2-[tert-butyl(phenylmethyl)amino]-1-[4-hydroxy-3-(hydroxymethyl)phenyl] hydrochloride

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25-09-2014 to 23-11-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Test guideline study conducted to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2016

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:

Deviations were considered to have no adverse impact on the scientific integrity of the study

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-[tert-butyl(phenylmethyl)amino]-1-[4-hydroxy-3-(hydroxymethyl)phenyl] hydrochloride

EC Number:

246-014-2

EC Name:

2-[tert-butyl(phenylmethyl)amino]-1-[4-hydroxy-3-(hydroxymethyl)phenyl] hydrochloride

Cas Number:

24085-08-3

Molecular formula:

C20H25NO3.HCl

IUPAC Name:

2-[tert-butyl(phenylmethyl)amino]-1-[4-hydroxy-3-(hydroxymethyl)phenyl] hydrochloride

Test material form:

other: solid

Test animals

Species:

rat

Strain:

other: Wistar Han™:RccHan™:WIST strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.
- Age at study initiation: 12 weeks
- Weight at study initiation: males weighed 302 to 342g, the females weighed 185 to 224g
- Fasting period before study: none
- Housing: Initially, all animals were housed in groups of four in solid floor polypropylene cages with
stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the
pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays
lined with absorbent paper on a one male: one female basis within each dose group. Following
evidence of successful mating, the males were returned to their original cages. Mated females
were housed individually during gestation and lactation in solid floor polypropylene cages with
stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): ad libitum- A pelleted diet (Rodent 2018C Teklad
Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used
- Water (e.g. ad libitum): ad libitum
- Acclimation period: The animals were acclimatized for five days during which time their health status was assessed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least fifteen
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours

IN-LIFE DATES: From: To: 25-09-2014 to 23-11-2014

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

400

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations as a
suspension in Polyethylene glycol 400. The stability and homogeneity of the test item
formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services.
Results show the formulations to be stable for at least fifteen days in the dark at 4 °C.
Formulations were therefore prepared weekly and stored at approximately 4 °C in the dark.


VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Results show the formulations to be stable for at least fifteen days in the dark at 4 °C.
Formulations were therefore prepared weekly and stored at approximately 4 °C in the dark.

Samples of the test item formulation were taken and analyzed for concentration of Glycyl
(CAS: 24085-08-3) at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The
method used for analysis of formulations and the results obtained are given in Appendix 25. The
results indicate that the prepared formulations were within acceptable ranges for the purpose of
this study.

Duration of treatment / exposure:

Males- 43 days
Females- 40-54 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000/750/500 mg/kg bw/day
Basis:
other: nitial treatment at 1000 mg/kg bw/day resulted in the termination of one m; dose lowered to 750 mg/kg bw/day from Day 2. Termination of 1 f on day 3 - dose reduced to 500 mg/kg bw/day from day 4.

No. of animals per sex per dose:

12 (twelve)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based upon dose range finding study
- Rationale for animal assignment (if not random):
- Rationale for selecting satellite groups: 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.

- Section schedule rationale (if not random): Samples of tissues were removed from five selected males and five selected
females from each dose group and preserved in buffered 10% formalin,

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioral change
immediately before dosing, soon after dosing, and one hour after dosing during the working
week, at weekends (except for females during parturition where applicable). All observations
were recorded.

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

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes
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 AND COMPOUND INTAKE (if drinking water study): Not applicable
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 42 for males and Day 4 postpartum for females
- Anaesthetic used for blood collection: No
- How many animals: 5/sex
- Parameters checked in table [1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 42 for males and Day 4 postpartum for females
- Animals fasted: No
- How many animals: 5/sex
- Parameters checked in table [2] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and at weekly intervals
- Dose groups that were examined: All
- Battery of functions tested: sensory activity / grip strength / motor activity / other: behavioral

OTHER:

Sacrifice and pathology:

Surviving adult males were killed by intravenous overdose of suitable barbiturate agent followed
by exsanguination on Day 43 or Day 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 were killed on or after Day 25 post coitum.

GROSS PATHOLOGY: Yes (see table 3)

HISTOPATHOLOGY: Yes (see table 4)

Statistics:

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

Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during
gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex
Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body
Weight, Offspring Body Weight Change, Offspring Surface Righting, Hematology, Blood
Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.

