[6R-[6α,7β(Z)]]-7-[2-furyl(methoxyimino)acetamido]-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

dose preparation instruction issued as amendment; delay in identifcation of reserve females; delay in observations during dose range finding study. None had impact on integity of study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

[6R-[6α,7β(Z)]]-7-[2-furyl(methoxyimino)acetamido]-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

EC Number:

260-086-2

EC Name:

[6R-[6α,7β(Z)]]-7-[2-furyl(methoxyimino)acetamido]-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Cas Number:

56271-94-4

Molecular formula:

C15H15N3O7S

IUPAC Name:

[6R-[6α,7β(Z)]]-7-[2-furyl(methoxyimino)acetamido]-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Identification: Furoxy Hydroxy (OK)
Appearance: Cream powder
Batch: G316533
Purity/Composition: See Certificate of Analysis (see Appendix 3)
Test item storage: In refrigerator (2-8°C)
Stable under storage conditions
until: 25 March 2018 (retest date)

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent
species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and
reproduction/developmental historical data in this species from the same strain and source.
This animal model has been proven to be susceptible to the effects of reproductive toxicants.
The total number of animals used in this study was considered to be the minimum required to
properly characterize the effects of the test item. This study has been designed such that it
does not require an unnecessary number of animals to accomplish its objectives.
At this time, studies in laboratory animals provide the best available basis for extrapolation to
humans and are required to support regulatory submissions. Acceptable models which do not
use live animals currently do not exist.
This type of study plan was reviewed and agreed by the Laboratory Animal Welfare Officer
and the Ethical Committee of Charles River Den Bosch as required by the Dutch Act on
Animal Experimentation (February 1997).

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System: Animal Receipt
On 01 Nov 2017, female Crl: WI(Han) rats were received and on 15 Nov 2017, male
Crl:WI(Han) rats were received from Charles River Deutschland, Sulzfeld, Germany. At
initiation of dosing, males were 10-12 weeks old and weighed between 260 and 303 g and
females were 12-14 weeks old and weighed between 205 and 245 g.
A health inspection was performed before the initiation of dosing.

Animal Identification
Prior to start of the pretest period (females) or treatment period (males), each animal was
identified using earmark and tattoo. Prior to the pretest period, reserve females were
numbered R1 through R8 at random by indelible marker (see deviation in Appendix 9).
Pups were identified on postnatal day (PND) 1. They were randomized per litter and
individually identified by means of subcutaneous injection of Indian ink. When general hair
growth blurred the identification, the pups were identified by tattoo on the feet.

Environmental Acclimation
The animals were allowed to acclimate to the Test Facility toxicology accommodation for 7
days prior to start of the pretest period (females) or 6 days before the commencement of
dosing (males).

Selection, Assignment, Replacement, and Disposition of Animals
A total of 40 females was selected at randomization before initiation of the pretest phase. As
none of the selected females was classified as not having regular estrous cycles during the
pretest phase, no replacement was performed before initiation of dosing. A total of 40
females with regular estrous cycles continued in the study. The supernumerary females were
removed from the study, and their estrous cycle results were kept in the raw data but not
reported.
Animals were assigned to groups by a computer-generated random algorithm according to
body weights, with all animals within ± 20% of the sex mean. Males and females were
randomized separately.

Husbandry
Housing
On arrival and following the pretest (females only) and pre-mating period, animals were
group housed (up to 5 animals of the same sex and same dosing group together) in
polycarbonate cages (Macrolon, MIV type, height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon
plastic cages (MIII type, height 18 cm).
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages,
MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually
housed in Macrolon plastic cages (MIII type, height 18 cm).
During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height
18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the
dams, when the pups were kept warm in their home cage using bottles filled with warm water.
In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled
with warm water for longer than 30-40 minutes.
During locomotor activity monitoring, animals were housed individually in a Hi-temp
polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage enrichment,
bedding material,food andwater.

The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne
GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The rooms
in which the animals were kept were documented in the study records.
Animals were separated during designated procedures/activities.
Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study
No., group, animal number(s), and sex.

