Reaction mass of 2-[[[2-[(2-aminoethyl)amino]ethyl]amino]carbonyl]-benzoic acid and 2,2' -[iminobis(2,1-ethanediyliminocarbonyl)]bis-benzoic acid and 7,8,9,10,11,12,20,21,22,23,24,25-dodecahydrodibenzo[i,t] [1,4,7,12,15,18] hexaazacyclodocosine-5,13,18,26(6H,19H)-tetrone

Endpoint summary

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

Study period:

The study was conducted between 14 August 2014 and 29 June 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

See below

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:

Animals and Animal Husbandry
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were
obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the
animals were examined for signs of ill-health or injury. The animals were acclimatized for
six days during which time their health status was assessed. A total of ninety six animals (forty
eight males and forty eight females) were accepted into the study. At the start of treatment the
males weighed 292 to 327g, the females weighed 185 to 211g, and were approximately twelve
weeks old.
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.
The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad
Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used. Mains drinking water was
supplied from polycarbonate bottles attached to the cage. Environmental enrichment was
provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd.,
Cheshire, UK) except for paired animals and mated females during gestation and lactation.
Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.
The diet, drinking water, bedding and environmental enrichment was considered not to contain
any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd.,
Shardlow, UK 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 computerized system, and print-outs of hourly temperatures and humidities are
included in the study records. The Study Plan target ranges for temperature and relative
humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets
were considered not to have affected the purpose or integrity of the study; see deviations from
Study Plan.
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.

Justification
The rat was selected for this study as it is a readily available rodent species historically used in
safety evaluation studies and is acceptable to appropriate regulatory authorities.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in distilled water; formulation at the high dose appeared to be homogenous on visual inspection. Since the method used for formulation analysis was considered to be non stability
indicating, test item formulation stability was not determined and therefore, fresh formulations were prepared each day and dosed within two hours of preparation. It is assumed that the formulation was stable for this duration. As stability was not determined, this is an exception with regard to GLP and has been reflected in the GLP compliance statement.
Samples of the test item formulation were taken and analyzed on a weekly basis for concentration of 1,3-Isobenzofurandione, reaction products with diethylenetriamine at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services.
The results indicate that the prepared formulations were within 92% to 108% of the nominal concentration.

Details on mating procedure:

On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Introduction
The test item concentration in the test samples was determined gravimetrically.

Test item
The test item described in the main part of this study was also used as the analytical standard.

Analytical procedure
Analysis of samples
Due to the complex nature of the test item and its limited solubility in organic and aqueous media, a substance specific quantitative method of analysis could not be developed. The concentration of test item in the formulations was determined using a gravimetric technique and homogeneity at the high dose was confirmed by visual assessment.
The test item formulations were weighed into tared beakers and then 5 mL of water was added. The samples were then dried in an oven at approximately 80 °C before allowing to cool over silica gel in a desiccator and re-weighed.

Preparation of accuracy samples
Samples of distilled water were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These samples were then prepared for analysis as the test samples.

Study samples and storage
Representative samples were dispatched to the analytical laboratories internally (under ambient conditions) and stored at room temperature until analysis.

Results
Validation of analytical method
Accuracy
The fortified samples of distilled water were found to have a recovery value of ± 10 % of the fortification.

Test item formulation
The formulations investigated during the study were found to comprise test item in the range of 92 – 108 % and, thus the required content of ± 10 % with reference to the nominal content was met.

Discussion
The analytical procedure had acceptable recoveries of test item in the vehicle. The method of analysis was validated and proven suitable for use.

Duration of treatment / exposure:

Up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females)

Frequency of treatment:

Daily

Details on study schedule:

Chronological Sequence of Study
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. The first six males in each
dose group were dosed up to Day 42 whilst the remaining males were dosed up to
Day 43.
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 and
blood chemical assessments on Day 42. The male dose groups were killed and examined
macroscopically on Day 43 or 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 surviving females and surviving offspring were killed and examined
macroscopically. Any female which did not produce a pregnancy was also killed and
examined macroscopically on Day 26 post coitum. One female from the intermediate
dose group died during the bleeding procedure on Day 4 post partum and was
subsequently examined macroscopically.

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

The test item was administered daily by gavage using a stainless steel cannula attached to a
disposable plastic syringe. Control animals were treated in an identical manner with 5 mL/kg of
distilled water.
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.

Positive control:

None

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 (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); see deviations from Study Plan.
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 the 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.

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.

Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt changes.

Reproductive Performance
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. Mated females were housed individually during the
period of gestation and lactation.

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 08:30 AM 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

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.

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.

Oestrous cyclicity (parental animals):

Not assessed

Sperm parameters (parental animals):

Not assessed

Litter observations:

Litter Data
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
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):

Pathology
Necropsy
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by
exsanguination on Days 43 or 44. Surviving 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 Day 26 post coitum. Female
70 died during the bleeding procedure on Day 4 post partum; the litter was also killed on the
same day.
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 including those dying during the study, were subjected to a full
external and internal examination, and any macroscopic abnormalities were recorded.

