DL-α-(aminomethyl)-p-hydroxybenzylic alcohol hydrochloride

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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:

minor deviations in observation periods which did not affect the results of the study

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

1

Reference substance name:

DL-α-(aminomethyl)-p-hydroxybenzylic alcohol hydrochloride

EC Number:

212-216-4

EC Name:

DL-α-(aminomethyl)-p-hydroxybenzylic alcohol hydrochloride

Cas Number:

770-05-8

Molecular formula:

C8H11NO2.ClH

IUPAC Name:

4-(2-amino-1-hydroxyethyl)phenol hydrochloride

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Identification: Octopamine hydrochloride
Appearance: White to off white powder (determined by
Charles River Den Bosch)
Batch: D151-1710037
Purity/Composition: 99.8%
Test item storage: At room temperature protected from light
Stable under storage conditions
until: 26 October 2019 (retest date)

Test animals

Species:

rat

Strain:

Wistar

Remarks:

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 study plan was reviewed and agreed by the Animal Welfare Body of Charles River
Laboratories Den Bosch B.V. within the framework of project license AVD2360020172866
approved by the Central Authority for Scientific Procedures on Animals (CCD) as required
by the Dutch Act on Animal Experimentation (December 2014).

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
On 12 Sep 2018, female Crl: WI(Han) rats were received and on 26 Sep 2018, male
Crl:WI(Han) rats were received from Charles River Deutschland, Sulzfeld, Germany. At
initiation of dosing, males were 10-11 weeks old and weighed between 268 and 310 g and
females were 13-14 weeks old and weighed between 200 and 236 g.
A health inspection was performed before the initiation of dosing.

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

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 cageenrichment, bedding material, food and water.
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 room in
which the animals were kept was 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.

DETAILS OF FOOD AND WATER QUALITY:

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.
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 was 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.

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 20 to 23°C with
an actual daily mean relative humidity of 41 to 70%. 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.
For psychological/environmental enrichment and nesting material, animals were provided
with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).


IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration was selected because this is a possible route of human
exposure during manufacture, handling or use of the test item.
The dose levels were selected based on the results of a 10-day dose range finder with oral
administration of Octopamine hydrochloride in rats (Test Facility Study No. 20152396), and in an attempt to produce graded responses to the test item.

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at
appropriate concentrations to meet dose level requirements. The dosing formulations were
prepared at least weekly as a solution, filled out in daily portions and stored in the
refrigerator. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing.
Details of the preparation and dispensing of the test item have been retained in the Study
Records.
Test item dosing formulations were kept at room temperature until dosing. If practically
possible, the dosing formulations and vehicle were continuously stirred until and during
dosing.
No adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.
Any residual volumes were discarded

VEHICLE
- Justification for use and choice of vehicle (if other than water):
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 was complete. These trial preparations have a non-GLP status and were carried out in the quality assured environment of the Test Facility.

- Concentration in vehicle: 0, 20 , 60 and 200/120 mg/mL
- Amount of vehicle (if gavage): 5mL
- Lot/batch no. (if required): N/A
- Purity: N/A

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

All samples to be analyzed were transferred (at room temperature under normal laboratory
light conditions) to the analytical laboratory at the Test Facility.
Residual samples were discarded after completion of the sample analysis.
4.5.2.1. Analytical Method
Analyses were performed using a validated analytical procedure (Test Facility Study No.
20152395).
4.5.2.2. Concentration Analysis
Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time
point were sent to the analytical laboratory. Concentration results were considered acceptable
if mean sample concentration results were within or equal to ± 10% for solutions of target
concentration.
4.5.2.3. Homogeneity Analysis
Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time
point were sent to the analytical laboratory. Homogeneity results were considered acceptable
if the coefficient of variation (CV) of concentrations was ≤ 10%.
4.5.2.4. Stability Analysis
Stability analyses performed previously in conjunction with the method development and
validation study (Test Facility Study No. 20152395) 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. 20152395.