Environmental Conditions
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were
maintained. The actual daily mean temperature during the study period was 19 to 22°C with
an actual daily mean relative humidity of 41 to 54%. A 12-hour light/12-hour dark cycle was
maintained, except when interrupted for designated procedures. Ten or greater air changes
per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was
provided ad libitum throughout the study, except during designated procedures. During
motor activity measurements, animals had no access to food for a maximum of 2 hours.
The feed was analyzed by the supplier for nutritional components and environmental
contaminants. Results of the analysis were provided by the supplier and are on file at the Test
Facility.
It is considered that there were no known contaminants in the feed that would interfere with
the objectives of the study.

Water
Municipal tap water was freely available to each animal via water bottles. During motor
activity measurements, animals had no access to water for a maximum of 2 hours.
Periodic analysis of the water is performed, and results of these analyses are on file at the Test
Facility.
It is considered that there were no known contaminants in the water that would interfere with
the objectives of the study.

Animal Enrichment
For psychological/environmental enrichment and nesting material, animals were provided
with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).

Veterinary Care
Veterinary care was available throughout the course of the study; however, no examinations
or treatments were required.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% w/v

Details on exposure:

The dose levels were selected based on the results of a 14-day dose range finder with oral
administration of Furoxy Hydroxy (OK) in rats. and in an attempt to produce graded responses to the test item.

Rationale for Vehicle
Trial preparations were performed at the Test Facility to select the suitable vehicle and to
establish a suitable formulation procedure. Trial preparation formulations were not used for
dosing and were discarded after the assessment is complete. These trial preparations have a
non-GLP status and were carried out in the quality assured environment of the Test Facility.

Details on mating procedure:

After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment
group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in
the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was
designated Day 0 post-coitum. Once mating had occurred, the males and females were
separated.
A maximum of 14 days was allowed for mating, after which females who had not shown
evidence of mating were separated from their males.
Detection of mating was not confirmed in first instance for female No. 46 (Group 1). The
actual mating date was determined based on a re-evaluation of the vaginal lavage for presence
of sperm cells, which was 21 days prior to the actual delivery date. Consequently, this couple
was separated 8 days after the actual mating date. The actual mating date was designated Day
0 post-coitum. Apparently, mating was overlooked in the assessment of the vaginal lavage,
which explains the continuation of di-estrus during the mating in this female.
For female no. 77 (Group 4), mating was not detected by the presence of sperm in the vaginal
lavage (sperm remnants were observed on one day of the mating period) or by a copulatory
plug but obtained by palpation and by presence of sperm remnants. However, she did not
deliver a litter and had no implantation sites. The mating date could not be determined.
Based on the palpation outcome and presence of sperm remants, this female was regarded as
non-pregnant.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical Method
Analyses were performed by using a validated analytical procedure (Test Facility Study No.
520299, ABL No. 17287).

. Concentration Analysis
Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory.
Concentration results were considered acceptable if mean sample concentration results were
within or equal to ± 15% for suspensions of target concentration.

Homogeneity Analysis
Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory.
Homogeneity results were considered acceptable if the coefficient of variation (CV) of
concentrations was <= 10%.

Stability Analysis
Stability analyses performed previously in conjunction with the method development and
validation study (Test Facility Study No. 520299, ABL No. 17287) demonstrated that the test
item is stable in the vehicle when prepared and stored under the same conditions at
concentrations bracketing those used in the present study. Stability data have been retained in
the study records for Test Facility Study No. 520299, ABL No. 17287.

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals by once daily oral
gavage 7 days a week for a minimum of 28 days. Males were treated for 29 days, up to and
including the day before scheduled necropsy. This included a minimum of 14 days prior to
mating and during the mating period. Females that delivered were treated for 50-56 days, i.e.
14 days prior to mating (with the objective to cover at least two complete estrous cycles), the
variable time to conception, the duration of pregnancy and 13-15 days after delivery, up to
and including the day before scheduled necropsy. Females which failed to deliver were
treated for 41, 44 or 54 days. The first day of dosing was designated as Day 1.
Female nos. 42, 49 (Group 1), 57, 60 (Group 2), 64 (Group 3) and 71 (Group 4), were not
dosed on one occasion as these females were littering at the moment of dosing. The omission
of one day of dosing over a period of several weeks was not considered to affect the
toxicological evaluation.
Animals were dosed approximately at the same time each day with a maximum of 6 hours
difference between the earliest and latest dose. The dose volume for each animal was based
on the most recent body weight measurement. The doses were given using a plastic feeding
tube. The dosing formulations were stirred continuously during dose administration.
A dose control system (DCS) was used as additional check to verify the dosing procedure
according to Standard Operating Procedures.
Pups were not treated directly but were potentially exposed to the test item in utero, via
maternal milk, or from exposure to maternal urine/feces.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