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. T
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).

Tissues shown below were weighed from all remaining animals:
Prostate, Seminal vesicles, Epididymides, Testes, Ovaries, Uterus (weighed with Cervix), Pituitary (post fixation).

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
# = lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before
immersion in fixative

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 1000 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 1000 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 1000 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, examination was subsequently
extended to include similarly prepared sections of liver from males in the low and intermediate
dose groups and the Peyer's patches from females in the low and intermediate dose groups; see
deviations from Study Plan.
Microscopic examination was conducted by the Study Pathologist (Roger Alison at Roger Alison
Ltd.) and a histopathology peer review was performed by another pathologist (W Henderson at
Harlan Laboratories Ltd., Shardlow).

Postmortem examinations (offspring):

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

Statistics:

See below

Reproductive indices:

See below

Offspring viability indices:

See below

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Adult Reponses
Mortality
Female 70 given the test item at 300 mg/kg bw/day was found dead during the blood sampling
procedure on Day 4 post partum. There were no clinical signs for this animal prior to death and
macroscopic findings were confined to the sloughing of the glandular region in the stomach;
histopathology examination of the stomach from this animal did not reveal any abnormal
observations. Although a cause of death could not be established, it may be due to the stress
associated with blood sampling procedure and was considered not to be treatment-related.
There were no other unscheduled deaths during the study.

Clinical Observations
Clinical signs during the dosing period were confined to a small number of males and females
treated with 1000 mg/kg bw/day showing sporadic instances of increased post-dose salivation
from Day 13 of dosing and one male given 300 mg/kg bw/day showing generalised fur loss from
Day 26 of the study; these were considered not to be related to the toxicity of the test item.

Functional Observations
Behavioral Assessments:
No treatment-related changes in the behavioral parameters were detected at any dose level.
Functional Performance Tests:
Any intergroup differences in functional performance were considered not related to treatment
with the test item.

Sensory Reactivity Assessments:
Sensory reactivity scores across all treated groups were similar to controls.

Body Weight
There was no detrimental effect of treatment on male body weight development throughout the
study. Males from the high dose group showed slightly but statistically significantly higher body
weight gain during the second week of dosing in comparison with controls (p<0.05); this
increase was considered to be of no toxicological significance.
There was no effect of treatment on body weight development in females during the pre-pairing,
gestation or lactation phases of the study.

Food Consumption
There was no effect of treatment with the test item on food intake or food efficiency in male
groups at any dose level.
There was no effect of treatment with the test item on food consumption or food efficiency in
females during the pre-pairing phase of the study. During gestation, dietary intake in females
receiving 1000 mg/kg bw/day was marginally lower than controls over Days 0 to 7 and 7 to 14,
with differences from controls achieving statistical significance in the first instance (p<0.01), but
thereafter it was similar to controls. As there was no associated effect on body weight
development in these females and food intake showed an improvement towards the end of the
gestation period, this finding was considered to be of no toxicological relevance. During
lactation, food intake across all test item-treated females was comparable with controls.

Water Consumption
Visual inspection of water bottles did not indicate any differences for the animals given the test
item in comparison with controls.

Reproductive Performance
Mating
There was no effect of treatment on mating performance.
With the exception of one female each from the control and low dose groups, the remaining
animals mated within four days after pairing; Female 17 (control) and Female 47 (low dose
group) respectively mated on Days 13 and 12 following pairing. Female 65 from the
intermediate dose group showed signs of uncertain mating (copulation plug found in the cage but
no sperm present in the vaginal smear) on Day 1 following pairing; this female was left with the
male in the cage for the duration of the pairing period, but it was found to be non-pregnant at
necropsy and as such was assumed to have failed to mate.

Fertility
Fertility as assessed by pregnancy index was unaffected by treatment with the test item at any
dose level.
Two females each from the low (Females 37 and 40) and intermediate (Females 65 and 69) dose
groups and one female from the high dose group (Female 88) did not achieve pregnancy. Of
these, Female 65 only showed signs of uncertain mating whilst evidence of successful mating
was present for the remaining animals. With the exception of Male 53 from the low dose group
(paired with Female 65) showing marked tubular atrophy in the testes and marked reduction in
spermatozoa in the epididymides, no histopathological correlates were evident in reproductive
tissues from the remaining animals which could have been considered to be the cause of the
infertility. Due to the isolated nature of the above microscopic findings in Male 53, they were
considered to be incidental.

Gestation Length
Gestation lengths were between 22 and 23½ days and the distribution of gestation lengths for
treated females was similar to controls.

Laboratory Investigations
Hematology
No toxicologically significant effects were detected in animals of either sex receiving the test
item at any dose level.
Females receiving the test item at all dose levels showed slightly but statistically significantly
higher group mean activated partial thromboplastin time when compared with controls (p<0.01
at 1000 mg/kg bw/day and p<0.05 at 100 or 300 mg/kg bw/day). The corresponding values in
males from the intermediate and high dose levels were similar to controls, but the mean value for
the low dose males was statistically significantly lower than controls (p<0.05). There was no
dose-relationship and individual values from these animals were within the background data
ranges. In the absence of any histopathology correlates, these findings were considered to be of
no toxicological significance.

