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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-03-27 to 2015-06-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
adopted 21 September 1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Guanidinhydrochloride

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Wistar rats, Crl: WI(Han) (Full Barrier)
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: females: 7-8 weeks old, males: 7-8 weeks old
- Weight at study initiation: females: 137 -170 g; males: 147 -193 g
- Housing: The animals were housed in groups (5 animals/sex/cage) in type IV cages
- Diet (e.g. ad libitum): Altromin 1324 maintenance diet for rats and mice (lot no. 1526) ad libitum
- Water (e.g. ad libitum): Free access to tap water, sulphur acidified to a pH of approximately 2.8
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 55 +/- 10 %
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark

IN-LIFE DATES: males From: 2014-03-25 to 2014-08-05 females From: 2014-03-25 to 2014-08-04

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE
-aqua ad injectionem (AlleMan Pharma)
- Concentration in vehicle: low dose: 5 mg/L; medium dose 20 mg/mL; high dose: 60 mg/mL
- Amount of vehicle (if gavage): dose volume for all groups was 5 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For determination of the concentration of test item in dosing formulations, samples of at least 5 mL were retained from all groups in weeks 1, 5, 9 and 13
during the treatment period and stored between -15 and -35 °C. In total 16 samples.
Stability of the dosing formulations was tested once at the beginning of the treatment period.
From the low, medium and high dose group, samples of dosing formulations were frozen after 0 hours and after 10 days (at room temperature) after the preparation and stored at -15 to -35 °C until analysis. In total 6 samples.
Duration of treatment / exposure:
90 days
Frequency of treatment:
7 days per week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
25 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
control: 15 animals per sex
high dose: 15 animals per sex
low dose: 10 animals per sex
mid dose: 10 animals per sex
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: according to the results of a previous dose range finding study
- Rationale for animal assignment (if not random): Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively, while ensuring to keep each animal with its initial cage partners.
- Rationale for selecting satellite groups: In order to allow a detection of possible delayed occurrence or persistence of or recovery from toxic effects, the animals in the recovery groups were observed for a period of 28 days (females) or 29 days (males) following the last administration.

Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day, preferably at the same time each day. The health condition of the animals was recorded.
Twice daily all animals were observed for morbidity and mortality, except on weekends and public holidays when observations were made once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first administration and at least once a week thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: once before the assignment to the experimental groups,
on the first day of administration and weekly during the treatment and recovery period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Food consumption was measured weekly during the treatment and recovery period.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

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: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the first administration and in the last week of the treatment period
as well as in the last week of the recovery period in the recovery animals.
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment and recovery period prior to or as part of the sacrifice
- Anaesthetic used for blood collection: No
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters examined, refer to table below

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment and recovery period prior to or as part of the sacrifice
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters examined, refer to table below

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the treatment and recovery period prior to or as part of the sacrifice
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes, overnight
- Parameters examined, refer to table below, additionally urine colour and appearance was recorded

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once before the first exposure and once in the last week of exposure
as well as in the last week of the recovery period
- Dose groups that were examined: all animals
- Battery of functions tested: changes in gait, posture, response to handling as well as the presence of clonic or tonic movements,
stereotypies or bizarre behaviour was recorded

OTHER: Examination of Fertility Parameters
- estrous cycle: Daily over a period of 8 days, the estrous cycle of all female animals was examined 4, 8 and 12 weeks after the first administration.
In the recovery animals the estrous cycle was examined during the last week of the recovery period.
- sperm parameters:
At necropsy (one day after the last administration) and at the end of the recovery period, left epididymis, left testis and left vas deferens were
separated and used for evaluation of sperm parameters.
Epididymal sperm motility and testicular sperm count as well as sperm morphology were evaluated in all male animals.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes, refer to table under “any other information on materials and method”
One day after the last administration (study day 91) all surviving animals of the treatment period and 4 weeks after the last administration all animals of the recovery period (study day 119 females, study day 120 males) were sacrificed using anesthesia (ketamine, Pharmanovo, lot no: 24863, expiry date: 10/2015 and xylazine, Serumwerk, lot no. 00513, expiry date: 05/2015) and were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