vehicle

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

10 per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were selected based on the results of a 14-day dose range finder with oral
administration of Furoxy Hydroxy (OK) in rats (Test Facility Study No. 520295, see
Appendix 6), and in an attempt to produce graded responses to the test item.
A total of 40 females was selected at randomization before initiation of the pretest phase. As
none of the selected females was classified as not having regular estrous cycles during the
pretest phase, no replacement was performed before initiation of dosing. A total of 40
females with regular estrous cycles continued in the study. The supernumerary females were
removed from the study, and their estrous cycle results were kept in the raw data but not
reported.
Animals were assigned to groups by a computer-generated random algorithm according to
body weights, with all animals within ± 20% of the sex mean. Males and females were
randomized separately.

Positive control:

NOne

Examinations

Parental animals: Observations and examinations:

Mortality/Moribundity Checks – F0-Generation
Throughout the study, animals were observed for general health/mortality and moribundity
twice daily, in the morning and at the end of the working day. Animals were not removed
from cage during observation, unless necessary for identification or confirmation of possible
findings.

Clinical Observations – F0-Generation
Clinical observations were performed once daily, beginning during the first administration of
the test item and lasting throughout the dosing periods up to the day prior to necropsy.
During the dosing period, these observations were performed at no specific time point, but
within a similar time period after dosing for the respective animals (no peak effect of
occurrence of clinical signs was observed in the dose range finder (Test Facility Study No.
520295, see Appendix 6)).
The time of onset, grade and duration of any observed sign was recorded. Signs were graded
for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight
(grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs,
only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored
grades were reported, as well as the percentage of animals affected in summary tables.

Arena Observations – F0-Generation
Clinical observations were conducted in a standard arena beginning before the first
administration of the test item and then once weekly throughout treatment. These
observations were conducted after dosing.

. Body Weights – F0-Generation
Animals were weighed individually on the first day of treatment (prior to dosing) and weekly
thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and
during lactation on PND 1, 4, 7, and 13.
A terminal weight was recorded on the day of scheduled necropsy.

Food Consumption – F0-Generation
Food consumption was quantitatively measured weekly, except for males and females which
were housed together for mating and for females without evidence of mating. Food
consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum
and during lactation on PND 1, 4, 7, and 13.

Water Consumption – F0-Generation
Subjective appraisal was maintained during the study, but no quantitative investigation was
introduced for the test item groups as no effect was suspected.
From Day 10 of treatment (i.e. 01 December 2017) onwards, water consumption was
measured daily in control animals only. This data was used for collection of historical control
data only and will be kept in the raw data, but not evaluated or reported in this study. No
water consumption was measured for males and females which were housed together for the
mating period.

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by
vaginal lavage.
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment
(pretest period), the first 14 days of treatment and during mating until evidence of copulation
was observed. Vaginal lavage was continued for those females with no evidence of
copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
testis weight, epididymis weight, coagulation gland/seminal vesicle weight; histology of epididymis, coagulation gland, prostate gland, seminal vesicles, testis

Litter observations:

Mortality/Moribundity Checks – F1-Generation
Pups were observed daily for general health/mortality. The number of live and dead pups
were determined on PND 1 and daily thereafter. Pups were not removed from the cage
during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations – F1-Generation
Clinical observations were performed at least once daily for all pups. Only days on which
clinical signs were present between the first and last litter check were given in the respective
report tables.

Body Weights – F1-Generation

Live pups were weighed individually on PND 1, 4, 7 and 13.

Sex – F1-Generation
Sex was externally determined for all pups on PND 1 and 4.

