Registration Dossier

Administrative data

Description of key information

Subacute (28 day) study oral (gavage), rat (Wistar-Han) m/f (OECD guideline 422, GLP): NOAEL >=100mg/kg bw/day (actual dose received)(m/f)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with GLP and OECD guideline 422
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
target range for relative humidity exceeded for extended periods due to adverse weather conditions. It is considered that these have had no adverse impact on the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.
- Age at study initiation:approximately twelve weeks old
- Weight at study initiation: males 306 to 363g, females 207 to 235g,
- Fasting period before study: No
- Housing: Initially, all animals were housed in groups of three in solid floor polypropylene cages with
stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the
pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays
lined with absorbent paper on a one male: one female basis within each dose group. Following
evidence of successful mating, the males were returned to their original cages. Mated females
were housed individually during gestation and lactation in solid floor polypropylene cages with
stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK. ad libitum
- Water (e.g. ad libitum): Mains drinking water ad libitum

- Acclimation period: seven days during which time their health status was assessed

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.

IN-LIFE DATES: From: To: 10 June 2014-
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
For the purpose of this study the test item was prepared at the appropriate concentrations as a
suspension in Arachis oil BP. The stability and homogeneity of the test item formulations were
determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services as part of this study.
Results show the formulations to be stable for at least twenty days. Formulations were therefore
prepared weekly and stored at approximately 4 °C in the dark.

Samples of the test item formulation were taken and analyzed for concentration of Diacetate
(CAS: 24085-06-1) at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The
method used for analysis of formulations and the results obtained are given in Appendix 25. The
results indicate that the prepared formulations were within 100-106% of the nominal
concentration.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Standard gavage solvent- formulations found to be stable for at least twenty days.
- Concentration in vehicle: 0, 10, 15, 20 mg/mL
- Amount of vehicle (if gavage): 5mL/kg
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the test samples was determined using HPLC with UV detection using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.
The test item described in the study was also used as the analytical standard.

Preparation of standard solutions: Stock solutions of test iitem in acetonitrile were prepared for external standard calibration: 50 mg in 100100 acetonitrile to yield a concentraiton of 0.5mg/mL. Aliquots of this stock solution were used to prepare working standard solutions of 0.1mg/mL in acetonitrile.

Sample Analysis: received formulaitons were extracted into acetonitrile; aliquots of formulaiton were weighed and brought to volume using acetonitrile before sonication (15 mins) and centrifugation (4500rpm, 10 min). the samples were diluted further with acetonitrile if neccessary to acheive working concentraitons.

Accuracy samples: smaples of arachis oil were fortified with known amounts of the test substance to the highest and lowest anticipated dose concnetraitons; then prepared as for sample analysis above.

Linearity Standards: a range of standard solutions were prepared from 0.598 mg/mL by serial dilution to cover 0-0.1794 mg/mL

Instrument setup:
HPLC: Agilent Technologies 1200 w autosampler
Column: Prodigy 5µODS 2 (250x4.6 mm id)
column temp 40oC
Mobiile phase: Acetonitrile water (60:40 v/v)
Flow rate: 1mL/min
UV detector: 246 nm
injeciton volume 5µL
Retention time ~4.5 min

samples stored at room temperature until analysis

Duration of treatment / exposure:
Males: 44 days
Females: 41-55 days
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
50 mg/kg
Basis:
other: The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Remarks:
Doses / Concentrations:
75 mg/kg
Basis:
other: The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Remarks:
Doses / Concentrations:
100 mg/kg
Basis:
other: The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
No. of animals per sex per dose:
12 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were chosen, in collaboration with the sponsor, based on available toxicity data
including a rat fourteen day ranger finder toxicity study (Harlan Laboratories Study No.:
41401388). In this preliminary study, macroscopic stomach findings indicating gastric irritation
were observed at 125 and 250 mg/kg bw/day and also for one animal at 75 mg/kg bw/day. It
was considered that these stomach findings precluded dosages of 125 and 250 mg/kg bw/day
from being used as the high dosage for further repeat dose toxicity studies. A dosage of
100 mg/kg bw/day was considered suitable when utilized in combination with an increase in
dosage volume to 5 ml/kg body weight in order to minimize the potential local irritation within
the stomach.
- Rationale for animal assignment (if not random): The animals were randomly allocated to treatment groups using a stratified body weight
randomization procedure and the group mean body weights were then determined to ensure
similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system
routinely used in these laboratories.