Data were analyzed using the decision tree from the Provantis Tables and Statistics Module as detailed as follows:

Where appropriate, data transformations were performed:
homogeneity of variance from mean values : Bartlett’s test
Intergroup variances:ANOVA, or if required, ANCOVA
transformed data were analyzed for significant effect using the Williams Test (parametric) or Shirley Test (nonparametric)
non-homogeneity of means: stepwise Dunnett’s (parametric) or Steel (non-parametric) for significant difference from the control group.
pair-wise tests : Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Data not analyzed by the Provantis data capture system were assessed separately using the R
Environment for Statistical Computing.
distribution of the data : Shapiro-Wilk normality test,
assessment of data homogeneity :Bartlett's test.
parametric analysis:(ANOVA), which if significant, was followed by pair-wise
comparisons using Dunnett's test.
non-parametric analysis of the data was performed incorporating the Kruskal-Wallis
test which if significant was followed by the Mann-Whitney "U" test

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were four unscheduled deaths on the study.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were four unscheduled deaths on the study.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

increase in absolute and body weight relative liver weights compared with control, with differences attaining statistical significance

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic necropsy of surviving animals revealed enlargement of the liver for the majority of males at 1000/750/500 mg/kg bw/day.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic findings considered to be treatment related were observed for the liver, kidneys and thyroid glands.

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY

Mortality
There were four unscheduled deaths on the study.

At 1000 mg/kg bw/day, male number 79 showed clonic convulsions and staining around the
mouth on the first day of dosing, the severity of the convulsions observed were considered
sufficient for this animal to be terminated for animal welfare considerations. Necropsy and
subsequent microscopic evaluation of the tissues did not reveal any obvious underlying reason
for the condition of the animal.

At 1000/750 mg/kg bw/day, female number 90 showed clonic convulsions on Day 3; the severity
of the convulsions observed were considered sufficient for this animal to be terminated for
animal welfare considerations. Necropsy and subsequent microscopic evaluation of the tissues
did not reveal any obvious underlying reason for the condition of the animal.

At 300 mg/kg bw/day, male number 55 was found dead on Day 13, no clinical signs had been
apparent for this animals prior to its’ death. Necropsy findings included sloughing of the
glandular region of the stomach, enlarged liver, reddened adrenals, reddened lungs, reddened
mandibular lymph nodes, reddened salivary glands, reddened mammary gland, gaseous
distension of the caecum and green discoloration of the skin (hind limb). Microscopic findings
included congestion in the lungs, mammary gland, mandibular lymph nodes and salivary glands,
slight granulolymphocytic inflammatory infiltrate in the trachea, slight lymphoid hyperplasia for
the mesenteric lymph node and autolysis of the stomach but did not reveal any obvious cause for
the death of the animal.

At 300 mg/kg bw/day, male number 56 showed piloerection, hunched posture and staining
around the mouth on Day 25. These clinical signs persisted to the following morning when
lethargy, decreased respiratory rate, pallor of the extremities and disrrhoea were also apparent
and the animal was terminated early for animal welfare considerations. Necropsy revealed
reddened lungs and black contents in the stomach, jejunum, ileum and caecum but no
microscopic findings were apparent for these tissues and histopathological evaluations did not
determine the cause of the decline in the clinical condition for this animal.

Clinical Observations
At 1000 mg/kg bw/day, treatment on Day 1 was associated with clonic convulsions for four
males, with one of these males having to be terminated early. Two of the males showing
convulsion also showed hunched posture. Following the lowering of the dosage to 750 mg/kg
bw/day on Day 2 and then to 500 mg/kg bw/day on Day 4, no similar signs were apparent for the
high dosage males. For females at 1000 mg/kg bw/day, treatment on Day 1 was associated with
clonic convulsions for one animal and hunched posture for five animals. Following the lowering
of dosage to 750 mg/kg bw/day, hunched posture persisted for two of these affected females,
with one also showing clonic convulsions on Day 2. A further female showed hunched posture
and clonic convulsions on Days 2 and 3 and lethargy, hunched posture and piloerection on
Day 3. Additionally on Day 3, another female at this dosage showed clonic convulsions and had
to be terminated early. Following the lower of the dosage to 500 mg/kg bw/day, no similar signs
were apparent for high dosage females.