Anogenital Distance – F1-Generation
Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was
normalized to the cube root of body weight.

Areola/Nipple Retention – F1-Generation
All male pups in each litter were examined for the number of areola/nipples on PND 13.

Postmortem examinations (parental animals):

Unscheduled Deaths
No animals died during the course of the study.

Scheduled Euthanasia
Animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using
isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
Scheduled necropsies were conducted on the following days:
-Males: Following completion of the mating period.
-Females which delivered: PND 14-16.
-Females which failed to deliver : With evidence of mating: Post-coitum Days 26 or 27
(nos. 47, 51, 53, 61, 69, 72 and 77).
Without evidence of mating: 26 days after the last day
of the mating period (no. 44).
All males were fasted overnight with a maximum of 24 hours before necropsy. Water was
available. F0- females were not fasted overnight.

Necropsy – F0-Generation
All animals were subjected to a full post mortem examination, with special attention being
paid to the reproductive organs.
Necropsy procedures were performed by qualified personnel with appropriate training and
experience in animal anatomy and gross pathology. A veterinary pathologist, or other
suitably qualified person, was available.
The numbers of former implantation sites were recorded for all paired females.
In case no macroscopically visible implantation sites were present, nongravid uteri were
stained using the Salewski technique in order to detect any former implantation sites and the
number of corpora lutea was recorded in addition.

Organ Weights – F0-Generation
The organs identified in the table below were weighed at necropsy for all scheduled
euthanasia animals. Paired organs were weighed together. In the event of gross
abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken
and recorded in the raw data. Organ to body weight ratios (using the terminal body weight)
were calculated.
Brain
Cervix (weighed together with uterus)
Epididymis (paired organ weight)
Gland, adrenal (paired organ weight)
Gland, coagulation (paired organ weight, together with seminal vesicles)
Gland, parathyroid (weighed with thyroid)
Gland, prostate
Gland, seminal vesicle (paired organ weight)
Gland, thyroid
Heart
Kidney (paired organ weight)
Liver
Ovaries (paired organ weight)
Spleen
Testes(paired organ weight)
Thymus
Uterus

Tissue Collection and Preservation – F0-Generation
Representative samples of the tissues identified below were collected from all
animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4%
formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated.
Animal identification
Artery, aorta
Body cavity, nasopharynx
Bone marrow
Bone, femur
Bone, sternum
Brain (seven levels)
Cervix
Epididymis
Esophagus
Eye
Gland, adrenal
Gland, coagulation
Gland, harderian
Gland, lacrimal
Gland, mammary
Gland, parathyroid
Gland, pituitary
Gland, prostate
Gland, salivary
Gland, seminal vesicle
Gland, thyroid
Gross lesions/masses
Gut-associated lymphoid tissue
Heart
Kidney
Large intestine, cecum
Large intestine, colon
Large intestine, rectum
Larynx
Liver
Lung
Lymph node (mandibular and mesenteric site)
Muscle, skeletal
Nerve, optic
Nerve, sciatic
Ovaries
Pancreas
Skin
Small intestine, duodenum
Small intestine, ileum
Small intestine, jejunum
Spinal cord
Spleen
Stomach
Testes
Thymus
Tongue
Trachea
Urinary bladder
Uterus
Vagina

Histology – F0-Generation
The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and
stained with hematoxylin and eosin:
Selected animals: Tissues identified above (except animal identification, aorta,
nasopharynx, esophagus, harderian gland, lacrimal gland, salivary gland, larynx, optic nerve, pancreas, skin
and tongue).
Males that failed to sire (except for
males which were selected) and
females that failed to deliver pups: Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina.

Postmortem examinations (offspring):

Terminal Procedures – F1-Generation
Method of Euthanasia - F1-Generation
Pups, younger than 7 days were euthanized by decapitation.
All remaining pups (PND 7-16), except for the two pups per litter selected for blood
collection were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol®
20%).
The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane
followed by exsanguination.

Unscheduled Deaths - F1-Generation
Pups that died before scheduled termination were examined externally and sexed (both
externally and internally).
Pups found dead during the weekend were fixed in identified containers containing 70%
ethanol as they were not necropsied on the same day.
The stomach of pups not surviving to the scheduled necropsy date was examined for the
presence of milk, if possible. If possible, defects or cause of death were evaluated.

