Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
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 OECD guideline 422 and GLP
Qualifier:
according to
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. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals 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.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on 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
Details on study schedule:
i. Groups of twelve male and twelve female animals were treated daily at the appropriate
dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii. Prior to the start of treatment and once weekly thereafter, all animals were observed for
signs of functional/behavioral toxicity.
iii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a
maximum of fourteen days.
iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned
to their original cages and females were transferred to individual cages.
v. On completion of the pairing phase (during Week 6), five selected males per dose group
were evaluated for functional/sensory responses to various stimuli.
vi. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post
partum. Litter size, offspring weight and sex, surface righting and clinical signs were
also recorded during this period.
vii. At Day 4 post partum, five selected females per dose group were evaluated for
functional/sensory responses to various stimuli.
viii. Blood samples were taken from five males from each dose group for hematological andblood chemical assessments on Day 42. The male dose groups were killed and examined
macroscopically on Day 43 or Day 44.
ix. Blood samples were taken from five randomly selected females from each dose group for
hematological and blood chemical assessment on Day 4 post partum. At Day 5 post
partum, all females and surviving offspring were killed and examined macroscopically.Any female which did not produce a pregnancy was also killed and examinedmacroscopically.
Remarks:
Doses / Concentrations:
100 mg/kg bw
Basis:
actual ingested
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 bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
50 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
12 (twelve) per sex per group
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.

Mating: Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to
fourteen days. Cage tray-liners were checked each morning for the presence of ejected
copulation plugs and each female was examined for the presence of a copulation plug in the
vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of
sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ
was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently
returned to their original holding cages (unless required for additional pairing). Mated females
were housed individually during the period of gestation and lactation.



Positive control:
Not applicable
Parental animals: Observations and examinations:
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.

Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as
late as possible during the normal working day) around the period of expected parturition.
Observations were carried out at approximately 0830 and as late as possible at weekends and
public holidays. The following was recorded for each female:


i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition






Oestrous cyclicity (parental animals):
Yes, during mating a vaginal smear was prepared for each female and the stage of estrus or the presence of
sperm was recorded.
Sperm parameters (parental animals):
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.

Litter observations:
On completion of parturition (Day 0 post partum), the number of live and dead offspring was
recorded. Offspring were individually identified within each litter by tattoo on Day 1 post
partum.
For each litter the following was recorded:

i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated
retrospectively from this data)


Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Postmortem examinations (parental animals):
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)

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.

Postmortem examinations (offspring):
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.
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)
Reproductive indices:
Mating Performance and Fertility

The following parameters were calculated from the individual data during the mating period of
the parental generation:

i. Pre-coital Interval

Calculated as the time elapsing between initial pairing and the observation of positive
evidence of mating.

ii. Fertility Indices

For each group the following were calculated:

Mating Index (%) = (Number of animals mated/Number of animals paired) x100

Pregnancy Index (%) = (Number of females pregnant/Number of mated animals) x 100

Gestation and Parturition Data

The following parameters were calculated from individual data during the gestation and
parturition period of the parental generation:

i. Gestation Length

Calculated as the number of days of gestation including the day for observation of mating
and the start of parturition.

ii. Parturition Index
The following was calculated for each group:
Parturition Index (%) = ( Number of females delivering live offspring/Number of Pregant females) x100
Offspring viability indices:
Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were first
calculated for each litter and the group mean was calculated using their individual litter values.
Group mean values included all litters reared to termination (Day 5 of age).

i.
Implantation Losses (%)

Group mean percentile pre-implantation and post-implantation loss were calculated for
each female/litter as follows:

Pre–implantation loss (%) = (number of corpora lutea - number of implantation sites)/number of corpora lutea ) x100

Post–implantation loss (%) = (Number of implantation sites- total number of offspring born)/number of implantation sites )x100

ii.
Live Birth and Viability Indices

The following indices were calculated for each litter as follows:

Live Birth Index (%) = (Number of offspring alive on Day 1/number of offspring born) x100

Viability Index (%) = (Number of offspring alive on Day 4/Number of offspring alive on day 1) x100

iii.
Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1 and 4
post partum, using the
following formula:

(Number of male offspring/total number of offspring) x100
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
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):
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.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
glandular and non-glandular stomach
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
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.