Blood Chemistry
No toxicologically significant effects were detected in animals of either sex receiving the test
item at any dose level.
At all dose levels, group mean total protein, albumin and calcium plasma concentrations in males
were slightly and statistically significantly higher than controls (p<0.05). There were no clear
dose-relationships and individual values from the test item-treated males were within the
background data ranges. Group mean plasma concentrations of bile acids in males and females
treated with 300 or 1000 mg/kg bw/day were slightly higher than controls in a dose-related
manner, albeit without achieving statistical significance. With the exception of one female from
the intermediate dose group and two females from the high dose group, all individual values
from the test item-treated animals were within the background data ranges. Microscopic
examination of the liver from males across all dose groups including controls revealed
incidences of hepatocyte hypertrophy, which were deemed incidental in the absence of any dose
response. As such, the observations mentioned above were considered to be of no toxicological
significance.
Any other intergroup differences for blood chemistry parameters did not achieve statistical
significance and were considered to be incidental findings.

Pathology
Necropsy
Offspring
Macroscopic necropsy findings for offspring on the study were typical for the age observed and
neither the incidence or distribution of these observations indicated any adverse effect of
maternal treatment on offspring development at 100, 300 or 1000 mg/kg bw/day.
Adults
There were no treatment-related macroscopic findings at necropsy.
Any macroscopic findings seen across the dose groups were typical of rats of this strain and age,
and were considered to be incidental.

Organ Weights
There were no toxicologically significant effects of treatment on the organ weights.
Group mean absolute and body weight-related liver weights in males given 1000 mg/kg bw/day
were slightly higher than controls, albeit without achieving statistical significance. There was no
dose-relationship and individual values from these animals were within the background data
ranges. Histopathological evaluation of the liver from males revealed a small increase in the
incidence of hepatocyte hypertrophy; however, in the absence of a dose-response this finding
was considered to be incidental, and therefore the intergroup differences in liver weight were
considered not to be treatment-related.
The corresponding values in females treated with the test item at this dose level were similar to
controls.

Histopathology
The following treatment-related microscopic abnormalities were detected:
Peyer's patches: There was an increase in incidence of lipid in the Peyer's patches of females
receiving the high dose of 1000 mg/kg bw/day. A possible relationship to treatment could not be
excluded, however this is a minor finding. Lipid is commonly seen in the Peyer’s patches of
control animals of this strain and age, and the severity grade in the present study did not exceed
the normal range, therefore this finding was considered to be of limited toxicological
significance.
Liver: A small increase in incidence of hepatocellular hypertrophy in high dose males correlated
with an equivocal increase in liver weight, therefore the histopathological examination was
extended to the low and intermediate dose males. As there was no relationship to dose after the
examination of low and intermediate dose animals, these findings were considered to be
incidental.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No statistically significant toxicologically relevant effects were observed in any of the parameters tested at any dose level.

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:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Litter Responses
In total twelve, ten, nine and eleven females from control, 100, 300 or 1000 mg/kg bw/ day dose
groups respectively gave birth to a live litter and successfully reared young to Day 5 of age. In
addition, Female 70 from the 300 mg/kg bw/day dose group gave birth to a live litter; however,
this animal was found dead during the blood sampling procedure on Day 4 post partum and the
litter from this dam was killed on the same day. The following assessment of litter response is
based on all litters reared to termination on Day 4/5 of lactation/age; as the death of Female 70
was considered unrelated to treatment with the test item, litter data from this dam has also been
included in this assessment, where applicable.

Offspring Litter Size, Sex Ratio and Viability
The mean number of corpora lutea, implantation counts or pre- and post-implantation losses for
females given the test item at all dose levels was similar to controls.
Of the litters born, litter size at birth and subsequently on Days 1 and 4 post partum from animals
treated with the test item were comparable with controls. One litter from the 300 mg/kg bw/day
dose group was killed on Day 4 post partum due to the early death of the dam.
There were no intergroup differences in sex ratio (percentage male offspring) for litters from
treated groups when compared with controls.

Offspring Growth and Development
Offspring bodyweight gain and litter weights on Days 1 and 4 post partum were comparable with
controls.
Surface righting reflex data did not reveal any intergroup differences when compared with
controls.
Clinical signs for the offspring included small size, going missing, found dead, open wound on
the back, scab on the snout, tail damage and no milk in the stomach; these were mainly confined
to the control and low dose groups and, were considered to be low incidence findings observed
in offspring in studies of this type.

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No statistically significant toxicologically relevant effects were observed in any of the parameters tested at any dose level.