Positive control:
Not applicable
Observations and examinations performed and frequency:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change
immediately before dosing, soon after dosing, and one hour after dosing during the working
week, at weekends (except for females during parturition where applicable). All observations
were recorded.

Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for
signs of functional/behavioral toxicity. Functional performance tests were also performed on
five selected males and females from each dose level, prior to termination, together with an
assessment of sensory reactivity to various stimuli.


Behavioral 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 color
Twitches Respiration
Convulsions Palpebral closure
Bizarre/Abnormal/Stereotypic behavior Urination
Salivation Defecation
Pilo-erection Transfer arousal
Exophthalmia Tail elevation
Lachrymation

This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The
scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral
Assessments and Sensory Reactivity Tests.


Functional Performance Tests
Motor Activity. Purpose-built 44 infra-red beam automated activity monitors were used to assess
motor activity. Animals were randomly allocated to the activity monitors. The tests were
performed at approximately the same time on each occasion (at least two hours after dosing),
under similar laboratory conditions. The evaluation period was thirty minutes for each animal.
The percentage of time 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, Reiter and Macphail, 1979).

Forelimb/Hindlimb Grip Strength. An automated 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. The assessment was developed
from the method employed by Meyer et al (1979).

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and
proprioceptive stimuli. This assessment was developed from the methods employed by Irwin
(1968) and Moser et al (1988).

The following parameters were observed:

Grasp response Touch escape
Vocalization Pupil reflex
Toe pinch Blink reflex
Tail pinch Startle reflex
Finger approach


Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males
until termination and weekly for females until pairing. During pairing phase females were
weighed daily until mating was confirmed. Body weights were then recorded for females on
Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Body weights were also
recorded at terminal kill.

Normal range data for body weight changes in pregnant and lactating females are shown in
Appendix 32.


Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults.
This was continued for males after the mating phase. For females showing evidence of mating,
food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20.
For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively
for males throughout the study period (with the exception of the mating phase) and for females
during the pre-pairing phase. Due to offspring growth and milk production, food efficiency
could not be accurately calculated for females during gestation and lactation.

Normal range data for pregnant and lactating females are presented in Appendix 32.

Water Consumption
Water intake was measured daily during the pre-pairing phase of the study.

Laboratory Investigations
Hematological and blood chemical investigations were performed on five males and five females
selected from each test and control group prior to termination (Day 42 for males and Day 4 post
partum for females). Blood samples were obtained from the lateral tail vein. Where necessary
repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to
sampling.

The methods used for hematological and blood chemical investigations are presented in
Appendix 34.


Hematology
The following parameters were measured on blood collected into tubes containing potassium
EDTA anti-coagulant:

Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices - mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes
were not assessed

Prothrombin time (CT) was assessed by ‘Innovin’ 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)
Bile acids



Sacrifice and pathology:
3.4.8.1 Necropsy
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by
exsanguination on Day 43 or Day 44. Adult females were killed by intravenous overdose of
suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving
offspring were terminated via intracardiac overdose of suitable barbiturate agent. Any females
which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum

For all females, the uterus was examined for signs of implantation and the number of uterine
implantations in each horn was recorded. This procedure was enhanced; as necessary, by
staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora
lutea were also counted.

All adult animals and offspring, including those dying during the study, were subjected to a full
external and internal examination, and any macroscopic abnormalities were recorded.

3.4.8.2 Organ Weights
The following organs were dissected free from fat and weighed before fixation from five
selected males and five selected females from each dose group. Tissues shown in bold were
weighed from all remaining animals:

Adrenals
Prostate
Brain
Seminal vesicles
Epididymides
Spleen
Heart
Testes
Kidneys
Thymus
Liver
Thyroid (weighed post-fixation with Parathyroid)
Ovaries
Uterus (weighed with Cervix)
Pituitary (post fixation)


Normal ranges for organ weights are given in Appendix 35.


