A mixture of: 2-ethylhexyl mono-D-glucopyranoside; 2-ethylhexyl di-D-glucopyranoside

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 February 2002 - 03 June 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: study performed according to standard guideline and under GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of: 2-ethylhexyl mono-D-glucopyranoside; 2-ethylhexyl di-D-glucopyranoside

EC Number:

414-420-0

EC Name:

A mixture of: 2-ethylhexyl mono-D-glucopyranoside; 2-ethylhexyl di-D-glucopyranoside

Cas Number:

125590-73-0

Molecular formula:

(C6H10O5)n, C8H18O n = 1,2

IUPAC Name:

Reaction mass of 2-ethylhexyl mono-D-glucopyranoside and 2-ethylhexyl di-D-glucopyranoside

Details on test material:

Identification: 2-Ethylhexylglucoside
Description: Dark brown liquid
Batch: 0081 43
Purity: Solids: 63.5%; Water: 36.5%
Test substance storage: At room temperature in the dark
Stability under storage conditions: Not Indicated
Expiry date: 16 February 2003

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: females 153-157g, males 191-193g
- Fasting period before study: not applicable
- Housing: 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors (55 x 34 x 21.5 cm height)
- Diet (e.g. ad libitum): Free access to standard pelleted laboratory animal diet (from Altromin (code VRF-1, Lage,Germany). Each batch is analysed for nutrients and contaminants are analysed on a regular basis. Results are examined and archived.
- Water (e.g. ad libitum): Free access to tap water. Certificates of analysis (performed quarterly) were examined and archived.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 05 February 2002 - 03 June 2002

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Method: Formulations (w/w) were prepared daily within 4 hours prior to dosing. The vehicle was heated to approximately 30°C before addition to the test substance, to dissolve crystal formation in the formulation. Once the crystals had dissolved (determined visually) the formulations were left at room temperature until dosing. Adjustment was made for specific gravity of the test substance and purity of the test substance (63.5%).
Vehicle: Water (Milli-U).
Stability in vehicle: At least 4 hours (determined at NOTOX as part of NOTOX project 321751 (a one generation reproduction toxicity study in the
rat)).
Storage conditions: At ambient temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

see NOTOX project 321751, a one generation reproduction toxicity study in the rat

Duration of treatment / exposure:

90 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females. An extra 10 animals per sex in the control and high dose group were allowed 28 days of recovery.

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
50-150-450 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: 28day study
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: no data
- Post-exposure recovery period in satellite groups: 28day
- Section schedule rationale (if not random): no data

Positive control:

not appicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily.
- Cage side observations : Mortality / Viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals, this was
also performed outside the home cage in a standard arena.
The time of onset, degree and duration were recorded.
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: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable, gavage study

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: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.


OPHTHALMOSCOPIC EXAMINATION: Yes
Following instillation of tropicamide solution (5 mglml), both eyes were examined by means of an ophthalmoscope:
at pretest : All animals
at week 13 : Groups 1 and 4
Since no treatment-related findings were noted, no examination was performed on the eyes of group 2 and 3 rats and at the end of the recovery period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled postmortem examination, between 7.30 and 9.30 a.m
- Anaesthetic used for blood collection: Yes (identity), iso-flurane
- Animals fasted: Yes, The animals were fasted overnight (with a maximum of 24 hours) before blood sampling.
- How many animals: all
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled postmortem examination, between 7.30 and 9.30 a.m
- Anaesthetic used for blood collection: Yes (identity), iso-flurane
- Animals fasted: Yes, The animals were fasted overnight (with a maximum of 24 hours) before blood sampling.
- How many animals: all
- Parameters checked in table 1 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 12-1 3 of treatment
- Dose groups that were examined: controla nd high dose recovery group animals
- 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). This test was also performed on group 2 and 3 animals (males and females of each group on separate days) since a dose-related effect was suspected.

OTHER:
ORGAN WEIGHTS
The following organ weights and terminal body weight were recorded from the surviving animals on the scheduled day of necropsy:
Adrenal glands, Ovaries, Brain, Spleen, Epididymides, Testes, Heart , Thymus, Kidneys, Uterus, Liver

Sacrifice and pathology:

GROSS PATHOLOGY: Yes,
All animals surviving to the end of the observation period and all moribund animals were deeply anaesthetised using iso-flurane and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in a 4% formaldehyde solution:
Adrenal glands, Aorta, Brain (cerebellum, mid-brain, cortex), Caecum, (Cervix), (Clitoral gland), Colon, Duodenum, Epididymides, (Eyes with optic nerve and Harderian gland), Female mammary gland area, (Femur including joint), Heart, Ileum, Jejunum, Kidneys, (Larynx), (Lacrimal gland, exorbital), Liver, Lung, infused with formalin, Lymph nodes - mandibular, mesenteric, (Nasopharynx), Oesophagus, 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 including parathyroid, (Tongue), Trachea, Urinary bladder, Uterus, (Vagina), All gross lesions