Reproductive effects observed:

not specified

Discussion

The oral administration of 1,3-Isobenzofurandione, reaction products with diethylenetriamine to rats for a period of up to eight weeks (including two weeks pre-pairing, gestation and early lactation for females) at dose levels of up to 1000 mg/kg bw/day, was well tolerated. Sporadic instances of increased salivation were seen in some animals of both sexes given the high dose however, these were considered likely to be related to the taste of the test item formulation rather than an indication of its systemic toxicity. Hematological and blood chemical assessments revealed some statistically significant intergroup differences but these were considered to be of no toxicological significance. Group mean absolute and body weight-related liver weights in males treated with 1000 mg/kg bw/day were slightly higher than controls at the end of the dosing period, albeit without achieving statistical significance. There were no treatment-related microscopic findings in the liver from males at any dose level and this slight increase in liver weight was considered not treatment-related. A small increase in incidence of lipid in the Peyer's patches of females from the high dose group was observed, but this was deemed to be of limited toxicological importance.

Mating performance and fertility remained unaffected by treatment with the test item at all dose levels. In addition, there was no effect of treatment on pre- and post-implantation losses, viability indices, sex ratios and offspring growth and development. There were no treatmentrelated macroscopic findings for any of the pups.

Conclusions:

The oral administration of 1,3-Isobenzofurandione, reaction products with diethylenetriamine to rats by gavage, at dose levels of 100, 300 or 1000 mg/kg bw/day, was well tolerated. Based on the available data, the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.

Executive summary:

Introduction

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 100, 300 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (distilled water). 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. Adult males were terminated on Day 43 or 44 of the study, followed by the termination of all surviving females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on Day 26 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results…….

Adult Responses

Mortality

Female 70 given the test item at 300 mg/kg bw/day was found dead during the blood sampling procedure on Day 4 post partum. Although a cause of death could not be established, it may be due to the stress associated with blood sampling procedure and was considered not to be treatment-related. There were no other unscheduled deaths during the study.

Clinical Observations

Throughout the study, there were no clinical signs considered to be related to the toxicity of the test item.

Behavioral Assessment

There were no treatment-related changes in the behavioral parameters measured.

Functional Performance Tests

There was no effect of treatment with the test item at any dose level on functional performance in animals of either sex.

Sensory Reactivity Assessments

Sensory reactivity scores across all treated groups were similar to controls.

Body Weight There was no detrimental effect of treatment on male body weight development during the study. There was no effect of treatment on body weight development in females during the pre-pairing, gestation or lactation phases of the study.

Food Consumption

Food intake and food efficiency across all groups of test item-treated males remained similar to controls during the study. There was no effect of treatment with the test item on food consumption and food efficiency in females during the pre-pairing phase of the study. During gestation, dietary intake in females receiving 1000 mg/kg bw/day was slightly lower than controls up to Day 14 of gestation, but thereafter it was similar to controls and as such considered to be of no toxicological significance. During lactation, food intake across all test item-treated females was comparable with controls.

Water Consumption

Visual inspection of water bottles did not indicate any differences for the animals given the test item in comparison with controls.

Reproductive Performance

Mating

There was no effect of treatment on mating performance. The majority of animals mated within four days of pairing.

Fertility

There were no treatment-related effects in conception rates for test item-treated animals.

Gestation Lengths

There were no differences in gestation lengths in animals receiving the test item when compared with controls.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There was no effect of treatment on corpora lutea count, pre-implantation loss, numbers of implantations, post-implantation loss, litter size, sex ratio and subsequent offspring survival to Day 5 of age at 100, 300 or 1000 mg/kg bw/day.

Offspring Growth and Development

There was no effect of treatment indicated by offspring body weight or body weight gain and litter weights, surface righting ability on Day 1 or clinical signs to Day 5 of age at 100, 300 or 1000 mg/kg bw/day.

Laboratory Investigations

Hematology

No toxicologically significant effects were detected in animals of either sex at any dose level.

Blood Chemistry

No toxicologically significant effects were detected in animals of either sex at any dose level. Pathology Necropsy Neither the type, incidence or distribution of macroscopic observations in adult animals or offspring indicated any adverse effect of treatment at 100, 300 or 1000 mg/kg bw/day.

Organ Weights

There were no toxicologically significant effects of treatment on the organ weights.

Histopathology

The following treatment-related microscopic abnormalities were detected:

Peyer's patches: There was an increase in incidence of lipid in the Peyer’s patches of females receiving the high dose of 1000 mg/kg bw/day. A possible relationship to treatment could not be excluded, however this is a minor finding. Lipid is commonly seen in the Peyer’s patches of control animals of this strain and age, and the severity grade in the present study did not exceed the normal range, therefore this finding was considered to be of limited toxicological significance.

Liver: A small increase in incidence of hepatocellular hypertrophy in high dose males correlated with an equivocal increase in liver weight, therefore the histopathological examination was extended to the low and intermediate dose males. As there was no relationship to dose after the examination of low and intermediate dose animals, these findings were considered to be incidental.