HISTOPATHOLOGY: Yes, refer to table under “any other information on materials and method”
The listed organs were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining for the animals of groups 1 and 4 sacrificed at the end of the treatment period and one male animal from HD (37) which was euthanised on study day 6 which was before the planned day of sacrifice.
For testis, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
These examinations were extended to animals of all other dosage groups for treatment-related changes observed in the kidneys, stomach, adrenals and thymus of high dose group. Only kidneys, stomach, adrenals and thymus of the other dosage groups and animals subjected to necropsy at the end of the recovery period were examined.
Any gross lesion macroscopically identified was examined microscopically in all animals.
Statistics:
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights and sperm analysis were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. Statistical comparisons of data acquired during the recovery period were performed with a Student’s t-Test. These statistics were performed with GraphPad Prism V.6.01 software (p<0.05 was considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
One male animal of the HD group was euthanized in a moribund condition for animal welfare reasons on study day 6. No macroscopic or histopathological abnormalities were seen as cause of this animal’s morbidity. Thus, observed clinical findings were considered as incidental and not related to the treatment with the test item. With this exception, all animals survived the treatment and recovery period.

During the treatment period, slight to severe salivation was noted in a dose-dependent pattern occasionally in some males and females of the HD
group and in a few of the MD group. Furthermore, moving the bedding was observed transitorily in all males and all females of the HD group, in some males and females of the MD group and in one female of the LD group and one male of the control group. As the symptoms of salivation and moving
the bedding were noted mainly immediately after administration, these signs were considered to be a sign of discomfort due to a local reaction to the test item.
During the weekly detailed clinical observation, no significant changes or differences between the groups were found.

BODY WEIGHT AND WEIGHT GAIN
In both, males and females, the mean body weight increased with the progress of the study in the control, the LD, the MD and the HD group. No biologically or statistically significant effects of Guanidinhydrochloride were found in the LD and the MD groups of both males and females. In males, a tendency towards a marginally lower mean body weight was observed in the HD group after the first week of treatment compared to the control group and the body weight was noted to be further slightly reduced until the end of the treatment period but without achieving statistical significance. During the recovery period, mean body weight of the male HD group remained slightly, statistically insignificantly lower compared to the control group. Over the treatment period, mean daily weight gain was slightly and statistically significantly lower in the male HD group when compared to the respective controls. During the recovery period, no such effect was seen. In females, mean body weight was marginally higher in the HD group compared to the control group. Body weight remained marginally higher during the recovery period without achieving statistical significance. Overall mean daily weight gain during the treatment period was slightly and statistically significantly higher in the female HD group when compared to the respective controls.

During the recovery period, mean daily body weight gain was slightly and statistically significantly reduced compared to the control group. As differences were marginal and values were within the normal range of variation for animals of this strain, effects on female body weight were not considered as an adverse effect of the test item.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There were no biologically or statistically significant effects on food consumption during the treatment period and recovery period in both males and females of the dose groups when compared to the respective controls.

FOOD EFFICIENCY
Not examined

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Not examined

OPHTHALMOSCOPIC EXAMINATION
No ophthalmologic findings were observed in any of the animals of this study.

HAEMATOLOGY
In males and females, no test item-related effect of toxicological relevance was observed for haematology and coagulation parameters. Statistically
significant differences between individual dose groups and the control group for mean corpuscular haemoglobin concentration and mean
corpuscular haemoglobin, platelet count, lymphocytes, neutrophils, mean corpuscular volume and prothrombin time were within the normal range of biological variation and not assumed to be biologically relevant.

CLINICAL CHEMISTRY
In male animals, at the end of the treatment period, total bile acids were observed to be marginally, statistically significantly higher in the HD group
when compared to the control group. In female animals, total bile acids and alkaline phosphatase were marginally but statistically significantly higher
in the HD group at the end of the treatment period compared to the control group. These findings can be considered to be related to the treatment
with the test item. Marginal but statistical significant differences for cholesterol in the male and female HD group compared to controls are not
assumed to be biologically relevant and values were within the normal range of variation. At the end of the recovery period, blood biochemistry values of the male and female HD group were comparable to the respective controls. Marginally and statistically significantly higher total bilirubin in the
male recovery HD group was considered to be incidental.

URINALYSIS
Slightly higher leukocyte levels were found in the urine of few male animals of the MD and the HD group at the end of the treatment period compared
to the control animals. No such trend was observed in the female animals. At the end of the recovery period, slightly higher leukocyte levels were
observed in few male and also female animals of the HD group compared to the control group. Higher leukocyte levels in the urine might be related to the histopathologically observed inflammatory changes in the kidneys.
However, as the individual findings within the dose groups lacked consistency and as a nephropathy was only observed in the HD group and not the MD group at histopathology, effects on leukocyte levels were not conclusive and not considered as adverse.