Scheduled Euthanasia - F1-Generation
On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two
surplus pups per litter, blood was collected, if possible. For details see also section 4.11.1.
All remaining pups were euthanized on PND 14-16. Sex was determined both externally and
internally. Descriptions of all external abnormalities were recorded. Particular attention was
paid to the external reproductive genitals to examine signs of altered development. External
abnormalities were collected and fixed in 10% buffered formalin at discretion of the Study
Director.
In addition, blood was collected from two pups per litter (see also section 4.11.1), and the
thyroid from two pups per litter (one male and one female pup, if possible) was preserved in
10% buffered formalin. Pups selected for blood sampling were the same pups as selected for
thyroid preservation.

Statistics:

ll statistical tests were conducted at the 5% significance level. All pairwise comparisons
were conducted using two sided tests and were reported at the 1% and or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as
indicated according to sex and occasion. Descriptive statistics number, mean and standard
deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
Inferential statistics were performed according to the matrix below when possible, but
excluded semi-quantitative data, and any group with less than 3 observations.

The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were
compared using Dunnett-test (many-to-one-t-test).

Non-Parametric
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
The motor activity data set was compared using an overall Kruskal-Wallis.

Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level.
The above pairwise comparisons were conducted using Fisher’s exact test whenever the
overall test is significant.

Reproductive indices:

Mating index (%):
(Number of females mated /Number of females paired ) x 100

Precoital time: Number of days between initiation of cohabitation and
confirmation of mating

Fertility index (%):
( Number of pregnant females/Number of females mated) x 100

Gestation index (%):
(Number of females with living pups on Day 1 /Number of pregnant females ) x 100

Duration of gestation: Number of days between confirmation of mating and the
beginning of parturition

Offspring viability indices:

Post-implantation survival index (%):
(Total number of offspring born/ Total number of uterine implantation sites ) x100

Live birth index (%):
(Number of live offspring on Day 1 after littering/Total number of offspring born) x100

Percentage live males at
First Litter Check (%):
(Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x100

Percentage live females at
First Litter Check (%):
(Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x100

Viability index (%):
(Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering ) x100

Lactation index (%):
(Number of live offspring on Day 13 after littering/ Number live offspring on Day 4 (after culling) ) x100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Fluid feces at a slight severity was noted in all cages (both sexes) at 300 and 1000 mg/kg on
treatment Days 7-15.
Any other clinical signs noted incidentally occurred within the range of background findings
to be expected for rats of this age and strain which are housed and treated under the
conditions in this study and showed no dose-related trend. At the incidence observed, these
were considered to be unrelated to treatment.
No clinical signs were noted during the weekly arena observations.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weights and body weight gain were considered not to be affected by treatment.
A finding of note at 300 mg/kg was the reduced body weight gain or slight weight loss which
occurred in half of the males and most females in the first week of the treatment period. As a
result, mean body weights at 300 mg/kg were about 5% lower compared to controls from
treatment Day 8 onwards (statistically significant in females on two occasions). After the
first treatment week, 300 mg/kg animals grew normally. As weight gain at 1000 mg/kg was
normal, the transiently reduced weight gain at 300 mg/kg was considered not to be related to
treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically relevant changes in food consumption before or after
correction for body weight.
Slightly higher food consumption values were noted in females at 1000 mg/kg from lactation
Day 4 towards the end of the treatment period, correlating with slightly higher body weight
gain. Relative food consumption differed up to 9% when compared to controls. As values
remained within the normal range, this finding was regarded as non-adverse.