Conclusion

The oral administration of 1,3-Isobenzofurandione, reaction products with diethylenetriamine to rats by gavage, at dose levels of 100, 300 or 1000 mg/kg bw/day, was well tolerated. Based on the available data, the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In the study, there is no statistically significant toxicologically relevant effects observed in any of the parameters tested at any dose level.

Short description of key information:
In an Oral (Gavage) Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422) the NOAEL for reproductive toxicity was found to be 1000 mg/kg/day.

Justification for selection of Effect on fertility via oral route:
Only this study is available and the study is Klimisch 1.

Effects on developmental toxicity

Description of key information

In an Oral (Gavage) Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422) the NOAEL for developmental toxicity was found to be 1000 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 14 August 2014 and 29 June 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD 422

Deviations:

yes

Remarks:

See below

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Animals and Animal Husbandry
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were
obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the
animals were examined for signs of ill-health or injury. The animals were acclimatized for
six days during which time their health status was assessed. A total of ninety six animals (forty
eight males and forty eight females) were accepted into the study. At the start of treatment the
males weighed 292 to 327g, the females weighed 185 to 211g, and were approximately twelve
weeks old.
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.
The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad
Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used. Mains drinking water was
supplied from polycarbonate bottles attached to the cage. Environmental enrichment was
provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd.,
Cheshire, UK) except for paired animals and mated females during gestation and lactation.
Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.
The diet, drinking water, bedding and environmental enrichment was considered not to contain
any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd.,
Shardlow, UK 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 computerized system, and print-outs of hourly temperatures and humidities are
included in the study records. The Study Plan target ranges for temperature and relative
humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets
were considered not to have affected the purpose or integrity of the study; see deviations from
Study Plan.
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.

Justification
The rat was selected for this study as it is a readily available rodent species historically used in
safety evaluation studies and is acceptable to appropriate regulatory authorities.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in distilled water; formulation at the high dose appeared to be homogenous on visual inspection. Since the method used for formulation analysis was considered to be non stability indicating, test item formulation stability was not determined and therefore, fresh formulations were prepared each day and dosed within two hours of preparation. It is assumed that the formulation was stable for this duration. As stability was not determined, this is an exception with regard to GLP and has been reflected in the GLP compliance statement. Samples of the test item formulation were taken and analyzed on a weekly basis for concentration of 1,3-Isobenzofurandione, reaction products with diethylenetriamine at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services.
The results indicate that the prepared formulations were within 92% to 108% of the nominal concentration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Introduction
The test item concentration in the test samples was determined gravimetrically.

Test item
The test item described in the main part of this study was also used as the analytical standard.

Analytical procedure
Analysis of samples
Due to the complex nature of the test item and its limited solubility in organic and aqueous media, a substance specific quantitative method of analysis could not be developed. The concentration of test item in the formulations was determined using a gravimetric technique and homogeneity at the high dose was confirmed by visual assessment.
The test item formulations were weighed into tared beakers and then 5 mL of water was added. The samples were then dried in an oven at approximately 80 °C before allowing to cool over silica gel in a desiccator and re-weighed.

Preparation of accuracy samples
Samples of distilled water were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These samples were then prepared for analysis as the test samples.

Study samples and storage
Representative samples were dispatched to the analytical laboratories internally (under ambient conditions) and stored at room temperature until analysis.

Results
Validation of analytical method
Accuracy
The fortified samples of distilled water were found to have a recovery value of ± 10 % of the fortification.

Test item formulation
The formulations investigated during the study were found to comprise test item in the range of 92 – 108 % and, thus the required content of ± 10 % with reference to the nominal content was met.

Discussion
The analytical procedure had acceptable recoveries of test item in the vehicle. The method of analysis was validated and proven suitable for use.

Details on mating procedure:

On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
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.

Duration of treatment / exposure:

Up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females)

Frequency of treatment:

Daily

Duration of test:

Up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females)

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

The test item was administered daily by gavage using a stainless steel cannula attached to a
disposable plastic syringe. Control animals were treated in an identical manner with 5 mL/kg of
distilled water.
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.

Maternal 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 (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); see deviations from Study Plan.
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 the 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.

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.

Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt changes.

Reproductive Performance
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. Mated females were housed individually during the
period of gestation and lactation.

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 08:30 AM 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

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.

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.

Pathology
Necropsy
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by
exsanguination on Days 43 or 44. Surviving 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 Day 26 post coitum. Female
70 died during the bleeding procedure on Day 4 post partum; the litter was also killed on the
same day.
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 including those dying during the study, were subjected to a full
external and internal examination, and any macroscopic abnormalities were recorded.

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. T
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).

Tissues shown below were weighed from all remaining animals:
Prostate, Seminal vesicles, Epididymides, Testes, Ovaries, Uterus (weighed with Cervix), Pituitary (post fixation).