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 of Groups 1-3 were treated for 29
days, i.e. 14 days prior to mating, during mating and up to and including the day before
scheduled necropsy. Females of Groups 1-3 (all with healthy offspring) were treated for 50-
54 (most females) or 64 days (one female of Group 2), 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. Due to severe toxicity, resulting in two premature deaths, the surviving
Group 4 males were sacrificed for ethical reasons on study Day 10 (last dose on Day 9).
Consequently, the Group 4 females were not mated and sacrificed on Day 30 (last dose on
Day 29).
The first day of dosing was designated as Day 1 (exception: alternate animals used for
replacement after Day 1 was assumed the day of the animal being replaced).
Animals were dosed approximately at the same time each day. 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:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

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)

Remarks:

reduced to 600 mg/kg bw/day from Day 3 onwards due to unexpected severe toxicity in dosed animals on day 1 and 2

No. of animals per sex per dose:

10 (ten)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
A total of 40 females was selected at randomization before initiation of the pretest phase.
Selected females classified as not having regular estrous cycles during the pretest phase were replaced before initiation of dosing by reserve females having regular estrous cycles. 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.
On Day 1 of treatment, after dosing, male No. 37 and female No. 74 were sacrificed in
extremis and female No. 77 was found dead. Due to the unexpected toxicity, reserve animals
were used from Study Day 2 onwards as replacements.

Positive control:

no positive control

Examinations

Observations and examinations performed and frequency:

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.
Animals showing pain, distress or discomfort which was considered not transient in nature or
is likely to become more severe, were sacrificed for humane reasons based on OECD
guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of
any death were recorded in detail.

Clinical Observations – F0-Generation
Clinical observations were performed once daily, beginning during the first administration of
the test item and lasting throughout the dosing period up to the day prior to necropsy.
Initially, these observations were performed one hour post dose (based on the peak effect of
occurrence of clinical signs observed in the dose range finder (Test Facility Study No.
20152396, see Appendix 6). From Study Day 9 onwards, animals were observed at least
twice daily, up to the day prior to necropsy, during the following observation intervals: prior
to dosing (to monitor overnight recovery) and 3 hours (± 30 minutes) after dosing. On one
occasion, animals were also observed approximately 4 hours after dosing.
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 before dosing.

Body Weights – F0-Generation
Animals were weighed individually on the first day of treatment (prior to dosing), and weekly
thereafter. Additional body weights were collected for all animals on Day 3 of the study.
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.
Terminal body weights were recorded on the day of necropsy (fasted for males and Group 4
females (non-lactating), non-fasted for females of Groups 1-3 (lactating).

Food Consumption – F0-Generation
Food consumption was quantitatively measured weekly, except for males and females which
were housed together for mating. Additionally, food consumption was determined for all
cages on Day 3 of the study. 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 as no effect was expected or noted at visual inspection of the water bottles.

Functional Tests – F0-Generation
Functional tests were performed on the selected 5 males of Groups 1-3 during Week 4 of
treatment and on the selected 5 females of Groups 1-3 during the last week of lactation (i.e.
PND 6-13). The first 5 females of Group 4 (non-lactating) were tested towards the end of
their 29-day treatment period (on Day 24). Males of Group 4 (terminated on Day 10) were
not subjected to functional tests. All functional tests were performed before dosing.
The following tests were performed (abbreviations mentioned in the respective tables are
indicated between brackets):
• Hearing ability (HEARING) (Score 0 = normal/present, score 1 = abnormal/absent).
• Pupillary reflex (PUPIL L/R) (Score 0 = normal/present, score 1 = abnormal/absent).
• Static righting reflex (STATIC R) (Score 0 = normal/present, score 1 =
abnormal/absent).
• Fore- and hind-limb grip strength, recorded as the mean of three measurements per
animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
• Locomotor activity (recording period: 1-hour under normal laboratory light
conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway,
USA). Total movements and ambulations were reported. Ambulations represent
movements characterized by a relocation of the entire body position like walking,
whereas total movements represent all movements made by the animals, including
ambulations but also smaller or more fine movements like grooming, weaving or
movements of the head.