3.4.8.3 Histopathology
Samples of the following tissues were removed from five selected males and five selected
females from each dose group and preserved in buffered 10% formalin, except where stated.
Tissues shown in bold were preserved from all remaining animals:

Adrenals Muscle (skeletal)
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
Coagulating gland
Salivary glands (submaxillary)
Colon Sciatic nerve
Duodenum
Seminal vesicles
Epididymides
Skin (hind limb)
Esophagus
Spinal cord (cervical, mid-thoracic and lumbar
Eyes*
Gross lesions
Spleen
Heart Stomach
Ileum (including peyer’s patches) Thyroid/parathyroid
Jejunum Trachea
Kidneys
Testes
Liver Thymus
Lungs (with bronchi) #
Urinary bladder
Lymph nodes (mandibular and mesenteric)
Uterus/Cervix
Mammary gland Vagina

 = preserved in Modified Davidsons fluid
* = eyes fixed in Davidson’s fluid

Tissues were dispatched to the Test Site (Propath UK Ltd, Willow Court, Netherwood Road,
Rotherwas, Hereford, HR2 6JU) for processing (Principal Investigator: N Fower). The tissues
from five selected control and 100 mg/kg bw/day dose group animals, any animals dying during
the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as
paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin
for subsequent microscopic examination. The tissues shown in bold from the remaining control
and 100 mg/kg bw/day animals and animals which did not achieve a pregnancy were also
processed. In addition, sections of testes from all control and 100 mg/kg bw/day males were also
stained with Periodic Acid-Schiff (PAS) stain and examined.

Detailed qualitative examination of the testes was undertaken, taking into account the tubular
stages of the spermatogenic cycle. The examination was conducted in order to identify
treatment-related effects such as missing germ cell layers or types, retained spermatids,
multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.
Any cell-or stage-specificity of testicular findings was noted.

Since there were indications of treatment-related changes in the stomach of adult animals in the
high dosage group at necropsy, examination was extended to include similarly prepared sections
of the stomach from all adult animals where retained.

Microscopic examination was conducted by the Study Pathologist (J Wilson at Propath GmbH,
Muttenzerstrasse 30, 4133 Pratteln, Switzerland). A peer review of the results was conducted by
the Test Facility. A complete histopathology phase report is presented in Appendix 24 and
represents the consensus opinion of the relevant pathologists.

Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the
significance of intergroup differences from control; statistical significance was achieved at a
level of p<0.05. Statistical analysis was performed on the following parameters:

Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during
gestation and lactation,, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.

Data were analyzed using Provantis TM Tables and Statistics Module:

Appropriate data transformations were performed using the most suitable method. The
homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup
variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate
covariates. Any transformed data were analyzed to find the lowest significant treatment level:
Williams Test - parametric data
Shirley Test- nonparametric data.

Data not analyzed by the Provantis data capture system were assessed using the R
Environment for Statistical Computing:
the distribution of the data was assessed by the Shapiro-Wilk normality test, and the homogeneity of the data using
Bartlett's test.
Parametric analysis of the data was applied using analysis of variance (ANOVA), with Dunnett's post test. Non-parametric analysis of the data was performed incorporating the Kruskal-Wallis test with a Mann-Whitney "U" posttest. Where the data was unsuitable for these analyses then 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)
Clinical signs:
no effects observed
Description (incidence and severity):
post dosing salivation for both sexes at 100 mg/kg bw/day, and to a lesser extent, males at 75 mg/kg bw/day
Mortality:
no mortality observed
Description (incidence):
post dosing salivation for both sexes at 100 mg/kg bw/day, and to a lesser extent, males at 75 mg/kg bw/day
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
100 mg/kg bw/day: Overall body weight gain at the end of treatment was 17 % lower than controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no obvious effect of treatment on food consumption or food conversion efficiency for both sexes at 50, 75 or 100 mg/kg bw/day.
Food efficiency:
no effects observed
Description (incidence and severity):
There was no obvious effect of treatment on food consumption or food conversion efficiency for both sexes at 50, 75 or 100 mg/kg bw/day.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Gravimetric assessment of water bottles during the pre-pairing phase did not reveal any consistent intergroup differences that indicated an effect of treatment on water consumption.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There was no clear effect of treatment on blood chemistry parameters at 50, 75 or 100 mg/kg bw/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no treatment-related changes in the behavioral, functional performance or sensory reactivity parameters at 50, 75 or 100 mg/kg bw/day.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
These were adverse effects of treatment on absolute or body weight relative organ weights for either sex at 50, 75 or 100 mg/kg bw/day.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
A plethora of macroscopic observations in the stomach of both sexes at all dosages were apparent at necropsy.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Non-Glandular and Glandular Stomach:
Histopathological findings: neoplastic:
not examined
Details on results:
Mortality
At 100 mg/kg bw/day, one male (number 80) was killed for animal welfare considerations due to
the severity of clinical signs apparent on Day 20. Clinical signs included laboured respiration,
decreased respiratory rate, prostration and clonic convulsions; no clinical signs had been
observed prior to this event. Necropsy observations for this animal consisted of thickening of
the glandular region of the stomach and multiple raised areas of the non-glandular region of the
stomach.