NEUROBEHAVIOUR
In the functional observation battery in males and females at the end of the treatment period, the grip strength of the hind legs was noted to be dose-dependently decreased in the dose groups compared to the control group. The hind limb reflex was moderately impaired in two males and all females of the HD group as well as in each three females of the LD and the MD group. One male of the HD group was observed with severely impaired grip
strength of the front legs and severely increased positional passivity. At the end of the recovery period, no relevant effects were observed in these
parameters, but in one male recovery animal of the HD group the grip strength of the hind legs and in another one the grip strength of the front legs was slightly decreased which was also noted at the end of the treatment period. As these findings for grip strength and limb reflex were only observed at the end of the treatment period and did not persist throughout the recovery period except for slight findings in two single male animals, they
were not considered to be adverse. No relevant effects were observed in any of the remaining parameters of the functional observation battery at the end of the treatment period and at the end of the recovery period.

ORGAN WEIGHTS
The kidney weight was significantly affected in male animals of the HD group at the end of the treatment period. The total weight of the kidneys was
38% higher than in the control group concerning the absolute weight, 51% higher when related to body weight and 39% when related to brain weight. A statistically significant increase of kidney weight was also noted in female animals of the HD group. Kidney weight was markedly higher in the female HD group compared to the control group (absolute 36%, relative to body weight 30% and relative to brain weight 36%). In the male HD recovery group, mean kidney weight was statistically significantly higher compared to the respective controls (absolute 23%, relative to body weight 31% and relative
to brain weight 25%). In female animals, mean kidney weight was slightly higher in the HD group than in the respective control group at the end of the recovery period but without achieving statistical significance.
At the end of the treatment period, liver weight was moderately, statistically significantly higher in male and female animals of the HD group compared to the respective controls. At the end of the recovery period, there were no statistically or biologically significant effects on the absolute and relative
liver weights in the male and female dose groups when compared to the respective controls. No histological correlate was found to the increased liver weights.
There were no test-item related effects of toxicological relevance on weight data for the remaining organs.

GROSS PATHOLOGY
Predominant macroscopic findings observed in male animals sacrificed at the end of treatment period were bilateral rough surfaces of the kidneys in 3 males of the HD group. After the recovery period, the same finding was observed in 3/5 males of the HD recovery group.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathologically, a nephropathy was observed in HD animals from both sexes. Few more gross pathological changes were recorded in male and
female animals at the end of the treatment period and the recovery period irrespective of the treatment group which were considered as normal
background lesions after histopathological evaluation.
The histopathologically observed nephropathy in HD animals from both sexes consisted mainly of an interstitial inflammation with related peritubular (interstitial) fibrosis at a minor severity. Males were more affected than females. The lesion was associated with tubular cell necrosis in two males, but with tubular basophilia (indicator for regeneration) and tubular dilation as an indicator for architectural changes due to inflammatory and
degenerative changes in almost all animals. All findings in the LD and MD groups were within the range of normal renal background alterations and
hence not related to treatment.

In the glandular stomach of the main test animals from both sexes, there were inflammatory lesions at a very minor incidence in all dose groups
consisting of submucosal inflammation and/or focal glandular erosions. The findings were randomly distributed throughout the groups, however,
the severity was slight to moderate in HD males, whereas in all other groups/sexes, only minimal findings were encountered. The test item is a highly basic compound that is deemed to be the trigger for local irritation.
In adrenal glands from MD and HD males, the fatty change in the zona fasciculata increased in incidence and/or severity. A minimal to slight diffuse
hypertrophy was noted in the male and female HD group. These were considered as typical secondary stress-related lesions.

OTHER FINDINGS
Examination of Fertility Parameters:
Guanidinhydrochloride had no effect on epididymal sperm motility or testicular sperm count analyzed at the end of the treatment or recovery period
of this study. Sperm staging and evaluation of sperm morphology did not reveal any indicator for toxicity induced by the test item.
Guanidinhydrochloride had no biologically significant effect on the estrous cycle analyzed 4, 8 and 12 weeks after the first administration and in the
last week of the recovery period.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Based on a nephropathy at a dose level of 300 mg/kg body weight/day
Dose descriptor:
LOEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Due to the local irritant effects of Guanidine hydrochloride in the glandular stomach of all dose groups

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Concentration analysis:

Nominal concentrations were confirmed for all dose groups, as measured concentration did not differ from nominal concentration by more than 15%.

Stability of formulation:

All samples were stable, as concentration after storage did not differ from start value by more than 15%.

Homogeneity of formulation:

All samples were homogenous, as coefficients of variation (COV) was below or equal 15%.