Clinical Pathology
.Sample Collection
Blood of F0-animals (except for Group 4 males) was collected on the day of scheduled
necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus
under anesthesia using isoflurane in the animal facility. After collection all samples were
transferred to the appropriate laboratory for analysis.
F0-males of Groups 1-3 and F0-females of Group 4 were fasted overnight with a maximum of
24 hours before blood sampling, but water was available. F0-females of Groups 1-3 were not
fasted.

Hematology
Blood samples at a target volume of 0.5 mL were collected into tubes containing K3-EDTA as
anticoagulant. Samples were analyzed for the parameters specified in the following table:
White blood cells (WBC); Red Blood Cell Distribution Width (RDW)
Neutrophils (absolute); Haemoglobin
Lymphocytes (absolute); Haematocrit
Monocytes (absolute); Mean corpuscular volume (MCV)
Eosinophils (absolute); Mean corpuscular haemoglobin (MCH)
Basophils (absolute); Mean corpuscular haemoglobin concentration (MCHC)
Red blood cells; Platelets
Reticulocytes (absolute)
A blood smear was prepared from each hematology sample. Blood smears were labeled,
stained, and stored. In case additional examination of blood smears was deemed necessary,
the smears were subsequently evaluated.

Coagulation
Blood samples at a target volume of 0.45 mL were collected into tubes containing Citrate as
anticoagulant. Samples were processed for plasma, and plasma was analyzed for prothrombin time (PT) and activated partial Thromboplastin time (APTT).

Clinical Chemistry
Blood samples at a target volume of 0.5 mL (plasma) were collected into tubes containing LiHeparin as anticoagulant. Serum samples at a target volume of 1.0 mL were collected in
tubes without anticoagulant. Blood samples were processed for plasma or serum (bile acids),
which was analyzed for the parameters specified in the following table.
Alanine aminotransferase (ALAT); Creatinine
Aspartate aminotransferase (ASAT); Glucose
Alkaline Phosphatase (ALP); Cholesterol
Total protein; Sodium
Albumin; Potassium
Total Bilirubin; Chloride
Bile Acids; Calcium
Urea; Inorganic Phosphate (Inorg. Phos)

Thyroid hormone
Blood samples at a target volume of 1.0 mL (F0-animals), were collected into tubes without anticoagulant. Blood samples
were processed for serum, and serum was analyzed for total Thyroxine (T4). Measurement of
total T4 was conducted for F0-males.
For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH;
both sexes) was considered not relevant because there were no treatment-related changes in
T4 in F0-males, or in the weight and morphology of the thyroid in both sexes.
Serum samples retained for possible future analysis were maintained by the Test Facility in
the freezer (≤-75°C). Under these storage conditions, samples are stable for 6 months. Any
remaining sample will be discarded.

Sacrifice and pathology:

A necropsy was conducted for animals that died on study, and specified tissues were saved
(Group 4 female No. 77 found dead and replaced on Day 1, and Group 4 male No. 37 found
dead on Day 10).
Three animals were euthanized for humane reasons as per Test Facility SOPs (Group 4
animal Nos. 37 and 74 sacrificed and replaced on Day 1, and No. 36 sacrificed on Day 8).
These animals were deeply anaesthetized using isoflurane and subsequently exsanguinated.
They underwent necropsy, and specified tissues were retained but not weighed.
On Day 10 of the study, remaining Group 4 males were euthanized for ethical reasons. At
necropsy, a terminal body weight was determined, animals were subjected to a full post
mortem examination and specified tissues were saved, but not weighed.

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 of Groups 1-3: Following completion of the mating period (29 days
of administration).
Females of Group 4: Following a 29-day treatment period.
Females which delivered (Group 1-3): PND 14-16.
All males surviving to scheduled necropsy and females of Group 4 were fasted overnight with
a maximum of 24 hours before necropsy. Water was available. Other F0- females were not
fasted.

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.

Organ Weights – F0-Generation
The organs identified in the table below were weighed at necropsy for all scheduled
euthanasia animals (males of Groups 1-3, females of Groups 1-4). Organ weights were not
recorded for Group 4 males. Paired organs were weighed together. Organ to body weight
ratios (using the terminal body weight) were calculated.