In the absence of similar adverse clinical reactions to treatment in the remaining animals at this
dosage, this death was most probably incidental and unrelated to treatment; although an
association with the local irritation effects in the stomach cannot be discounted.
There were no further unscheduled deaths.

Clinical Observations
At 100 mg/kg bw/day, all surviving males and all females showed increased post-dosing
salivation from Day 12 onwards.
At 75 mg/kg bw/day, eight males also showed episodes of increased post-dosing salivation
between Days 12 and 14.
Increased post-dosing salivation is frequently observed when a slightly unpalatable or irritant test
item is administered via the oral gavage route and is considered to reflect distaste of the dosing
formulations rather than any adverse systemic effect of treatment.

Functional Observations

Behavioral Assessments
There were no treatment-related changes in the behavioral parameters at 50, 75 or 100 mg/kg
bw/day.

Functional Performance Tests
There were no changes in functional performance considered to be related to treatment at 50, 75
or 100 mg/kg bw/day.
For males at 100 mg/kg bw/day, lower forelimb grip strength during trial one attained statistical
significance when compared with control. In the absence of a dosage relationship or any similar
statistically significant differences from control in the two subsequent trials for forelimb grip
strength, this finding was considered incidental and unrelated to treatment.

Sensory Reactivity Assessments
There were no toxicologically significant changes in the sensory reactivity assessments at 50, 75
or 100 mg/kg bw/day.

All inter and intra group differences in sensory reactivity scores were considered to be a result of
normal variation for rats of the strain and age used and were of no toxicological importance.

Body Weight
t 100 mg/kg bw/day, body weight gains for males were lower throughout much of the
treatment period with differences from control attaining statistical significance during Week 5.
Overall body weight gain at the end of treatment was 17 % lower than controls.
No such effects were detected in males treated with 50 or 75 mg/kg bw/day.
There was no adverse effect of treatment on body weight gain of females during the pre-pairing,
gestation and lactation phases of the study at 50, 75 or 100 mg/kg bw/day.



Food Consumption and Food Conversion Efficiency
There was no obvious effect of treatment on food consumption or food conversion efficiency for
both sexes at 50, 75 or 100 mg/kg bw/day.

Water Consumption
Gravimetric assessment of water bottles during the pre-pairing phase did not reveal any
consistent intergroup differences that indicated an effect of treatment on water consumption.

Hematology
Hematology parameters for both sexes were unaffected by treatment at 50, 75 and 100 mg/kg
bw/day.


Clinical Chemistry
There was no clear effect of treatment on blood chemistry parameters at 50, 75 or 100 mg/kg
bw/day.
For males at 75 and 100 mg/kg bw/day, higher creatinine levels attained statistical significance
when compared with control but no dosage relationship was apparent. Additionally for males at
100 mg/kg bw/day also higher glucose levels and lower aspartate aminotransferase levels
attained statistical significance when compared with controls.
For females at 100 mg/kg bw/day, higher bile acid levels attained statistical significance when
compared with controls. Additionally for females at 100 mg/kg bw/day, higher aspartate
aminotransferase levels attained statistical significance.
The majority of individual values for treated animals were within the historical control ranges
and in the absence of any histopathological correlates, the intergroup differences were
considered to be of no toxicological importance.


