D-Xylopyranose, oligomeric, 2-octyldodecyl xylosides

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

APX 20P

IUPAC Name:

APX 20P

Details on test material:

Sponsor's identification : APX20P
Description : dark orange solid block
Purity : >99%
Batch number : 1137JG
Label : APX 20 P Lot: 1137JG
Date received : 20 November 2006
Storage conditions : room temperature in the dark

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

A sufficient number of male and female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats were
obtained from Charles River (UK) Limited, Margate, Kent. On receipt the animals were
examined for signs of ill-health or injury. The animals were acclimatised for 8 days during which
time their health status was assessed. A total of forty animals (twenty males and twenty females)
were accepted into the study. At the start of treatment the males weighed 156 to 185g, the
females weighed 139 to 167g, and were approximately 6 to 8 weeks old.
The animals were housed in groups of five by sex in polypropylene grid-floor cages suspended
over trays lined with absorbent paper. The animals were allowed free access to food and water.
A pelleted diet (Rodent 5LF2 (Certified) Diet, BCM IPS Limited, London, UK) was used.
Certificates of analysis of the batches of diet used are given in Appendix 14. Mains drinking
water was supplied from polycarbonate bottles attached to the cage. The diet and drinking water
were considered not to contain any contaminant at a level that might have affected the purpose or
integrity of the study. Environmental enrichment was provided in the form of wooden chew
blocks (B & K Universal Ltd., Hull, UK) and cardboard fun tunnels (Datesand Ltd., Cheshire,
UK).
The animals were housed in a single air-conditioned room within the Safepharm Barrier
Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and
the low intensity fluorescent lighting was controlled to give twelve hours continuous light and
twelve hours darkness. Environmental conditions were continuously monitored by a
computerised system, and print-outs of hourly mean temperatures and humidities were included in
the study records. The temperature and relative humidity controls were set to achieve target
values of 21 ± 2ºC and 55 ± 15% respectively.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

The test material was administered daily, for twenty-eight consecutive days, by gavage using a
stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an
identical manner with 4 ml/kg/day of Arachis oil BP.
The volume of test and control material administered to each animal was based on the most recent
bodyweight and was adjusted at weekly intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Summary
The concentration of APX20P in the test material formulations was determined by gas
chromatography (GC) using an external standard technique.

Samples
The test material formulations were extracted with methanol to give a final, theoretical test
material concentration of approximately 0.1 mg/ml.

Standards
Standard solutions of test material were prepared in methanol at a nominal concentration of 0.1
mg/ml.

Procedure
The standard and sample solutions were analysed by GC using the following conditions:
GC system : Agilent Technologies 5890, incorporating
autosampler and workstation
Column
: DB-1 (30 m x 0.32 mm id x 0.25 μm film)
Oven temperature program
: initial 180 ºC for 0 mins
rate 10 ºC/min
final 260 ºC for 0 mins
Injection temperature
: 250 ºC
Flame ionisation detector
temperature : 250ºC
Injection volume
: 1 μl
Retention time : ~ 4 mins

Homogeneity Determinations
The test material formulations were mixed thoroughly and samples were taken from the top,
middle and bottom of the container, shaking between sampling. Sampling was performed in
triplicate.

Stability Determinations
The test material formulations were sampled and analysed initially and then after storage at
approximately +4ºC in the dark for fourteen days.

The test material formulations were sampled and analysed within three days of preparation.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
15 mg/ kg bw/day
Basis:
other: dose level

Doses / concentrations 2

Remarks:

Doses / Concentrations:
150 mg/ kg bw/day
Basis:
other: dose level

Doses / concentrations 3

Remarks:

Doses / Concentrations:
750 mg /kg bw /day
Basis:
other: dose level

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

Clinical Observations
All animals were examined for overt signs of toxicity, ill-health or behavioural change
immediately before dosing, immediately post dosing and one and five hours after dosing during
the working week. Animals were observed immediately before and after dosing and one hour
after dosing at weekends and public holidays. All observations were recorded.