For surviving animals at 1000/750/500 mg/kg bw/day, treatment was associated with increased
post-dosing salivation, with all animals being affected to some extent although the individual
incidence showed great variation. Such salivation is frequently observed when a slightly irritant
or unpalatable test item is administered via the oral gavage route and is generally considered to
reflect distaste of the dosing formulations rather than any systemic effect of treatment. Two
males at 1000 mg/kg bw/day showed noisy respiration on Day 1 and a further high dosage male
also showed noisy respiration on Day 30, when receiving 500 mg/kg bw/day. This finding most
probably represents accidental inspiration of the test item formulations during the dosing
procedure and at the incidence and duration observed on this study was considered not to
indicate a systemic effect of treatment.

At 300 mg/kg bw/day, clinical signs were mainly restricted to that previously described for
decedent male number 56. One female showed scabbing during the study, otherwise there were
no clinical signs apparent for surviving animals at this dosage.

No clinical signs were apparent for animals treated at 100 mg/kg bw/day.


BODY WEIGHT AND WEIGHT GAIN
There was considered to be no effect of treatment on body weight gain for males at 100, 300 or
1000/750/500 mg/kg bw/day. Body weight gains of the treated animals were generally superior
to control and the differences in body weight gain that did attain statistical significance were
considered to reflect normal biological variation.

There was considered to be no effect of treatment on body weight gain for females during the
pre-pairing, gestation and lactation phases of the study at 100, 300 or 1000/750/500 mg/kg
bw/day.

FOOD CONSUMPTION AND FOOD EFFICIENCY
There was considered to be no effect of treatment on food consumption or food conversion
efficiency for males at 100, 300 or 1000/750/500 mg/kg bw/day. Food intake for treated males
appeared higher than control during the post-pairing phase but was consistent with the body
weight gain and in the absence of any accompanying effect on food conversion efficiency, this
finding was considered to reflect normal biological variation.

There was considered to be no effect of treatment on food consumption or food conversion
efficiency for females during the pre-pairing, gestation and lactation phases of the study at 100,
300 or 1000/750/500 mg/kg bw/day.

WATER CONSUMPTION
Visual assessment of water bottle residues throughout the study did not indicate any obvious
effect on water intake for either sex at 100, 300 or 1000/750/500 mg/kg bw/day.

HAEMATOLOGY
There were no obvious effects of treatment on hematology parameters for either sex at 100, 300
or 1000/750/500 mg/kg bw/day.

For males at all dosages, lower mean cell hemoglobin concentration attained statistical
significance when compared with control, although mean values showed no dosage relationship.
Additionally mean hematocrit was statistically significantly higher than control at 100 mg/kg
bw/day. For both parameters, individual values for treated animals were with the historical
control range and, in the absence of any supporting histopathological change, these findings were
considered to incidental and unrelated to treatment.

For males at all dosages, higher mean platelet attained statistical significance when compared
with control, additionally clotting time at 1000/750/500 mg/kg bw/day was statistically
significantly shorter than control. For treated animals, only one value for platelet count at 100,
300 or 1000/750/500 mg/kg bw/day exceeded the historical control range while one control
platelet value was below this historical range; in the absence of any supporting histopathological
change, these findings were considered to incidental and unrelated to treatment.


CLINICAL CHEMISTRY
There were no adverse effects of treatment on blood chemical parameters for either sex at 100,
300 or 1000/750/500 mg/kg bw/day.

For males at all dosages, higher levels of total cholesterol and lower levels of urea and glucose
attained statistical significance compared to control. For treated animals, only total cholesterol
values for two males at 300 mg/kg bw/day and one male at 1000/750/500 mg/kg bw/day
exceeded the historical control range; one control value for glucose also exceeded the historical
range. Additionally for males at all dosages, higher total protein and albumin levels attained
statistical significance compared to control; there was no accompanying statistical significant
increase in albumin/globulin ratios and only one value for total protein at 1000/750/500 mg/kg
bw/day exceeded the historical control range. While the differences observed for these
parameters may be related to altered metabolism indicated by microscopic changes observed for
the liver, at the level observed on this study, they were considered not to indicate an adverse
effect of treatment.

For males at all dosages, there was a statistically significant increase in calcium and inorganic
phosphorus levels although mean values showed no dosage relationship. All individual values
were with the historical control range and, while they may have been influenced by microscopic
changes observed for the kidneys, the differences observed were considered to be of no
toxicological significance.