HISTOPATHOLOGY: Yes,
Tissues mentioned within brackets (above) were not examined as there were no signs of toxicity or target organ involvement.
- all tissues and organs collected at the scheduled sacrifice from all main group 1 and 4
animals;
- all tissues and organs from the animals that died spontaneously;
- all gross lesions of all animals;
were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of thetreated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed tofollow a normal distribution.
- The Fisher-Exact test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. No statistical analysis was performed on motor activity data. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

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):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

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:

no effects observed

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
One recovery group male (no. 56) and one main group female (no. 104) receiving 450 mg/kg/day were found dead on day 29 and 21, respectively. These animals showed laboured respiration, hunched posture andlor piloerection prior to death. Necropsy findings consisted of beginning autolysis, red foci on the lungs andlor red discolouration of the mesenteric lymph nodes. Microscopic examination revealed severe necrosis with exudation of the tracheal mucosa

The following clinical signs that distinguished treated animals from controls:
- Rales (occasionally shown by four males and in eight females at 450 mg/kg/day, and by one female dosed at 150 mg/kg/day
- Laboured respiration (shown by male no. 56 and female no. 104 prior to death, and by two surviving females (nos. 106, 1 17) at 450 mg/kg/ day);
- Hunched posture (shown by two males (nos. 47, 53) during most of the treatment period and recovery phase and by three females (female no. 104 prior to death and by two surviving females (nos. 101, 105) during the last few weeks of treatment at 450 mg/kg/day);
- Lethargy (occasionally shown by two males (nos. 51, 53) at 450 mg/kg/day;
- Gasping (incidentally shown by one female (no. 118)) at 450 mg/kg/day;
- Piloerection (shown by one surviving male (no. 53) and by one female (no. 104) prior to death at 450 mg/kg/day.

All animals dosed at 450 mg/kg/day showed salivation during treatment and incidentally during recovery. This clinical sign is often noted in rats of this age and strain following oral gavage and considered to be related to multiple intra-oesophageal intubation andlor irritant or bad taste of the test substance. In the absence of a microscopic correlate, salivation was considered not to be a sign of toxicity. No reasonable explanation could be given for the occurrence of purple discolouration of the toes or ear shown by two control males and one group 2 male. Other (incidental) findings that were noted included alopecia, scabs, swelling of the ears, a wound on the mouth, focal erythema of the ear, and brown staining of the fur. At the incidence observed these findings are more often noted in rats of this age and strain which are housed and treated under the conditions in this study. Therefore these observations 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 study period.
The statistically significant increase of body weight gain of females dosed at 450 mg/kg/day at week 2 of treatment was considered to have occurred by chance since it was not consistently seen over time and the mean was within the normal range. This change was considered unrelated to treatment.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There were no differences in food consumption before or after allowance for body weight between treated and control animals that were considered to be related to treatment. Slight statistically significant changes occurring in food intake of males and females in week 1 of recovery were considered to have occurred by chance and were absent during treatment. These changes were considered to be of no toxicological significance.

OPHTHALMOSCOPIC EXAMINATION
There were no ophthalmoscopic findings at pre-dose and in week 13.

HAEMATOLOGY
Haematological parameters of treated rats were considered not to have been affected by treatment.

At the end of treatment, a statistically significant increase of mean neutrophil counts was measured for males dosed at 150 or 450 mg/kg/day. This was caused by neutrophilia in individual animals (e.g. nos. 49 and 50) and is a stress-response which is more often noted following administration of a test substance. Reduced mean corpuscular haemoglobin values noted in females dosed at 150 or 450 mg/kg/day were considered to have occurred by chance in the absence of any signs of anaemia. Other changes attaining statistical significance in groups 2 andlor 3 (i.e. prothrombin time values, lymphocyte counts and platelet counts of males, and erythrocyte counts of females) occurred in the absence of a clear dose related response andlor values were within the normal range.

The significant reduction of mean corpuscular volume and mean corpuscular haemoglobin values of high dose males at the end of recovery was considered incidental in the absence of similar changes at the end of the treatment period.

CLINICAL CHEMISTRY
Total protein values were significantly higher in males treated at 450 mg/kg/day.