Functional Observations
Prior to the start of treatment and on Days 5, 12, 19 and 27, all animals were observed for signs of
functional/behavioural toxicity. Functional performance tests were also performed on all animals
during Week 4, together with an assessment of sensory reactivity to different stimuli.
Observations were carried out from approximately two hours after dosing on each occasion.
- Behavioural Assessments
Detailed individual clinical observations were performed for each animal using a purpose built
arena. The following parameters were observed:
Gait Hyper/Hypothermia
Tremors Skin colour
Twitches Respiration
Convulsions Palpebral closure
Bizarre/Abnormal/Stereotypic behaviour Urination
Salivation Defecation
Pilo-erection Transfer arousal
Exophthalmia Tail elevation
Lachrymation
The scoring system used is outlined in The Key to Scoring System and Explanation for
Behavioural Assessments and Sensory Reactivity Tests.
- Functional Performance Tests
Motor Activity. Twenty purpose built 44 infra-red beam automated activity monitors were used to
assess motor activity. Animals of one sex were tested at each occasion and were randomly
allocated to the activity monitors. The evaluation period was thirty minutes for each animal. The
time in seconds each animal was active and mobile was recorded for the overall thirty minute
period and also during the final 20% of the period (considered to be the asymptotic period).
Forelimb/Hindlimb Grip Strength. An automated grip strength meter was used. Each animal was
allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by
the base of the tail until its grip was broken. The animal was drawn along the trough of the meter
by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of
the tail until its grip was broken. A record of the force required to break the grip for each animal
was made. Three consecutive trials were performed for each animal.
- Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and
proprioceptive stimuli. The scoring system used is outlined in The Key to Scoring System and
Explanation for Behavioural Assessments and Sensory Reactivity Tests.
The following parameters were observed:
Grasp response Touch escape
Vocalisation Pupil reflex
Toe pinch Blink reflex
Tail pinch Startle reflex
Finger approach

Bodyweight
Individual bodyweights were recorded on Day 1 and at weekly intervals thereafter. Bodyweights
were also performed prior to terminal kill.

Food Consumption
Food consumption was recorded for each cage group at weekly intervals throughout the study.

Water Consumption
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for
any overt changes, except during the final two weeks of the study when gravimetric
measurements were undertaken by the daily weighing of water bottles.

Sacrifice and pathology:

Pathology
On completion of the dosing period all animals were killed by intravenous overdose of sodium
pentobarbitone followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic
abnormalities were recorded.
- Organ Weights
The following organs, removed from animals that were killed at the end of the study, were
dissected free from fat and weighed before fixation:
Adrenals Liver
Brain Ovaries
Epididymides Spleen
Heart Testes
Kidneys Thymus

- Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10%
formalin:
Adrenals Oesophagus
Aorta (thoracic) Ovaries
Bone & bone marrow (femur including stifle joint) Pancreas
Bone & bone marrow (sternum) Pituitary
Brain (including cerebrum, cerebellum and pons) Prostate
Caecum Rectum
Colon Salivary glands (submaxillary)
Duodenum Sciatic nerve
Epididymides Seminal vesicles
Eyes Skin (hind limb)
Gross lesions Spinal cord (cervical)
Heart Spleen
Ileum Stomach
Jejunum Testes
Kidneys Thymus
Liver Thyroid/parathyroid
Lungs (with bronchi) # Trachea
Lymph nodes (cervical and mesenteric) Urinary bladder
Muscle (skeletal) Uterus
All tissues were despatched to HistologiX Ltd, BioCity Pennyfoot Street, Nottingham, UK for
processing (Principal Investigator: M Dow). The tissues shown in bold from all control
and 750 mg/kg/day dose group animals were prepared as paraffin blocks, sectioned at nominal
thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic
examination. Any macroscopically observed lesions were also processed together with the liver
and spleen from all 15 and 150 mg/kg/day dose group animals.
Microscopic examination was conducted by the Study Pathologist. All findings were entered into
the ROELEE Pathology computerisation system for tabulation and report production.