Body Weight
At 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.

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.

Reproductive Performance
Mating
There was no effect of treatment on mating performance at 50, 75 or 100 mg/kg bw/day.

Fertility
Fertility as assessed by pregnancy rate was unaffected by treatment at 50, 75, 100 mg/kg bw/day.

Gestation Length
The distribution of gestation lengths for treated females did not indicate any effect of treatment
at 50, 75 or 100 mg/kg bw/day.
Dose descriptor:
NOAEL
Effect level:
>= 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Litter Responses
One female at 75 mg/kg bw/day and one female at 100 mg/kg bw/day failed to achieve
pregnancy. Additionally one female at 50 mg/kg bw/day and one female at 100 mg/kg bw/day
showed total litter loss post partum. The following assessment is generally based on the 12, 11,
11 and 10 females successfully rearing offspring to Day 4 post partum at 0 (Control), 50, 75 and
100 mg/kg bw/day respectively.

Offspring Litter Size, Sex Ratio and Viability
Numbers of corpora lutea and implantations, pre- and post implantation losses, litter size at
birth/Day 1, subsequent offspring survival to Day 4 and sex ratio were unaffected by maternal
treatment at 50, 75 or 100 mg/kg bw/day.

One female at 50 mg/kg bw/day and one female at 100 mg/kg bw/day showed total litter loss
post partum. Both females only had a single offspring and it is not unusual for dams to fail to
maintain such small litters. In the absence of similar post natal losses for other litters at these
dosages, these total litter losses were considered incidental and unrelated to maternal treatment.


4.1.3.2
Offspring Growth and Development

Offspring body weight and assessment of surface righting on Day 1 and subsequent body weight
gain to Day 4 post partum were unaffected by maternal treatment at 50, 75 or 100 mg/kg bw/day.

At 50 and 75 mg/kg bw/day, surface righting reflex as superior to control, with differences
attaining statistical significance. An increase in this parameter is considered not to represent an
adverse effect of treatment and therefore the intergroup differences were considered to represent
normal biological variation.

Clinical signs apparent for offspring on the study were typical for the age observed and the
incidence/distribution of these observations did not indicate any effect of maternal treatment on
offspring development at 50, 75 or 100 mg/kg bw/day.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Reproductive 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.

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.

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.

Reproductive Parameters:

Numbers of corpora lutea and implantations, pre- and post implantation losses, litter size at

birth/Day 1, subsequent offspring survival to Day 4 and sex ratio were unaffected by maternal

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

Offspring body weight and assessment of surface righting on Day 1 and subsequent body weight

gain to Day 4 post partum were unaffected by maternal treatment at 50, 75 or 100 mg/kg bw/day.

Clinical signs apparent for offspring on the study were typical for the age observed and the

incidence/distribution of these observations did not indicate any effect of maternal treatment on

offspring development at 50, 75 or 100 mg/kg bw/day.

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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
A single screening study conducted to OECD 422 and GLP is available. The results of the study are considered adequate for hazard assessment.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Male and female rats were exposed to the test substance at 50, 75 and 100 mg/kg bw/day prior to mating, during pairing and subsequently during the period of gestation up to 5 days postpartum. No adverse effects on reproductive performance, gonad pathology/histopathology or fetus/pup development were observed at any dose tested. Testing at concentrations greater than 100 mg/kg bw/day was not considered possible due to the observation of gastric irritation at 125 and 250 mg/kg bw/day in the 14 day range finding study.


Short description of key information:
Reproductive Toxicity Screening: Subacute Reproductive Test (oral gavage), rat (Wistar-Han) m/f (OECD guideline 422, GLP): NOAEL: >100 mg/kg bw/day (parent and offspring).

Justification for selection of Effect on fertility via oral route:
A single screening study conducted to OECD 422 and GLP is available.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

At the maximum concentration tested, there was no evidence that the test substance had an adverse effect upon gonad (histo)pathology, reproductive performance or fetal/pup development.Based upon the results of the screening study the classification criteria for reproductive toxicity or development set out in Commission Regulation 1272/2008/EC are not met.