Pathology
Necropsy
A plethora of macroscopic observations in the stomach of both sexes at all dosages were
apparent at necropsy. Observations included multiple raised areas of the stomach, thickened nonglandular
region of the stomach, sloughing of the glandular o rnon-glandular region of the stomach and raised limiting ridge.
The remaining necropsy observations apparent on the study did not indicate any adverse effect of treatment at 50, 75 or 100 mg/kg bw/day.

Organ Weights
These were adverse effects of treatment on absolute or body weight relative organ weights for
either sex at 50, 75 or 100 mg/kg bw/day.

For females at all dosages, absolute and body weight relative liver and kidney weights were
higher than control with differences attaining statistical significance; however, there was no
dosage relationship and all individual values were within the historical control range. In the
absence of any histopathology changes for these organs, these increases were considered to be
incidental and unrelated to treatment.

Histopathology
The following treatment-related macroscopic abnormalities were detected:
Non-Glandular Stomach:
Submucosal edema:
at slight degree in one Group 3 (75 mg/kg bw/day) male, two females and
in one Group 4 female.
Granulolymphocytic infiltrate: at minimal or slight degree in Group 2 - four males, three
females, at slight or moderate in Group 3 - eight males, seven females and at the same severity in
Group 4 - all twelve males, ten females.
Erosion: at slight severity in Group 2 - one female, at minimal or slight in Group 3 - three males,
three females and in Group 4 at minimal to moderate - five males, three females.
Ulceration: at minimal or slight severity in Group 3 - three males, two females and in Group 4 at
minimal to moderate - four males, three females.
Submucosal granulation tissue: at slight severity in Group 2 - one female, at slight to moderate
in Group 3 - two males, four females and in Group 4 at minimal to moderate degree - four males,
eight females.
Glandular Stomach:
Granulolymphocytic infiltrate:
at minimal degree in Group 1 - two males, one female, minimal
or slight in Group 2 - nine males, four females, minimal to moderate in Group 3 - eleven males,
six females and in Group 4 at minimal to moderate - twelve males, five females.
Dose descriptor:
NOAEL
Effect level:
>= 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Whilst no overall adverse effect level was established in the study, as the effects were considered to be due to irritation rather than systemic toxicity, a NOAEL for systemic toxicity is probably 100 mg/kg bw/day and at least 75 mg/kg bw/day.
Critical effects observed:
not specified
Conclusions:
The ‘No-observed effect level’ for local irritation of the test formulation in the stomach was less
than 50 mg/kg bw/day. However, the (NOAEL) for systemic, or developmental toxicity of
'diacetate' is considered to be greater than 100 mg/kg bw/day in the rat.
Executive summary:

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996). This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). Methods……. The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 50, 75 and 100 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP) over the same treatment period. Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study. Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex. Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4 post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. Surviving adult males were terminated on Day 43 or Day 44, followed by the termination of all females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results

Mortality

At 100 mg/kg bw/day, one male was killed for animal welfare considerations due to the severity

of clinical signs apparent on Day 20. There were no further unscheduled deaths on the study.

Clinical Observations

At 100 mg/kg bw/day, all surviving males and all females showed increased post-dosing

salivation from Day 12 onwards. At 75 mg/kg bw/day, eight males also showed episodes of increased post-dosing salivation

between Days 12 and 14.

Behavioral Assessment

There were no treatment-related changes in the behavioral parameters at 50, 75 or 100 mg/kg

bw/day.

Functional Performance Tests

There were no changes in functional performance considered to be related to treatment at 50, 75

or 100 mg/kg bw/day.

Sensory Reactivity Assessments

There were no toxicologically significant changes in the sensory reactivity assessments at 50, 75

or 100 mg/kg bw/day.

Body Weight

At 100 mg/kg bw/day, body weight gains for males were lower throughout much of the

treatment period. No such effects were detected in males treated with 50 or 75 mg/kg bw/day.