Other examinations:

Laboratory Investigations
Haematological and blood chemical investigations were performed on all animals from each test
and control group at the end of the study (Day 28). Blood samples were obtained from the lateral
tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy
on Day 29. Animals were not fasted prior to sampling.
- Haematology
The following parameters were measured on blood collected into tubes containing potassium
EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices - mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Cresyl blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by ‘Thrombomax HS with calcium’ and Activated partial
thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium
citrate solution (0.11 mol/l).

- Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing
lithium heparin anti-coagulant:
Urea Calcium (Ca++)
Glucose Inorganic phosphorus (P)
Total protein (Tot.Prot.) Aspartate aminotransferase (ASAT)
Albumin Alanine aminotransferase (ALAT)
Albumin/Globulin (A/G) ratio (by calculation) Alkaline phosphatase (AP)
Sodium (Na+) Creatinine (Creat)
Potassium (K+) Total cholesterol (Chol)
Chloride (Cl-) Total bilirubin (Bili)

Statistics:

Evaluation of Data
Data were processed to give group mean values and standard deviations where appropriate.
All data was summarised in tabular form. Where appropriate, quantitative data were analysed by
the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation
of the data was found, the use of possible covariates checked and the homogeneity of means
assessed using ANOVA or ANCOVA and Bartlett’s test. The transformed data were analysed to
find the lowest treatment level that showed a significant effect, using the Williams Test for
parametric data or the Shirley Test for non-parametric data. If no dose response was found, but
the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett
(parametric) or Steel (non-parametric) test to determine significant differences from the control
group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or
the Mann-Whitney U test (non-parametric).
Probability values (p) are presented as follows:
p < 0.01 **
p < 0.05 *
p ≥ 0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no unscheduled deaths during the study.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No adverse effects on dietary intake was detected for control or treated animals.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Details on results:

Clinical Observations
Sporadic incidents of increased salivation were detected immediately after dosing for animals of either sex treated with 750 or 150 mg/kg/day, from Day 3 onwards for 750 mg/kg/day females and from Day 8 onwards for 750 or 150 mg/kg/day males and for 150 mg/kg/day females. This
was coupled, for 750 and 150 mg/kg/day animals with sporadic episodes of generalised red/brown staining plus isolated incidents of wet fur for animals of either sex treated with 750 mg/kg/day.
One female treated with 750 mg/kg/day had hunched posture on Day 21; another female of the same treatment group had a tip-toe gait on Day 27 only. As these observations were isolated in nature, they were considered incidental and not to be of any toxicological significance. One control male had a damaged tail tip from Day 17 onwards; this was a physical injury and is unrelated to treatment.
No such observations were detected for animals of either sex treated with 15 mg/kg/day.

Bodyweight
A statistical significant reduction in bodyweight development was detected for males treated with 750 mg/kg/day during Week 2 and Week 3 of the study when compared to controls.
No such observations were detected for females treated with 750 mg/kg/day or for animals of either sex treated with 150 or 15 mg/kg/day.

Water Consumption
Although visual inspection of water bottles did not reveal any overt intergroup differences, an increase in water consumption was detected at 750 mg/kg/day during gravimetric analysis carried out during the final two weeks of the treatment period. This comprised of approximately 14%
increase in water consumption detected for males treated at this dose and approximately 52% increase in water intake for 750 mg/kg/day females when compared to controls during Weeks 3 and 4.
No such observations were detected for animals of either sex treated with 150 or 15 mg/kg/day.