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
# = lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before
immersion in fixative

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 1000 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 1000 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 1000 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, examination was subsequently
extended to include similarly prepared sections of liver from males in the low and intermediate
dose groups and the Peyer's patches from females in the low and intermediate dose groups; see
deviations from Study Plan.
Microscopic examination was conducted by the Study Pathologist (Roger Alison at Roger Alison
Ltd.) and a histopathology peer review was performed by another pathologist (W Henderson at
Harlan Laboratories Ltd., Shardlow).

Ovaries and uterine content:

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.

Fetal examinations:

Litter Data
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
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.

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

Statistics:

See below

Indices:

See below

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Adult Reponses
Mortality
Female 70 given the test item at 300 mg/kg bw/day was found dead during the blood sampling
procedure on Day 4 post partum. There were no clinical signs for this animal prior to death and
macroscopic findings were confined to the sloughing of the glandular region in the stomach;
histopathology examination of the stomach from this animal did not reveal any abnormal
observations. Although a cause of death could not be established, it may be due to the stress
associated with blood sampling procedure and was considered not to be treatment-related.
There were no other unscheduled deaths during the study.

Clinical Observations
Clinical signs during the dosing period were confined to a small number of males and females
treated with 1000 mg/kg bw/day showing sporadic instances of increased post-dose salivation
from Day 13 of dosing and one male given 300 mg/kg bw/day showing generalised fur loss from
Day 26 of the study; these were considered not to be related to the toxicity of the test item.

Functional Observations
Behavioral Assessments:
No treatment-related changes in the behavioral parameters were detected at any dose level.
Functional Performance Tests:
Any intergroup differences in functional performance were considered not related to treatment
with the test item.

Sensory Reactivity Assessments:
Sensory reactivity scores across all treated groups were similar to controls.

Body Weight
There was no detrimental effect of treatment on male body weight development throughout the
study. Males from the high dose group showed slightly but statistically significantly higher body
weight gain during the second week of dosing in comparison with controls (p<0.05); this
increase was considered to be of no toxicological significance.
There was no effect of treatment on body weight development in females during the pre-pairing,
gestation or lactation phases of the study.

Food Consumption
There was no effect of treatment with the test item on food intake or food efficiency in male
groups at any dose level.
There was no effect of treatment with the test item on food consumption or food efficiency in
females during the pre-pairing phase of the study. During gestation, dietary intake in females
receiving 1000 mg/kg bw/day was marginally lower than controls over Days 0 to 7 and 7 to 14,
with differences from controls achieving statistical significance in the first instance (p<0.01), but
thereafter it was similar to controls. As there was no associated effect on body weight
development in these females and food intake showed an improvement towards the end of the
gestation period, this finding was considered to be of no toxicological relevance. During
lactation, food intake across all test item-treated females was comparable with controls.

Water Consumption
Visual inspection of water bottles did not indicate any differences for the animals given the test
item in comparison with controls.

Reproductive Performance
Mating
There was no effect of treatment on mating performance.
With the exception of one female each from the control and low dose groups, the remaining
animals mated within four days after pairing; Female 17 (control) and Female 47 (low dose
group) respectively mated on Days 13 and 12 following pairing. Female 65 from the
intermediate dose group showed signs of uncertain mating (copulation plug found in the cage but
no sperm present in the vaginal smear) on Day 1 following pairing; this female was left with the
male in the cage for the duration of the pairing period, but it was found to be non-pregnant at
necropsy and as such was assumed to have failed to mate.

Fertility
Fertility as assessed by pregnancy index was unaffected by treatment with the test item at any
dose level.
Two females each from the low (Females 37 and 40) and intermediate (Females 65 and 69) dose
groups and one female from the high dose group (Female 88) did not achieve pregnancy. Of
these, Female 65 only showed signs of uncertain mating whilst evidence of successful mating
was present for the remaining animals. With the exception of Male 53 from the low dose group
(paired with Female 65) showing marked tubular atrophy in the testes and marked reduction in
spermatozoa in the epididymides, no histopathological correlates were evident in reproductive
tissues from the remaining animals which could have been considered to be the cause of the
infertility. Due to the isolated nature of the above microscopic findings in Male 53, they were
considered to be incidental.

Gestation Length
Gestation lengths were between 22 and 23½ days and the distribution of gestation lengths for
treated females was similar to controls.

Laboratory Investigations
Hematology
No toxicologically significant effects were detected in animals of either sex receiving the test
item at any dose level.
Females receiving the test item at all dose levels showed slightly but statistically significantly
higher group mean activated partial thromboplastin time when compared with controls (p<0.01
at 1000 mg/kg bw/day and p<0.05 at 100 or 300 mg/kg bw/day). The corresponding values in
males from the intermediate and high dose levels were similar to controls, but the mean value for
the low dose males was statistically significantly lower than controls (p<0.05). There was no
dose-relationship and individual values from these animals were within the background data
ranges. In the absence of any histopathology correlates, these findings were considered to be of
no toxicological significance.