Organs Weighed at Necropsy for all selected animals
Brain; Cervix
Epididymis; Gland, adrenal
Gland, coagulation; Gland, parathyroid
Gland, prostate; Gland, seminal vesicle
Gland, thyroid; Heart
Kidney; Liver
Ovaries; Spleen
Testes; Thymus
Uterus;

Organs Weighed at Necropsy for all remaining animals
Epididymis; Gland, coagulation,
Gland, parathyroid; Gland, prostate
Gland, seminal vesicle; Gland, thyroid
Testes

Tissue Collection and Preservation – F0-Generation
Representative samples of the tissues identified in the table 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.

Tissue Collection and Preservation for all selected animals, all Group 4 males and the three Group 4 animals that
were found dead/sacrificed in extremis and replaced on Day 1.
Animal identification
Artery, aorta; Body cavity, nasopharynx
Bone marrow; Bone, femur
Bone, sternum; Brain (eight levels)
Cervix; Epididymidesa
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

Tissue Collection and Preservation for all remaining animals.
Animal identification; Cervix
Epididymis; Gland, coagulation
Gland, mammary; Gland, parathyroid
Gland, pituitary; Gland, prostate
Gland, seminal vesicle; Gland, thyroid
Gross lesions/masses; Ovaries
Testes; Uterus
Vagina

Histology – F0-Generation
Tissue processing and trimming was performed at the Test Facility. All other histology
procedures were performed by Den Bosch.
The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and
stained with hematoxylin and eosin:
Selected animals of Groups 1-3: Tissues identified in Text Table 11 (except animal
identification, aorta, nasopharynx, esophagus, harderian
gland, lacrimal gland, salivary gland, larynx, optic nerve,
pancreas, skin and tongue).
Remaining animals of Groups 1-3: Gross lesions/masses.
Histology was not performed for males and females of Group 4.

Histopathology – F0-Generation
All tissues as defined under Histology – F0-Generation (section 4.11.6) were examined by a
board-certified toxicological pathologist with training and experience in laboratory animal
pathology. Target tissues identified by the study pathologist during microscopic evaluation
were communicated to the Study Director; tissues were evaluated and reported.
For the testes of all selected males of Groups 1 and 3 a detailed qualitative examination was
made, taking into account the tubular stages of the spermatogenic cycle.
A peer review on the histopathology data was performed by a second pathologist

Other examinations:

CONSTRUCTED VARIABLES
All results presented in the tables of the report were calculated using values as per the raw
data rounding procedure and may not be exactly reproduced from the individual data
presented.
Parental Variables:
Body Weight Gain: Calculated against the body weight on Day 1 (premating, mating and lactation periods) or Day 0 (postcoitum period).
Relative Food Consumption: Calculated against the body weight for scheduled intervals.
Organ Weight Relative to Body Weight: Calculated against the terminal body weight.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons
were conducted using two sided tests and were reported at the 1% 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 were reported whenever possible. Inferential statistics were performed according to
the comparison 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. The Wilcoxon
Rank-Sum test was applied to compare the treated groups to the control group.

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.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment at 1000 mg/kg was associated with clinical signs of toxicity. On treatment Day 1,
all animals showed piloerection and quick breathing, and most animals showed a flat posture
and slight or moderate salivation. The next day, one male and one female dosed at
1000 mg/kg showed piloerection, quick breathing and a flat posture (the remaining Group 4
animals were not dosed on Day 2). Because of the severe toxicity, the highest dose level was
reduced to 600 mg/kg from Day 3 onwards.
After reduction of the highest dose level, piloerection persisted in males and females.
Additionally, on treatment Day 9, severe lethargy, a flat or hunched posture, quick breathing,
moderate salivation and/or chromodacryorrhoea were noted in a few 600 mg/kg males
(including No. 37 which was found dead the next day). To prevent further suffering, all
surviving high-dose males were sacrificed on Day 10. In females, additional clinical signs
consisted of slight lethargy on Day 12 (all females) and slight salivation on several days from
Day 15 onwards (in up to all females).
At 300 mg/kg, most animals showed piloerection after dosing from treatment Day 6 (males)
or Day 8 (females) onwards. Additionally, slight lethargy was noted on treatment Day 12 (in
most animals) and hunched posture was noted on treatment Day 32 (in most females).
No treatment-related clinical signs were noted at 100 mg/kg