Haematology
There were no toxicologically significant changes detected in the haematological parameters measured.
A statistically significant increase in erythrocyte count, haemoglobin and haematocrit was detected for females treated with 750 mg/kg/day. In the absence of histopathological evidence to suggest a treatment related effect of the heamopoetic system, these findings are considered not to be related to systemic toxicity; but are considered to be a consequence of treatment.
An increase in erythrocyte count was detected for all treated males. In addition platelet count was increased for males treated with 750 or 150 mg/kg/day and for females treated with 750 mg/kg/day, whilst mean cell haemoglobin concentration was decreased for 750 mg/kg/day males. All individual values for these parameters were within the normal range for rats of the strain and age employed, and in the absence of histopathological correlates, these findings were considered unrelated to test material toxicity.
A statistically significant decrease in total leucocyte count, specifically in the lymphocyte fraction, was detected for all treated females. Decreases in these parameters are not usually associated with test material toxicity and in the absence of any histopathological correlates to
suggest spleenic changes; these findings were considered incidental and unrelated to test material toxicity.

Blood Chemistry
Animals of either sex treated with 750 mg/kg/day showed statistically significant elevations in albumin, albumin/globulin ratio, aspartate aminotransferase and alanine aminotransferase. In addition females of this treatment group showed elevations in total protein. The elevation in albumin, albumin/globulin ratio and alanine aminotransferase was also detected for females treated with 150 mg/kg/day. A decrease in bilirubin was detected for animals of either sex treated with 750 mg/kg/day and for males treated with 150 mg/kg/day.
A statistically significant increase in creatinine and a decrease in cholesterol were detected for females treated with 750 mg/kg/day. The majority of values for creatinine and all individual values for cholesterol were within the normal range for rats of the strain and age used, as such;
these findings were considered unrelated to systemic toxicity.
A statistically significant decrease in creatinine was decreased for males treated with 15 mg/kg/day. As this decrease was not detected for 750 or 150 mg/kg/day males a dose related response was not detected, therefore, this finding was considered incidental and unrelated to
treatment.
No such effects were detected for females treated with 15 mg/kg/day.

Organ Weights
A statistically significant elevation in absolute liver weight and liver weight related to terminal
bodyweight was detected for animals of either sex treated with 750 and 150 mg/kg/day.
All treated males showed elevations in absolute adrenal weight and adrenal weight relative to
terminal bodyweight. All individual values for adrenal weight for males treated with 750 or
150 mg/kg/day and for the majority of individual values for males treated with 15 mg/kg/day were
within the normal range for rats of the strain and age employed. As such, and in the absence of a
dose related response, this finding was considered incidental and unrelated to treatment.
No such effects were detected for females treated with 150 or 15 mg/kg/day.

Histopathology
The following treatment related effects were detected:
LIVER: Hepatocyte enlargement, centrilobular in distribution for males and centrilobular or
generalised for females, was observed in relation to treatment for animal of either sex treated with
750 mg/kg/day or 150 mg/kg/day but not at 15 mg/kg/day.
All remaining morphological changes were those commonly observed in laboratory maintained
rats of the age and strain employed, and there were no differences in incidence or severity
between control and treatment groups that were considered to be of toxicological significance.

Effect levels

1

Key result

Dose descriptor:

NOEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

water consumption and compound intake

2

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

water consumption and compound intake

Remarks on result:

other: See "Remark"

Remarks:

Oral administration of the test material, to rats for a period of up to twenty eight consecutive days at dose levels of up to 750 mg/kg/day resulted in blood chemical changes; elevations in liver weight and microscopic evidence of adaptive changes in the liver for animals of either sex treated with 750 or 150 mg/kg/day. The ‘No Observed Effect Level’ for animals of either sex was considered to be 15 mg/kg/day. As the treatment related effects detected at 750 or 150 mg/kg/day were considered adaptive in nature, the ‘No Observed Adverse Effect Level’ (NOAEL) was considered to be 750 mg/kg/day for animals of either sex.

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (actual dose received)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Applicant's summary and conclusion

Conclusions:

Oral administration of the test material, APX20P, to rats for a period of up to twenty eight consecutive days at dose levels of up to 750 mg/kg/day resulted in blood chemical changes; elevations in liver weight and microscopic evidence of adaptive changes in the liver for animals of either sex treated with 750 or 150 mg/kg/day.
The ‘No Observed Effect Level’ for animals of either sex was considered to be 15 mg/kg/day. As the treatment related effects detected at 750 or 150 mg/kg/day were considered adaptive in nature, the ‘No Observed Adverse Effect Level’ (NOAEL) was considered to be 750 mg/kg/day for animals of either sex.

