the genus Enterococcus and differentiation of phylogenetically distinct enterococcal species and species groups.Journal of Applied Microbiology,75(5), 399-408.

.

Justification for classification or non-classification

In a Repeated Dose Toxicity Study according OECD 422 the source 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).

For the combined 28-days/reproductive/developmental screening Study a dose range finding study was conducted in order to set the dose levels for the main study.

Groups of 3 female Wistar rats (10-12 weeks old) were dosed at 500 mg/kg body weight/day (Group 1) and 1000 mg/kg body weight/day (Group 2) for 5 days by oral gavage. No mortality, clinical signs, macroscopic findings or organ weight effects were observed at 500 and 1000 mg/kg bw/day. Slightly decreased body weight gain and food consumption was noted at both dose levels (without a dose response).

Based on the available results the parental NOEL is 150 mg/kg bw/day, based on the findings noted at 1000 mg/kg bw/day in the study according to OECD 422.

The parental NOAEL for the source substance 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 during the main study and without any adverse effect during the dose range finding study.

The dose descriptor obtained from the existing combined repeated dose and reproduction/developmental screening study according to OECD 422 with the source substance is considered as an appropriate starting point for deriving a DNEL. In general, based on the diverging molecular weight of the target and the source substance this dose descriptor has to be adjusted. However, due to supporting information from varying sources adjustment of the dose descriptor is scientifically not justified. A justification is given below.

Although alpha methyl glucoside is the core structure of the source substance Isostearic acid, esters with α-methyl glucoside, it is also structurally almost identical to alpha D-glucopyranoside. Alpha D-glucopyranoside is the major carbon source and an indispensable part of the intermediary metabolism in many organisms including rodents and humans (common textbooks of Biochemistry). It was shown that both compounds are absorbed by the same transporter and both, alpha D-glucopyranoside and alpha methyl glucoside are known to be renally excreted. There are published information that indicate that higher doses (3000 mg/kg bw) of alpha methyl glucoside administered to rats via oral gavage do not result in adverse effects within 2h after administration (Demarest, K et al., 2012). Due to the structural similarity to a-D glucose, whose LD50 is > 20000 mg/kg bw in rabbits (Spector, 1955), the LD50 of alpha methyl glucoside, although non-metabolisable, is considered to be in the same order of magnitude. Data about the acute oral toxicity of α-D-glucopyranoside is mainly provided by studies conducted prior to implementation of GLP and/or OECD test-guidelines, i.e. collection of data/handbook data, thus, a detailed documentation of the occurring effects after administration of high doses of α-D-glucopyranoside is lacking. The exceptional high doses of glucose which are required to mediate a toxic effect (deterioration of physiological functions/mortality) suggest that toxicity results mainly from deterioration of cell functions mediated by dehydration. Based on the structural similarity it can be assumed that despite its non-metabolisable nature, alpha methyl glucoside causes the same dehydration symptoms at high doses. Furthermore, it is highly unlikely that other adverse effects occur at low doses of alpha methyl glucoside because it is considered to be excreted unchanged. Local effects were not detected during reliable and relevant irritation studies conducted according to OECD test guideline 439 and 437 with the target substance alpha methyl glucoside, thus, based on all available information an increased risk even during the intermittent workers exposure patterns or due to peak exposures during work is not expected.

Moreover, although alpha methyl glucoside is a non-metabolisable compound, information from other regulatory institutions like US EPA confirm a low toxicity. At the US EPA alpha methyl glucoside is listed on the ‘Safer Chemical Ingredients list’ as chemical that has been verified to be of low concern based on experimental and modeled data. Additionally, the FDA states that alpha methyl glucoside is considered to be safe in certain substances which are polyester resins and are safely used as articles which are intended for repeated use in contact with food.

In summary, the available information provides evidence that alpha methyl glucoside exhibits a low toxicity, thus, adjustment of the descriptor starting point is scientifically not justified.

 

 

References:

1)   Spector, W. S. (1955).HANDBOOK OF TOXICOLOGY. VOLUME 1. NATIONAL ACADEMY OF SCIENCES-NATIONAL RESEARCH COUNCIL WASHINGTON DC.

2)   Brot-Laroche, Edith, et al. "Characterization of the d-glucose/Na+ cotransport system in the intestinal brush-border membrane by using the specific substrate, methyl α-d-glucopyranoside."Biochimica et Biophysica Acta (BBA)-Biomembranes904.1 (1987): 71-80.

3)   Kimmich GA, Randles J: alpha-Methylglucoside satisfies only Na+-dependent transport system of intestinal epithelium. Am J Physiol 241:C227–C232, 1981

4)   Genel, M., London, D., Holtzapple, P. G., & Segal, S. (1971). Uptake of alpha-methylglucoside by normal and diabetic human jejunal mucosa.Translational Research,77(5), 743-750.

5)   Lostao, M. P., et al. "On the multiplicity of glucose analogues transport systems in rat intestine."Revista espanola de fisiologia47.4 (1991): 209-216.

6)   Segal, Stanton, et al. "Transport of alpha-methyl-D-glucoside by human kidney cortex."Metabolism22.1 (1973): 67-76.

7)   Demarest, Keith T., et al. "Methods of using alpha-methylglucoside (AMG) as an indicator for glucose absorption and excretion." U.S. Patent No. 8,147,801. 3 Apr. 2012.