Food efficiency:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to be affected by treatment.
Except for one 300 mg/kg female which had an irregular cycle during the premating period
(no. 65), all females had regular cycles of four days. Female no. 65 had a normal precoital
interval (evidence of mating after one day of cohabitation) and a normal litter.
Extended di-estrus during pairing was noted in two females which were non-mated (control
no. 44) or non-pregnant (no. 77 at 1000 mg/kg). These findings were not attributed to
treatment due to the incidental occurrence and lack of a dose-related trend.
9.3.2. Mating Index

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

stage aware evaluation of the testes did not show any indication
for abnormal spermatogenesis.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

In each group, 2/10 couples had no offspring. One control couple showed no evidence of
mating (female/male nos. 44/04), four females were not pregnant (female/male nos. 51/11 at
100 mg/kg, 61/21 and 69/29 at 300 mg/kg, and 77/37 at 1000 mg/kg), and three females had
implantation sites only (control female/male nos. 47/07, 53/13 at 100 mg/kg and 72/32 at
1000 mg/kg).
Female no. 61 (300 mg/kg) had marked bilateral cysts in the ovaries and female no. 77
(1000 mg/kg) showed inflammatory infiltrate with granulocytic cells in the vaginal lumen.
These lesions were considered to be the cause of the infertility of these females. For the other
couples histopathology did not reveal any changes in the reproductive organs that could
explain their lack of offspring.
There were no morphological findings in the reproductive organs of either sex which could be
attributed to the test item, and stage aware evaluation of the testes did not show any indication
for abnormal spermatogenesis.

Mating Index
Mating index was considered not to be affected by treatment. Except for one control female
(no. 44, paired with male no. 4) all females showed evidence of mating. The mating indices
were 90% for the control and 100% for the other groups.
For the couple of the control group (no. 4 and 44) no morphological changes were seen in
male or female reproductive organs that could explain their unsuccessful mating.

9.3.3. Precoital Time
Precoital time was not affected by treatment. All mated females showed evidence of mating
within four days.

Number of Implantation Sites
Number of implantation sites was considered not to be affected by treatment.
Two pregnant females treated with the test item had only one implantation site (nos. 53 and
72 at 100 and 1000 mg/kg, respectively; these females had no offspring). Such a low number
of implantation sites occasionally occurs in untreated controls (concurrent control female no.
47 had only two implantation sites and no offspring). The other treated females had normal
numbers of implantation sites. The low numbers of implantation sites in two treated females
were regarded as unrelated to treatment due to the incidental occurrence and lack of a doserelated
trend.

Fertility Index
Fertility index was considered not to be affected by treatment. Except for one female at
100 mg/kg (no. 51), two females at 300 mg/kg (nos. 61 and 69), and one female at 1000
mg/kg (no. 77), all mated females were pregnant. The fertility indices were 100, 90, 80 and
90% for the control, 100, 300 and 1000 mg/kg groups, respectively.
Female no. 77 showed inflammatory infiltrate with granulocytic cells in the vaginal lumen,
which was judged to be the cause of the unsuccessful mating of the 1000 mg/kg couple. This
finding was considered to be unrelated to treatment due to its incidental occurrence. Female
no. 61 showed marked bilateral cysts in the ovaries, which was judged to be the cause of her
infertility. The other non-pregnant females showed no related histopathology changes in the
reproductive organs.
In the absence of a dose-related trend, these incidental cases of non-pregnancy were
considered to be unrelated to treatment.

Gestation Index and Duration
Gestation index and duration of gestation were considered not to be affected by treatment.
Except for one control female (no. 47), one female at 100 mg/kg (no. 53) and one female at
1000 mg/kg (no. 72), all pregnant females had live offspring. The gestation indices were 89%
for the control, 100 and 1000 mg/kg groups and 100% for the 300 mg/kg group.
These cases of failed pregnancy, without supportive morphological changes in reproductive
organs, were judged to be unrelated to treatment due to the incidental occurrence and lack of
a dose-related trend.

Parturition/Maternal Care
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature
birth. No deficiencies in maternal care were observed.

Post-Implantation Survival Index
Post-implantation survival index (total number of offspring born as percentage of total
number of uterine implantation sites) was considered not to be affected by treatment. The
survival indices were 92, 92, 84 and 87% for the control, 100, 300 and 1000 mg/kg groups,
respectively.
At 300 and 1000 mg/kg, post-implantation survival index appeared lower compared to the
control group. For the 300 mg/kg group this could be explained by an abnormally high
number of unaccounted for sites in female no. 70 (7/9 implantation sites were unaccounted
for). This incidental high number of unaccounted for sites in a mid-dose female was regarded
as unrelated to treatment. As the indices remained within the range of historical control data
and as no dose-responserelationship was observed, these changes were considered not to reflect an effect of the test
item.