For females at 300 and 1000/750/500 mg/kg bw/day, higher levels of total cholesterol attained
statistical significance compared to control; three individual values at 300 mg/kg bw/day and two
individual values at 1000/750/500 mg/kg bw/day exceeded the historical control range. As
previously discussed for males, this finding may be associated with microscopic liver changes
observed for the liver, but at the level observed on this study, was considered not to indicate an
adverse effect of treatment.
or females at all dosages, lower albumin/globulin ratios attained statistical significance when
compared with control, but there was dosage relationship or accompanying statistically
significant difference observed for total protein or albumin values. All values for treated animals
were within the historical control range and, in isolation, this finding was considered to be of no
toxicological significance.

For females at all 300 and 1000/750/500 mg/kg bw/day, higher levels of alanine
aminotransferase attained statistical significance compared to control; there was no dosage
relationship and for treated animals only two individual values at 300 mg/kg bw/day exceeded
the historical control range. Although adaptive microscopic change was observed for the liver,
there was no evidence of cellular damage and this finding was considered to be incidental and of
no toxicological significance.



NEUROBEHAVIOUR
Behavioral Assessments

There were no treatment-related changes in the behavioural parameters at 100, 300 or
1000/750/500 mg/kg bw/day.
Functional Performance Tests

There were no changes in functional performance considered to be related to treatment at 100,
300 or 1000/750/500 mg/kg bw/day.

For males at 1000/750/500 mg/kg bw/day, mean values for the last 20% activity was lower than
control and attained statistical significance; however, in the absence of any significant difference
for overall activity this finding was considered to be incidental and of no toxicological
significance.

For females at 1000/750/500 mg/kg bw/day and 300 mg/kg bw/day, higher mean values for the
third test of forelimb grip strength attained statistical significance; however, mean values showed
no dosage relationships and, in the absence of a statistically significant differences in the two
previous trials of forelimb grip strength, these differences were considered to be incidental and
unrelated to treatment.

Sensory Reactivity Assessments

There were no inter-group differences in sensory reactivity scores.

ORGAN WEIGHTS
For males at 100 mg/kg bw/day and both sexes at 300 and 1000/750/500 mg/kg bw/day, there
was an increase in absolute and body weight relative liver weights compared with control, with
differences attaining statistical significance. For this organ, body weight relative values are
considered to be the best indicator of toxicological effect and the majority of individual body
weight values for treated animals exceeded the historical control range. For females at
100 mg/kg bw/day, liver weights were marginally higher than control and failed to attained
statistical significance, although two of the five individual body weight relative values exceeded
the historical control.

For females at 100 mg/kg bw/day and both sexes at 300 and 1000/750/500 mg/kg bw/day, there
was an increase in absolute and body weight relative kidney weights compared with control, with
differences attaining statistical significance. For females, absolute kidney values did not show
any consistent dosage relationship, although a dosage response was apparent for body weight
relative values. Body weight relative values are probably the better indicator of toxicological
effect; however the majority of individual body weight values for treated animals were within
the historical control range. For males at 100 mg/kg bw/day, kidney weights were marginally
higher than control but differences failed to attained statistical significance, and all individual
body weight relative values were within the historical range.

For males at 300 and 1000/750/500 mg/kg bw/day, there was an increase in absolute and body
weight relative heart weights compared with control, with differences attaining statistical
significance. Mean values showed no dosage relationship and, although a number of individual
values at 300 mg/kg bw/day exceeded the historical control range, all values at the high dosage
were within this historical range. In the absence of any supporting histopathology at
1000/750/500 mg/kg bw/day, it was considered that the observed differences from control for
heart weight were of no toxicological significance.

GROSS PATHOLOGY
Macroscopic necropsy of surviving animals revealed enlargement of the liver for the majority of
males at 1000/750/500 mg/kg bw/day. Similar enlargement of the liver was apparent for one
female at this dosage, three males and one female at 300 mg/kg bw/day and one male at
100 mg/kg bw/day.

Neither the type, incidence or distribution of other macroscopic abnormalities observed for
surviving animals at terminal necropsy indicated an effect of treatment at 100, 300 or
1000/750/500 mg/kg bw/day.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic findings considered to be treatment related were observed for the liver, kidneys and
thyroid glands.