Blood Chemistry
No toxicologically significant effects were detected in animals of either sex receiving the test
item at any dose level.
At all dose levels, group mean total protein, albumin and calcium plasma concentrations in males
were slightly and statistically significantly higher than controls (p<0.05). There were no clear
dose-relationships and individual values from the test item-treated males were within the
background data ranges. Group mean plasma concentrations of bile acids in males and females
treated with 300 or 1000 mg/kg bw/day were slightly higher than controls in a dose-related
manner, albeit without achieving statistical significance. With the exception of one female from
the intermediate dose group and two females from the high dose group, all individual values
from the test item-treated animals were within the background data ranges. Microscopic
examination of the liver from males across all dose groups including controls revealed
incidences of hepatocyte hypertrophy, which were deemed incidental in the absence of any dose
response. As such, the observations mentioned above were considered to be of no toxicological
significance.
Any other intergroup differences for blood chemistry parameters did not achieve statistical
significance and were considered to be incidental findings.

Pathology
Necropsy
Offspring
Macroscopic necropsy findings for offspring on the study were typical for the age observed and
neither the incidence or distribution of these observations indicated any adverse effect of
maternal treatment on offspring development at 100, 300 or 1000 mg/kg bw/day.
Adults
There were no treatment-related macroscopic findings at necropsy.
Any macroscopic findings seen across the dose groups were typical of rats of this strain and age,
and were considered to be incidental.

Organ Weights
There were no toxicologically significant effects of treatment on the organ weights.
Group mean absolute and body weight-related liver weights in males given 1000 mg/kg bw/day
were slightly higher than controls, albeit without achieving statistical significance. There was no
dose-relationship and individual values from these animals were within the background data
ranges. Histopathological evaluation of the liver from males revealed a small increase in the
incidence of hepatocyte hypertrophy; however, in the absence of a dose-response this finding
was considered to be incidental, and therefore the intergroup differences in liver weight were
considered not to be treatment-related.
The corresponding values in females treated with the test item at this dose level were similar to
controls.

Histopathology
The following treatment-related microscopic abnormalities were detected:
Peyer's patches: There was an increase in incidence of lipid in the Peyer's patches of females
receiving the high dose of 1000 mg/kg bw/day. A possible relationship to treatment could not be
excluded, however this is a minor finding. Lipid is commonly seen in the Peyer’s patches of
control animals of this strain and age, and the severity grade in the present study did not exceed
the normal range, therefore this finding was considered to be of limited toxicological
significance.
Liver: A small increase in incidence of hepatocellular hypertrophy in high dose males correlated
with an equivocal increase in liver weight, therefore the histopathological examination was
extended to the low and intermediate dose males. As there was no relationship to dose after the
examination of low and intermediate dose animals, these findings were considered to be
incidental.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter Responses
In total twelve, ten, nine and eleven females from control, 100, 300 or 1000 mg/kg bw/ day dose
groups respectively gave birth to a live litter and successfully reared young to Day 5 of age. In
addition, Female 70 from the 300 mg/kg bw/day dose group gave birth to a live litter; however,
this animal was found dead during the blood sampling procedure on Day 4 post partum and the
litter from this dam was killed on the same day. The following assessment of litter response is
based on all litters reared to termination on Day 4/5 of lactation/age; as the death of Female 70
was considered unrelated to treatment with the test item, litter data from this dam has also been
included in this assessment, where applicable.

Offspring Litter Size, Sex Ratio and Viability
The mean number of corpora lutea, implantation counts or pre- and post-implantation losses for
females given the test item at all dose levels was similar to controls.
Of the litters born, litter size at birth and subsequently on Days 1 and 4 post partum from animals
treated with the test item were comparable with controls. One litter from the 300 mg/kg bw/day
dose group was killed on Day 4 post partum due to the early death of the dam.
There were no intergroup differences in sex ratio (percentage male offspring) for litters from
treated groups when compared with controls.

Offspring Growth and Development
Offspring bodyweight gain and litter weights on Days 1 and 4 post partum were comparable with
controls.
Surface righting reflex data did not reveal any intergroup differences when compared with
controls.
Clinical signs for the offspring included small size, going missing, found dead, open wound on
the back, scab on the snout, tail damage and no milk in the stomach; these were mainly confined
to the control and low dose groups and, were considered to be low incidence findings observed
in offspring in studies of this type.

Abnormalities:

not specified

Developmental effects observed:

not specified

Discussion

The oral administration of 1,3-Isobenzofurandione, reaction products with diethylenetriamine to rats for a period of up to eight weeks (including two weeks pre-pairing, gestation and early lactation for females) at dose levels of up to 1000 mg/kg bw/day, was well tolerated. Sporadic instances of increased salivation were seen in some animals of both sexes given the high dose however, these were considered likely to be related to the taste of the test item formulation rather than an indication of its systemic toxicity. Hematological and blood chemical assessments revealed some statistically significant intergroup differences but these were

considered to be of no toxicological significance. Group mean absolute and body weight-related liver weights in males treated with 1000 mg/kg bw/day were slightly higher than controls at the end of the dosing period, albeit without achieving statistical significance. There were no treatment-related microscopic findings in the liver from males at any dose level and this slight increase in liver weight was considered not treatment-related. A small increase in incidence of lipid in the Peyer's patches of females from the high dose group was observed, but this was deemed to be of limited toxicological importance.