Litter Size
The mean number of living pups at first litter check (live litter size) was considered not to be
affected by treatment.

Live Birth Index
Live birth index (number of live offspring on PND 1 as percentage of total number of
offspring born) was not affected by treatment. The live birth indices were 97% for the
100 mg/kg group and 100% for the other groups.
At first litter check, three pups of the 100 mg/kg group (litter nos. 54 and 58) were found
dead. This incidental pup mortality in the lowest dose group was unrelated to treatment.

Viability Index
Viability index (number of live offspring on PND 4 before culling as percentage of number of
live offspring on PND 1) was considered not to be affected by treatment. The viability
indices were 99, 96, 100 and 99% for the control, 100, 300 and 1000 mg/kg groups,
respectively.
One pup of the control group (litter no. 41), four pups at 100 mg/kg (one in litter no. 54, three
in litter no. 55) and one pup at 1000 mg/kg (litter no. 75) went missing (presumed
cannibalized) on PND 2 or 3. This early postnatal loss was judged not to be related to
treatment as the incidence showed no dose-related trend and remained within the range
considered normal for pups of this age.

Lactation Index
Lactation index (number of live offspring on PND 13 as percentage of number of live
offspring after culling on PND 4) was not affected by treatment. The lactation indices were
98, 98, 100 and 100% for the control, 100, 300 and 1000 mg/kg groups, respectively.
One pup of the control group (litter no. 50) and one pup of the 100 mg/kg group (litter no. 56)
were found dead on PND 15 and PND 5, respectively). The death of a single pup in the
lowest dose group was regarded as unrelated to treatment.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

other: no treatment related adverse effects observed

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment.
The clinical signs observed incidentally remained within the range considered normal for
pups of this age, and were therefore considered to be unrelated to treatment.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Viability index (number of live offspring on PND 4 before culling as percentage of number of
live offspring on PND 1) was considered not to be affected by treatment. The viability
indices were 99, 96, 100 and 99% for the control, 100, 300 and 1000 mg/kg groups,
respectively.
One pup of the control group (litter no. 41), four pups at 100 mg/kg (one in litter no. 54, three
in litter no. 55) and one pup at 1000 mg/kg (litter no. 75) went missing (presumed
cannibalized) on PND 2 or 3. This early postnatal loss was judged not to be related to
treatment as the incidence showed no dose-related trend and remained within the range
considered normal for pups of this age.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights of pups were considered not to be affected by treatment.
The slightly lower mean pup body weights noted at 100 mg/kg were considered to reflect
normal background variation. The differences from control values, about 5% throughout the
lactation period, were not statistically significant.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 14-16 pups were not affected by treatment.

Sexual maturation:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to
treatment.
The nature and incidence of the few findings noted remained within the range considered
normal for pups of this age, and were therefore considered to be unrelated to treatment.

Other effects:

no effects observed

Description (incidence and severity):

Sex Ratio
Sex ratio was considered not to be affected by treatment.

Anogenital Distance
Anogenital distance (absolute and normalized for body weight) in male and female pups was
not affected by treatment.
It was noted that mean anogenital distances in all test item groups were lower compared to the
control means. These intergroup differences, which showed no dose-related response, were
considered to be the result of slightly high concurrent control values (values in two control
litters, nos. 45 and 46 exceeded the historical control ranges) rather than an effect of the test
item. Mean values in the test item groups were close to (male pups) or slightly lower than
(female pups) the historical control mean.

Areola/Nipple Retention
Treatment up to 1000 mg/kg had no effect on areola/nipple retention. For none of the
examined male pups nipples were observed at PND 13.

Effect levels (F1)

Dose descriptor:

NOAEL

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 treatment related adverse effect observed

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with
the reproduction/developmental toxicity screening test, the following No Observed Adverse
Effect Levels (NOAELs) of Furoxy Hydroxy (OK) were established:
Parental NOAEL: at least 1000 mg/kg.
Reproduction NOAEL: at least 1000 mg/kg.
Developmental NOAEL: at least 1000 mg/kg.