For the liver, diffuse midzonal/centrilobular hypertrophy at minimal/slight degree was recorded
in 4/12 males and 5/9 females at 100 mg/kg bw/day and in 8/12 males and 7/8 females at
300 mg/kg bw/day. This finding was apparent at minimal/slight/moderate in all males and at
mimimal/slight in 9/10 females at 1000/750/500 mg/kg bw/day. This finding was only observed
at a minimal level for one female in the control group.

For the kidneys, cortical hyaline droplets at minimal degree were recorded in 2/5 control males
and at minimal/slight degree in 5/5 males at 100 mg/kg bw/day and 6/7 males at 300 mg/kg
bw/day. This finding was apparent at slight degree in 5/6 males at 1000/750/500 mg/kg bw/day.

For the thyroid, diffuse follicular hypertrophy/hyperplasia at minimal/slight degree was recorded
in 2/7 males and 1/5 females at 300 mg/kg bw/day and in 5/6 males and 3/6 females at
1000/750/500 mg/kg bw/day. This finding was also observed at a minimal level for one male
and one female in the control group.

Testes morphology did not reveal any disturbances of spermatogenic staging.

The remainder of the recorded microscopic findings were within the range of background
pathology encountered in Han Wistar rats of this age in this type of study and occurred at similar
incidences and severity in both control and treated rats.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: death/termination of 2 males dosed at 300 mg/kk bw/day

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The oral administration of Glycyl (CAS: 24085-08-3) to rats by gavage, at dose levels of up to
1000 mg/kg bw/day resulted in adverse clinical signs at the high dosage (1000 and 750 mg/kg
bw/day). Following the final reduction of the high dosage to 500 mg/kg bw/day treatment was
better tolerated, however, there were two deaths at 300 mg/kg bw/day, the aetiology of which
was unknown. The No Observed Adverse Effect Level (NOAEL) for systemic toxicity on this
study was therefore considered to be 100 mg/kg bw/day. Due to the equivocal nature of findings
for isolated litters at the high dosage, it is difficult to state with certainty whether reproductive
effects have been observed at this dosage. The No Observed Effect Level (NOEL) for
reproduction, including the growth, survival and development of the offspring was therefore
considered to be 300 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 approximately six weeks (including a two

week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100,

300 and 500 mg/kg bw/day. Initial treatment at 1000 mg/kg bw/day resulted in adverse clinical

signs on Day 1 resulting in the termination of one male; the high dosage was lowered to

750 mg/kg bw/day from Day 2. Adverse clinical signs remained apparent at 750 mg/kg bw/day

resulting in the termination of one female due to animal welfare considerations. Consequently,

the high dosage was further lowered to 500 mg/kg bw/day from Day 4 of the study. A control

group of twelve males and twelve females was dosed with vehicle alone (Polyethylene glycol

400).

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 at least five selected males and females from each dose group.

Surviving adult males were terminated on Day 43 or Day 44, followed by the termination of all

surviving females and offspring on Day 5 post partum. Any female which did not produce

a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross

necropsy examination and histopathological evaluation of selected tissues was performed.

Results

Mortality

At 1000 mg/kg bw/day, one male was killed on Day 1 after showing clonic convulsions and

staining around the mouth. Following the reduction of this dosage to 750 mg/kg bw/day, one

female was killed after showing clonic convulsions on Day 3. Necropsy and subsequent

microscopic evaluation of the tissues did not reveal any obvious underlying reason for the

condition of these animals.

At 300 mg/kg bw/day, one male was found dead on Day 13 without any clinical signs being

apparent. Necropsy revealed sloughing of the glandular region of the stomach, enlarged liver,

reddened adrenals, lungs, mandibular lymph nodes, salivary glands and mammary gland,

gaseous distension of the caecum and green discoloration of the skin (hindlimb). Microscopic

findings included congestion in the lungs, mammary gland, mandibular lymph nodes and

salivary glands, slight granulolymphocytic inflammatory infiltrate in the trachea, slight lymphoid

hyperplasia for the mesenteric lymph node and autolysis of the stomach but did not reveal any

obvious cause for the death of the animal.

At 300 mg/kg bw/day, one male was killed on Day 26 after showing piloerection, hunched

posture, staining around the mouth, lethargy, decreased respiratory rate, pallor of the extremities

and diarrhoea. Necropsy revealed reddened lungs and black contents in the stomach, jejunum,

ileum and caecum although no microscopic findings were apparent for these tissues and

histopathological evaluations did not reveal any obvious underlying reason for the condition of

this animal.