Executive summary:

Introduction.

The study was designed to investigate the systemic toxicity of the test material. It complies with the requirements for notification of a new chemical substance in the EC and follows the testing method described in Commission Directive 96/54/EC (Method B7) and OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (Adopted 27 July 1995).

Methods.

The test material was administered by gavage to three groups, each of five male and five female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for twenty-eight consecutive days, at dose levels of 15, 150 and 750 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP). Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed.

Results.

Mortality. There were no treatment-related deaths during the study.

Clinical Observations. Sporadic incidents of increased salivation were detected immediately after dosing for animals of either sex treated with 750 or 150 mg/kg/day. This was coupled for 750 and 150 mg/kg/day animals with sporadic episodes of generalised red/brown staining and isolated incidents of wet fur for animals of either sex treated with 750 mg/kg/day. No such observations were detected for animals of either sex treated with 15 mg/kg/day.

Behavioural Assessment. No treatment-related effects were detected. Functional Performance Tests. No treatment-related effects were detected. Sensory Reactivity Assessments. There were no treatment-related changes in sensory reactivity.

Bodyweight. A reduced bodyweight development was detected for males treated with 750 mg/kg/day during Week 2 and Week 3 of the study when compared to controls. No such observations were detected for females treated with 750 mg/kg/day or for animals of either sex treated with 150 or 15 mg/kg/day.

Food Consumption. A reduction in food efficiency was detected for animals of either sex treated with 750 mg/kg/day from Week 2 for males and from Week 3 for females. No such observations were detected for animals of either sex treated with 150 or 15 mg/kg/day.

Water Consumption. An increase in water consumption was detected for animals of either sex treated with 750 mg/kg/day during the final two weeks of the treatment period for males and during the final week of treatment for the females. No such observations were detected for animals of either sex treated with 150 or 15 mg/kg/day.

Haematology. No toxicologically significant effects were detected.

Blood Chemistry. Animals of either sex treated with 750 mg/kg/day showed elevations in albumin, albumin/globulin ratio, aspartate aminotransferase and alanine aminotransferase. In addition females of this treatment group showed elevations in total protein. The elevation in albumin, albumin/globulin ratio and alanine aminotransferase was also detected for females treated with 150 mg/kg/day. In addition a decrease in bilirubin was detected for animals of either sex treated with 750 mg/kg/day and for males treated with 150 mg/kg/day. No treatment related effects were detected for animals of either sex treated with 15 mg/kg/day.

Organ Weights. An elevation in absolute liver weight and liver weight relative to terminal bodyweight was detected for animals of either sex treated with 750 or 150 mg/kg/day. No such observations were detected for animals of either sex treated with 15 mg/kg/day.

Necropsy. No treatment-related macroscopic abnormalities were detected.

Histopathology. The following treatment-related changes were observed: LIVER: Hepatocyte enlargement, centrilobular in distribution for males and centrilobular or generalised for females, was observed in relation to treatment for animal of either sex treated with 750 mg/kg/day or 150 mg/kg/day but not at 15 mg/kg/day.

Conclusion.

Oral administration of the test material, APX20P, to rats for a period of up to twenty eight consecutive days at dose levels of up to 750 mg/kg/day resulted in blood chemical changes; elevations in liver weight and microscopic evidence of adaptive changes in the liver for animals of either sex treated with 750 or 150 mg/kg/day.

The ‘No Observed Effect Level’ for animals of either sex was considered to be 15 mg/kg/day. As the treatment related effects detected at 750 or 150 mg/kg/day were considered adaptive in nature, the ‘No Observed Adverse Effect Level’ (NOAEL) was considered to be 750 mg/kg/day for animals of either sex.