Mortality:

mortality observed, treatment-related

Description (incidence):

Test item-related mortality occurred at 1000/600 mg/kg.
On treatment Day 1, after dosing, high-dose female No. 77 died spontaneously and two highdose animals were sacrificed for humane reasons (female No. 74 and male No. 37). Clinical
signs (which included piloerection, flat posture, quick breathing and salivation) and
macroscopic findings in these animals are presented below. These three decedents
were replaced by spare animals which were dosed from Day 3 onwards.
High-dose male No. 36 was sacrificed for humane reasons on treatment Day 8. A few hours
after dosing on Day 8, this animal lay in its cage without any movement apart from breathing,
and barely responded to touch or sound (reported in Study Daybook). The animal lost 3% of
its initial weight over Days 1-8. Findings at necropsy were limited to red-brown foci on the
kidneys.
High-dose male No. 37 was found dead on study Day 10 and showed advanced autolysis at
necropsy. After dosing on Day 9, this animal showed severe lethargy, a flat posture, quick
breathing and piloerection. Its body weight gain between Days 1-8 was normal.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Half of the males treated at 1000/600 mg/kg lost some weight over Days 1-3 (up to 4% of
their initial weight). Thereafter, most high-dose males gained weight, though less than
controls. Mean body weight gain of 1000-600 mg/kg males was statistically lower than that
of controls on treatment Days 3 and 8, but mean body weights did not differ statistically
significantly (relative difference at Day 8: -3%). In addition, five out of the eight males
sacrificed on Day 10 showed a slight body weight loss (up to -3.2%) between Day 8 and
Day 10.
Males at 300 mg/kg showed slightly reduced body weight gain from initiation of treatment
(achieving statistical significance after three weeks). The resulting reduction in mean body
weight at the end of their 29-day treatment period was 4% (not statistically significantly
different from the control mean).
Females treated at 1000/600 mg/kg showed normal body weight development during their 29-
day treatment period.
Females at 300 mg/kg showed slightly reduced mean body weights throughout gestation
(from -4% to -7% at post-coitum Days 0 and 20, respectively) and lactation (mostly -5%).
Statistical significance was reached from post-coitum Day 11. Mean body weight gain of
300 mg/kg females during gestation and lactation was not remarkably different from that of
controls.
Body weights and body weight gain of males and females treated at 100 mg/kg were
unremarkable.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of males and females at 1000/600 mg/kg was reduced by about 25-30% in
the interval Day 1-3. Thereafter, when the dose level was reduced from 1000 to 600 mg/kg,
their food consumption returned to normal.
Food consumption of rats treated at 100 or 300 mg/kg was not affected by treatment.