Clinical Observations

At 1000 mg/kg bw/day, treatment was associated with clonic convulsions and hunched posture

for some animals on Day 1. Following the lowering of the dosage to 750 mg/kg bw/day on

Day 2, no similar signs were apparent for males but clonic convulsions and hunched posture was

still observed for some females. Following the lowering of the dosage to 500 mg/kg bw/day, no

similar signs were apparent for either sex.

For surviving animals at 1000/750/500 mg/kg bw/day, treatment was associated with increased

post-dosing salivation, with all animals being affected to some extent.

There were no clinical signs considered to be of toxicological importance at 100 or 300 mg/kg

bw/day.

Behavioral Assessment

There were no treatment-related changes in the behavioral parameters at 100, 300 or

1000/750/500 mg/kg bw/day.

Functional Performance Tests

There were no changes in functional performance considered to be related to treatment at 100,

300 or 1000/750/500 mg/kg bw/day.

Sensory Reactivity Assessments

There were no inter-group differences in sensory reactivity scores.

Body Weight

There was considered to be no effect of treatment on body weight gain for either sex at 100, 300

or 1000/750/500 mg/kg bw/day.

Food Consumption and Food Conversion Efficiency

There was considered to be no effect of treatment on food consumption or food conversion

efficiency for either sex at 100, 300 or 1000/750/500 mg/kg bw/day.

Water Consumption

There was no obvious effect on water intake for either sex at 100, 300 or 1000/750/500 mg/kg

bw/day.

Laboratory Investigations

Hematology

There were no obvious effects of treatment on hematology parameters for either sex at 100, 300

or 1000/750/500 mg/kg bw/day.

Blood Chemistry

There were no adverse effects of treatment on the blood chemical parameters for either sex at

100, 300 or 1000/750/500 mg/kg bw/day.

Pathology

Necropsy

Macroscopic necropsy of surviving animals revealed enlargement of the liver for the majority of

males at 1000/750/500 mg/kg bw/day. Similar enlargement of the liver was apparent for one

female at this dosage, three males and one female at 300 mg/kg bw/day and one male at

100 mg/kg bw/day.

Organ Weights

For males at 100 mg/kg bw/day and both sexes at 300 and 1000/750/500 mg/kg bw/day, there

was a statistically significant increase in absolute and body weight relative liver weights

compared with control.

For females at 100 mg/kg bw/day and both sexes at 300 and 1000/750/500 mg/kg bw/day, there

was a statistically significant increase in absolute and body weight relative kidney weights

compared with control.

Histopathology

Microscopic findings, considered to be treatment related but adaptive in nature, were observed

for the liver, kidneys and thyroid glands.

For the liver, diffuse midzonal/centrilobular hypertrophy was observed for both sexes at 100, 300

and 1000/750/500 mg/kg bw/day; the incidence and severity tending to increase with dosage.

For the kidneys, an increase incidence of cortical hyaline droplets, compared with control, was

apparent for males at 100, 300 and 1000/750/500 mg/kg bw/day, with the highest severity being

observed in the high dosage group.

For the thyroid, an increase incidence of diffuse follicular hypertrophy/hyperplasia at

minimal/slight degree was observed for both sexes at 300 and 1000/750/500 mg/kg bw/day; the

incidence and severity tending to increase with dosage.

Conclusion

The oral administration of Glycyl (CAS: 24085-08-3) to rats by gavage, at dose levels of up to

1000 mg/kg bw/day resulted in adverse clinical signs at the high dosage (1000 and 750 mg/kg

bw/day). Following the final reduction of the high dosage to 500 mg/kg bw/day treatment was

better tolerated, however, there were two deaths at 300 mg/kg bw/day, the aetiology of which

was unknown. The No Observed Adverse Effect Level (NOAEL) for systemic toxicity on this

study was therefore considered to be 100 mg/kg bw/day. Due to the equivocal nature of findings

for isolated litters at the high dosage, it is difficult to state with certainty whether reproductive

effects have been observed at this dosage. The No Observed Effect Level (NOEL) for

reproduction, including the growth, survival and development of the offspring was therefore

considered to be 300 mg/kg bw/day.