The statistically significant reduction of glucose values in high dose males at the end of recovery was considered to be incidental in the absence of similar changes at the end of the treatment period. Also, high dose glucose values of males were within normal range at the end of recovery and treatment periods. Lower glucose values and increased potassium values of females dosed at 150 or 450 mg/kg/day at the end of treatment did not show a dose-related response and means were considered to be normal. Similarly, no dose-related trend was noted for the apparent increase of inorganic phosphate levels of treated females at the end of treatment, while control values of this parameter were slightly low. Statistically significant decreases of alkaline phosphatase activity, urea and creatinine values among females dosed at 50 or 150 mg/kg/day did not show a dose-response. The reduction of alanine and aspartate aminotransferase activity values in treated females at the end of treatment was considered to be related to increased individual control values. Several plasma samples among all dose groups were found to be haemolytic, which was considered to be due to the blood sampling procedure. Since the nature of this finding was slight, interpretation of the clinical biochemistry data was considered not to have been adversely affected

NEUROBEHAVIOUR
No changes were observed in hearing ability, pupillary reflex, static righting reflex and grip strength in the animals treated with 2-Ethylhexylglucoside, when compared to control animals. The variation in motor activity was not related to the dose and did not indicate a relation with treatment. Some slightly high motor activity values were obtained for some high dose females (nos 112, 115 and 117: low sensors). Since this did not form part of a group- and dose-response and did not correlate to clinical signs in this group, no toxicological significance was attributed to these changes.

ORGAN WEIGHTS
Organ weights and organ:body weight ratios of treated animals were considered to be similar to those of control animals.
The statistically significant reduction of absolute andlor relative liver weights of females dosed at 150 or 450 mg/kg/day was not related to the dose and had no morphological correlate. The reduced absolute thymus weight and increased relative brain weight in females at 150 mg/kg/day was considered incidental in the absence of similar changes at the highest dose level. The increased absolute and relative adrenal weights in females treated with 450 mg/kg/day at the end of the recovery period was absent at the end of treatment and supportive structural lesions in the adrenals were absent. These changes were therefore considered not to be toxicologically relevant.

GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
Incidental findings among surviving control and treated animals included red discolouration of the lungs, thymus andlor mandibular lymph nodes, an enlarged mandibular lymph node, flaccid testes, scabs, alopecia, greenish nodules on the epididymides, a reduced size of the epididymides or seminal vesicles, fluid in the uterus (related to a stage in the oestrous cycle) and watery cysts on the uterus. These findings are occasionally seen among rats used in these types of studies. At the incidence observed they were considered changes of no toxicological significance.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no microscopic findings recorded which could be attributed to treatment with the test substance.
All microscopic findings were within the range of background pathology encountered.in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

Effect levels

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Treatment-related findings observed were as follows: 50 mg/kg/day no treatment related findings were noted. At 150 mg/kg/day rales were observed in one female. At 450 mg/kg/day two animals were found dead. Increased total protein values were observed in females and breathing difficulties (rales, laboured respiration, gasping), hunched posture, piloerection, lethargy were observed in males and females. Rales shown by a single female at 150 mg/kg/day occurred in the absence of other functional or morphological disturbances. Therefore, a definitive No Observed Adverse Effect Level (NOAEL) for 2-Ethylhexylglucoside of 150 mg/kg/day was established.

Executive summary:

90 -Day oral toxicity study with 2-Ethylhexylglucoside by daily gavage in the rat followed by a 28- day recovery period. The dose levels for this 90-day oral gavage study were selected to be 0, 50, 150 and 450 mg/kg/day, based on results of a 28-day oral gavage study by daily gavage in the rat and on a one generation reproduction toxicity study in the rat (NOTOX project 321751) with 2-Ethylhexylglucoside. The study was based on the following guidelines. - EC Directive 67/548/EEC, B Repeated Dose (90 days) Toxicity (oral), 2001. - OECD 408, Repeated Dose 90-day Oral Toxicity Study in Rodents, 1998. - OPPTS 870.3100 "90-day oral toxicity", Public Draft June 1996. The test substance was administered daily for at least 90 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females. An extra 10 animals per sex in the control and high dose group were allowed 28 days of recovery. The following parameters were evaluated: Clinical signs, functional observations, ophthalmoscopy, body weight and food consumption. At termination: clinical pathology, macroscopy, organ weights and histopathology on a selection of tissues.

RESULTS Formulations: Accuracy, homogeneity and stability over at least 4 hours of formulations of test substance in water (Milli-U) were demonstrated by analyses. Treatment-related findings observed were as follows: 50 mg/kg/day no treatment related findings were noted. At 150 mg/kg/day rales were observed in one female. At 450 mg/kg/day two animals were found dead. Increased total protein values were observed in females and breathing difficulties (rales, laboured respiration, gasping), hunched posture, piloerection, lethargy were observed in males and females.

CONCLUSION Rales shown by a single female at 150 mg/kg/day occurred in the absence of other functional or morphological disturbances. Therefore, a definitive No Observed Adverse Effect Level (NOAEL) for 2-Ethylhexylglucoside of 150 mg/kg/day was established.