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematology parameters (red and white blood cell parameters, number of platelets) showed no
differences between control and treated rats that were considered to be toxicologically
significant.
The mean number of reticulocytes in females treated at 100 or 300 mg/kg was lower than that
in concurrent controls (-15 and -30%, respectively). These differences were considered not to
reflect an adverse effect of the test item on red blood cells as the main red blood cell
parameters (hemoglobin concentration, number of red blood cells) of treated females were not
affected. Moreover, no statistical significance was achieved and the concurrent control mean
was relatively high (at the upper limit of the historical range).
Hematology values in females treated at 1000/600 mg/kg generally remained close to the
historical control means.
An isolated, statistically significant variation noted in males was considered to be unrelated to
treatment due to the lack of a dose-related response (lower hemoglobin at 100 mg/kg).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following, statistically significant, changes distinguished animals treated up to 300 mg/kg
from control animals. Relative changes in mean values compared to the control group are
indicated between parentheses.
• Higher concentration of urea in males at 300 mg/kg (+39%).
• Higher concentration of creatinine in males at 300 mg/kg (+11%).
Mean urea and creatinine values in 300 mg/kg males remained in the historical control
ranges6
.
The other statistically significant variations noted were considered to be unrelated to
treatment due to the lack of a dose-related response (lower inorganic phosphate in 100 mg/kg
males) or minimal magnitude of the difference from controls (lower sodium in 300 mg/kg
males).
Females treated at 1000/600 mg/kg showed the following remarkable differences from
historical controls:
• Higher concentration of urea (+46% relative to the historical control mean).
• Higher concentration of bile acids (2.9x of the historical control mean).
• Higher concentration of calcium (+6% relative to the historical control mean).
Mean values (and most individual values) for urea, bile acids and calcium in 1000/600 mg/kg
females exceeded the P95-limits of the historical ranges.
Thyroid hormone analyses:
Serum levels of T4 in F0 males were considered not to be affected by treatment up to
300 g/kg.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation parameters were considered not to be affected by treatment up to
300 mg/kg (males) or 1000/600 mg/kg (females).
Hearing ability, pupillary reflex and static righting reflex were normal in all examined
animals. Forelimb and hind limb grip strength were not affected by treatment.
The variation in motor activity did not indicate a relation with treatment. All examined
groups showed a similar habituation profile with a decreasing trend in activity over the
duration of the test period. A higher mean value for ambulations noted in 300 mg/kg females
(+35% relative to controls) was regarded as unrelated to treatment since the difference was
not statistically significant and not accompanied by changes in other measures in the
neuromuscular domain (including total movements, air righting reflex and grip strength). In
addition, individual values generally remained within the historical control data range3
.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related organ weight changes.
A statistically significant difference noted in 300 mg/kg females (absolute spleen weight 21%
lower than controls) remained within the historical control range and was not accompanied
by a significant change in relative spleen weight. Therefore, this finding was not attributed to
treatment.
Mean organ weights (absolute and relative to body weight) of females treated at
1000/600 mg/kg remained within normal limits

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no test item-related gross observations in animals treated at 100 or 300 mg/kg.
The few macroscopic findings in these animals were within the range of background gross
observations encountered in rats of this age and strain. These findings were therefore
considered to be unrelated to treatment.
Macroscopic findings in the decedents of the 1000/600 mg/kg group are described in
mortality section above. Macroscopic findings in the eight high-dose males
that were sacrificed for ethical reasons on study Day 10 were limited to the presence of dark
red foci in the glandular stomach of one of these animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the adrenal gland of males at
300 mg/kg, as summarized and described below.
Vacuolation of the zona glomerulosa in the adrenal gland was present in 4/5 males at minimal
to slight degree.
The remainder of the recorded microscopic findings were within the range of background
pathology encountered in rats of this age and strain. There was no test item-related alteration
in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Coagulation parameters (PT (prothrombin time) and APTT (activated partial thromboplastin
time)) of rats treated at 100 or 300 mg/kg showed no differences between control and treated
rats that were considered to be toxicologically significant.
It was noted that APTT tended to be reduced in females treated at 300 mg/kg (-15% relative
to concurrent controls). Mean APTT at 300 mg/kg was slightly below the P5-limit of the
historical control range5
. Statistical significance was not achieved, values in treated females
were not critically low, and there were no associated adverse anatomic pathology alterations.
As such, the lower APTT in 300 mg/kg females was regarded as non-adverse.
Mean PT and APTT values in females at 1000/600 mg/kg differed by about 15% from the
historical control means (see Appendix 9 for historical control data for nulliparous female
Wistar Han rats). Mean PT slightly exceeded the historical range whereas APTT was at the
lower end of this range.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

Target system / organ toxicity

Critical 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 Octopamine hydrochloride were established:
Parental NOAEL: 300 mg/kg, based on mortality and clinical signs of toxicity at 1000/600 mg/kg which resulted in premature termination of the
males (on Study Day 10).