Mating performance and fertility remained unaffected by treatment with the test item at all dose levels. In addition, there was no effect of treatment on pre- and post-implantation losses,viability indices, sex ratios and offspring growth and development. There were no treatmentrelated macroscopic findings for any of the pups.

Conclusions:

The oral administration of 1,3-Isobenzofurandione, reaction products with diethylenetriamine to rats by gavage, at dose levels of 100, 300 or 1000 mg/kg bw/day, was well tolerated. Based on the available data, the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.

Executive summary:

Introduction

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 100, 300 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (distilled water). 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. Adult males were terminated on Day 43 or 44 of the study, followed by the termination of all surviving females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on Day 26 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results…….

Adult Responses

Mortality

Female 70 given the test item at 300 mg/kg bw/day was found dead during the blood sampling procedure on Day 4 post partum. Although a cause of death could not be established, it may be due to the stress associated with blood sampling procedure and was considered not to be treatment-related. There were no other unscheduled deaths during the study.

Clinical Observations

Throughout the study, there were no clinical signs considered to be related to the toxicity of the test item.

Behavioral Assessment

There were no treatment-related changes in the behavioral parameters measured.

Functional Performance Tests

There was no effect of treatment with the test item at any dose level on functional performance in animals of either sex.

Sensory Reactivity Assessments

Sensory reactivity scores across all treated groups were similar to controls.

Body Weight There was no detrimental effect of treatment on male body weight development during the study. There was no effect of treatment on body weight development in females during the pre-pairing, gestation or lactation phases of the study.

Food Consumption

Food intake and food efficiency across all groups of test item-treated males remained similar to controls during the study. There was no effect of treatment with the test item on food consumption and food efficiency in females during the pre-pairing phase of the study. During gestation, dietary intake in females receiving 1000 mg/kg bw/day was slightly lower than controls up to Day 14 of gestation, but thereafter it was similar to controls and as such considered to be of no toxicological significance. During lactation, food intake across all test item-treated females was comparable with controls.

Water Consumption

Visual inspection of water bottles did not indicate any differences for the animals given the test item in comparison with controls.

Reproductive Performance

Mating

There was no effect of treatment on mating performance. The majority of animals mated within four days of pairing.

Fertility

There were no treatment-related effects in conception rates for test item-treated animals.

Gestation Lengths

There were no differences in gestation lengths in animals receiving the test item when compared with controls.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There was no effect of treatment on corpora lutea count, pre-implantation loss, numbers of implantations, post-implantation loss, litter size, sex ratio and subsequent offspring survival to Day 5 of age at 100, 300 or 1000 mg/kg bw/day.

Offspring Growth and Development

There was no effect of treatment indicated by offspring body weight or body weight gain and litter weights, surface righting ability on Day 1 or clinical signs to Day 5 of age at 100, 300 or 1000 mg/kg bw/day.

Laboratory Investigations

Hematology

No toxicologically significant effects were detected in animals of either sex at any dose level.

Blood Chemistry

No toxicologically significant effects were detected in animals of either sex at any dose level. Pathology Necropsy Neither the type, incidence or distribution of macroscopic observations in adult animals or offspring indicated any adverse effect of treatment at 100, 300 or 1000 mg/kg bw/day.

Organ Weights

There were no toxicologically significant effects of treatment on the organ weights.

Histopathology

The following treatment-related microscopic abnormalities were detected:

Peyer's patches: There was an increase in incidence of lipid in the Peyer’s patches of females receiving the high dose of 1000 mg/kg bw/day. A possible relationship to treatment could not be excluded, however this is a minor finding. Lipid is commonly seen in the Peyer’s patches of control animals of this strain and age, and the severity grade in the present study did not exceed the normal range, therefore this finding was considered to be of limited toxicological significance.

Liver: A small increase in incidence of hepatocellular hypertrophy in high dose males correlated with an equivocal increase in liver weight, therefore the histopathological examination was extended to the low and intermediate dose males. As there was no relationship to dose after the examination of low and intermediate dose animals, these findings were considered to be incidental.

Conclusion

The oral administration of 1,3-Isobenzofurandione, reaction products with diethylenetriamine to rats by gavage, at dose levels of 100, 300 or 1000 mg/kg bw/day, was well tolerated. Based on the available data, the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In the study, there is no statistically significant toxicologically relevant effects observed in any of the parameters tested at any dose level.

Justification for selection of Effect on developmental toxicity: via oral route:
Only this study is available and the study is Klimisch 1.

Justification for classification or non-classification

Based on the above stated assessment the substance does not need to be classified as for reprotoxicity according CLP (Regulation (EC) No 1272/2008 Of The European Parliament And Of The Council)as implementation of UN-GHS in the EU.