There was no clear adverse effect of treatment on body weight gain of females during the prepairing,

gestation and lactation phases of the study at 50, 75 or 100 mg/kg bw/day.

Food Consumption

There was no obvious effect of treatment on food consumption or food conversion efficiency for

both sexes at 50, 75 or 100 mg/kg bw/day.

Water Consumption

Gravimetric assessment of water bottles during the pre-pairing phase did not reveal any clear

intergroup differences.

Laboratory Investigations

Hematology

Hematology parameters for both sexes were unaffected by treatment at 50, 75 and 100 mg/kg

bw/day.

Blood Chemistry

There was no clear effect of treatment on blood chemistry parameters at 50, 75 or 100 mg/kg

bw/day.

Pathology

Necropsy

A plethora of macroscopic observations in the stomach of both sexes at all dosages were

apparent at necropsy. Observations included multiple raised areas of the stomach, thickened nonglandular

region of the stomach, sloughing of the glandular or non-glandular region of the stomach and raised limiting ridge.

Organ Weights

There was no effect of treatment on organ weights at 50, 75 or 100 mg/kg bw/day.

Histopathology

The following treatment-related macroscopic abnormalities were detected in the stomach:

Submucosal edema: at slight degree in one Group 3 (75 mg/kg bw/day) male, two females and

in one Group 4 female.

Granulolymphocytic infiltrate: at minimal or slight degree in Group 2 - four males, three

females, at slight or moderate in Group 3 - eight males, seven females and at the same severity in

Group 4 - all twelve males, ten females.

Erosion: at slight severity in Group 2 - one female, at minimal or slight in Group 3 - three males,

three females and in Group 4 at minimal to moderate - five males, three females.

Ulceration: at minimal or slight severity in Group 3 - three males, two females and in Group 4 at

minimal to moderate - four males, three females.

Submucosal granulation tissue: at slight severity in Group 2 - one female, at slight to moderate

in Group 3 - two males, four females and in Group 4 at minimal to moderate degree - four males,

eight females.

Conclusion

The ‘No-observed effect level’ for local irritation of the test formulation in the stomach was less

than 50 mg/kg bw/day. However, the (NOAEL) for systemic, or developmental toxicity of

'diacetate' is considered to be greater than 100 mg/kg bw/day in the rat.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
A single repeat dose study conducted to OECD guideline 422 and GLP is available- this is considered adequate for hazard assessment purposes.

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

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item and is designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

At 100 mg/kg bw/day, one male was killed for animal welfare considerations due to the severity of clinical signs apparent on Day 20. There were no further unscheduled deaths on the study. At 100 mg/kg bw/day, body weight gains for males were lower throughout much of the treatment period. A plethora of macroscopic observations in the stomach of both sexes at all dosages were apparent at necropsy. Observations included multiple raised areas of the stomach, thickened nonglandular region of the stomach, sloughing of the glandular or non-glandular region of the stomach and raised limiting ridge. The following treatment-related macroscopic abnormalities were detected in the stomach in all dose groups: Granulolymphocytic infiltrate- minimal to moderate; Erosion- slight to moderate; Ulceration- slight to moderate and submucosal granulation tissue- slight to moderate with severity increasing with dose level. No other measured parameters were affected in a toxicologically significant manner. Given that the only toxicologically significant finding was local irritation of the gastric (sub)mucosa which occurs as a direct result of the route of dosing it was concluded that the ‘No-observed effect level’ for local irritation of the test formulation in the stomach was less than 50 mg/kg bw/day. However, the (NOAEL) for systemic, or developmental toxicity of 'diacetate' is considered to be greater than 100 mg/kg bw/day in the rat.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The study is relevant for the assessment of systemic toxicity hazard being conducted to OECD guideline 422 and GLP. No other study is available.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: stomach

Justification for classification or non-classification

With the exception of local irritation of the gastric mucosa, no significant toxicological effects are observed following oral exposure up to 100 mg/kg bw/day. The extent of gastric irritation observed is not sufficient to warrant classification, as in the opinion of the assessor it does not constitute "significant organ damage noted at necropsy and/or subsequently seen or confirmed at microscopic examination.", since the grading of the injury is maximally moderate rather than marked or severe.