Executive summary:

The objectives of this study were to determine the potential toxic effects of Octopamine hydrochloride when given orally by gavage for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated. The dose levels in this study were selected to be 0, 100, 300 and 1000 mg/kg, (Groups 1 to 4 respectively) based on the results of the dose range finder.

In Groups 1-3, males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days) and females were treated for two weeks prior to mating, during mating, during post-coitum, and 13-15 days of lactation (mostly for 50-54 days). In Group 4, males were treated for 9 days and sacrificed for ethical reasons on Study Day 10. Consequently, the Group 4 females were not mated and sacrificed on Day 30 (last dose on Day 29). These females were therefore excluded from the reproductive part of the study. As no concurrent controls were available for comparison with the Group 4 animals, no histopathologic examination was performed for these animals. Chemical analyses of formulations were conducted during the study to assess accuracy and homogeneity. The following parental parameters were evaluated in this study: mortality/moribundity, clinical signs, functional tests, body weight, food consumption, estrous cycle length and regularity, clinical pathology, serum level of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examination (Groups 1-3). In addition, the following reproduction/developmental parameters were determined for Groups 1-3: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio, live litter size and early postnatal pup development (mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, serum level of thyroid hormone T4 in PND 14-16 pups, and macroscopy). Formulation analysis showed that formulations were prepared accurately and homogeneously.

Parental results Treatment at 1000 mg/kg resulted in mortality (one male and two females died or were sacrificed for humane reasons on Day 1), clinical signs of toxicity (piloerection, quick breathing, flat posture and salivation) and reduced food consumption in both sexes and body weight loss in males. After reduction of the dose level to 600 mg/kg on Day 3, food consumption returned to normal and males gained weight, though less than controls. However, piloerection persisted in both sexes and, after dosing on Day 9, a few males showed severe lethargy, a flat or hunched posture, quick breathing, moderate salivation and/or chromodacryorrhoea. One of these males was found dead the next day, whereafter the surviving high-dose males were sacrificed for ethical reasons. Toxicity was less severe in females dosed at 600 mg/kg. These females survived until their scheduled sacrifice, showed normal growth and no obvious clinical signs of toxicity (in addition to piloerection, they showed slight lethargy on Day 12 and slight salivation from Day 15 onwards). At 300 mg/kg, all males and females showed piloerection after dosing, mostly starting after about one week of treatment and persisting until the end of the treatment period. This piloerection was not regarded as an adverse health effect as it was not consistently accompanied by other signs of toxicity and recovered overnight. Males at 300 mg/kg showed slightly reduced body weight gain from initiation of treatment, resulting in a 4% lower mean body weight at the end of the treatment period. Body weight gain of 300 mg/kg females remained close to that of controls throughout the study, but mean body weights were slightly reduced during gestation and lactation (6% at the end of the treatment period). These minimal reductions in body weight gain or mean body weight were regarded as non-adverse. Minimal to slight vacuolation in the zona glomerulosa of the adrenal gland was observed in males at 300 mg/kg (1000/600 mg/kg males were not examined). This finding was regarded as non-adverse because it was not accompanied by degenerative, inflammatory or proliferative changes. Clinical pathology showed increased plasma levels of urea and creatinine in males at 300 mg/kg (1000/600 mg/kg males were not examined). These changes were regarded as non-adverse since they were not associated with adverse anatomic pathology alterations and remained in the historical control ranges. Compared with historical control means1 , 1000/600 mg/kg females (non-lactating) showed a lower mean value for APTT (activated partial thromboplastin time) and higher mean values for PT (prothrombin time), urea, bile acids and calcium. APTT remained in the historical control range, but mean values for the other parameters exceeded the P95-limit of this range. Without information on associated anatomic pathology data (high-dose females were not subjected to histopathological examination), no adversity designation was made for these clinical pathology biomarkers.

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 Octopamine hydrochloride were established: Parental NOAEL: 300 mg/kg, based on mortality and clinical signs of toxicity at 1000/600 mg/kg which resulted in premature termination of the males (on Study Day 10).