Applicant's summary and conclusion

Conclusions:
The no observed adverse effect level (NOAEL) of Guanidine hydrochloride in this study is considered to be 100 mg/kg body weight/day.
Executive summary:

In a subchronic toxicity study according to OEDC Guideline 408 (adopted 21 September 1998), Guanidine hydrochloride was administered to 10 Wistar rats/sex/dose in water, by gavage at dose levels of 0, 50, 100 and 300 mg/kg bw/day.

In order to allow a detection of possible delayed occurrence or persistence of or recovery from toxic effects a satellite group of 5 rats/sex was exposed at dose levels of 0 and 300 mg/kg bw/day (control and HD).

There were no compound related effects in mortality, clinical signs, body weight, food consumption, ophthalmologic findings, haematology and coagulation parameters.

Predominant macroscopic findings observed in male animals sacrificed at the end of treatment period were bilateral rough surfaces of the kidneys in 3 males of the HD group. After the recovery period, the same finding was observed in 3/5 males of the HD recovery group. Histopathologically, a nephropathy was observed in HD animals from both sexes. Few more gross pathological changes were recorded which were considered as normal background lesions after histopathological evaluation.

In the glandular stomach of the main test animals from both sexes, there were inflammatory lesions at a very minor incidence in all dose groups consisting of submucosal inflammation and/or focal glandular erosions. The findings were randomly distributed throughout the groups, however, the severity was slight to moderate in HD males, whereas in all other groups/sexes, only minimal findings were encountered. The test item is a highly basic compound that is deemed to be the trigger for local irritation.

In adrenal glands from MD and HD males, the fatty change in the zona fasciculata increased in incidence and/or severity. A minimal to slight diffuse hypertrophy was noted in the male and female HD group. These were considered as typical secondary stress-related lesions.

The kidney weight was significantly affected in male animals of the HD group at the end of the treatment period. A statistically significant increase of kidney weight was also noted in female animals of the HD group. In the male HD recovery group, mean kidney weight was statistically significantly higher compared to the respective controls. In female animals, mean kidney weight was slightly higher in the HD group than in the respective control group at the end of the recovery period but without achieving statistical significance.

At the end of the treatment period, liver weight was moderately, statistically significantly higher in male and female animals of the HD group compared to the respective controls. At the end of the recovery period, there were no statistically or biologically significant effects on the absolute and relative liver weights in the male and female dose groups when compared to the respective controls. No histological correlate was found to the increased liver weights.

There were no test-item related effects of toxicological relevance on weight data for the remaining organs.

 

In male animals, at the end of the treatment period, total bile acids were observed to be marginally, statistically significantly higher in the HD group when compared to the control group. In female animals, total bile acids and alkaline phosphatase were marginally but statistically significantly higher in the HD group at the end of the treatment period compared to the control group. These findings can be considered to be related to the treatment with the test item.

Effects of remaining blood biochemistry parameters are not assumed to be biologically relevant and values were within the normal range of variation. At the end of the recovery period, blood biochemistry values of the male and female HD group were comparable to the respective controls. Marginally and statistically significantly higher total bilirubin in the male recovery HD group was considered to be incidental.

Higher leukocyte levels in the urine might be related to the histopathologically observed inflammatory changes in the kidneys. However, as the individual findings within the dose groups lacked consistency and as a nephropathy was only observed in the HD group and not the MD group at histopathology, effects on leukocyte levels were not conclusive and not considered as adverse.

Findings for grip strength and limb reflex were not considered to be adverse; effects were only observed at the end of the treatment period and did not persist throughout the recovery period except for slight findings in two single male animals. No relevant effects were observed in any of the remaining parameters of the functional observation battery at the end of the treatment period and at the end of the recovery period.

Guanidine hydrochloride had no effect on epididymal sperm motility or testicular sperm count analyzed at the end of the treatment or recovery period of this study. Sperm staging and evaluation of sperm morphology did not reveal any indicator for toxicity induced by the test item. Guanidine hydrochloride had no biologically significant effect on the estrous cycle analyzed 4, 8 and 12 weeks after the first administration and in the last week of the recovery period.

Nominal concentrations were confirmed for all dose groups by analytical verification, all samples were stable and homogenous.

No observed adverse effect level (NOAEL) of Guanidine hydrochloride: 100 mg/kg body weight/day

Based on a nephropathy at a dose level of 300 mg/kg body weight/day.

A no observed effect level (NOEL) could not be established, due to the local irritant effects of Guanidine hydrochloride in the glandular stomach of all dose groups.

Low observed effect level LOEL: 25 mg/kg body weight/day

This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirement for a subchronic oral study (OECD 408) in rats.