Registration Dossier

Administrative data

Description of key information

 A sub-acute study (oral; 28 days exposure) performed according to OECD/EC guidelines and GLP principles, is available for guanidine phosphate (1:1). Based on this study, a No Observed Adverse Effect Level (NOAEL) of 200 mg/kg was established. Based on a 90-day repeated dose study performed according to OECD guideline and GLP principles, the no observed adverse effect level (NOAEL) of analogue guanidinium hydrochloride is considered to be 100 mg/kg body weight/day. This value for sub-chronic exposure is read across to guanidine phosphate (1:1).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January - February 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Guideline:
other: - OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, 2000
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: appr. 6 weeks
- Weight at study initiation:
- Fasting period before study:
- Housing: group housing in Macrolon cages
- Diet: free excess to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements and overnight prior to necropsy, animals had no access to food.
- Water: Free access to tap water except during motor activity measurements.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): appr. 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 16JAN2013 To: 25FEB 2013
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing, and were homogenized to visually acceptable levels. No correction was made for the purity of the test substance.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion after the in-life phase of the study, according to a validated method (TNO project 20292/05). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration).

The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration for solutions, or 85-115% for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
28 days.
Frequency of treatment:
Once daily, 7 d/w.
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
800 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were based on the results of a 8-day range finding study (WIL Research Project 501084).
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily in the cage. Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly.

FOOD CONSUMPTION:
- Weekly.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked: According to test guidelines

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked: According to test guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
- Organ weights of surviving animals were recorded (according to test guide line)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- All animals were fasted overnight with a maximum of 20 hours prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- Dose groups that were examined:
- Tissues/organs checked: According to test guidelines (+ deviations)

ORGAN WEIGHTS:
Organs checked according to test guidelines (+deviations)

HISTOPATHOLOGY: Yes (see table) / No / No data
According to test guidelines (+ deviations)
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 3) was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The animals treated at 800 mg/kg bw showed lethargy, hunched posture, swelling of the abdomen, piloerection, pale faeces, lean appearance and salivation. Hunched posture, piloerection, slight salivation and lean appearance was noted for the male found dead on day 13 and no clinical signs were seen in the other male found dead on day 3. One female at 200 mg/kg showed piloerection and lean appearance (day 10), which was transient and therefore not considered toxicologically relevant. No further clinical signs were noted for any other animal. Two control animals, one male and one female, had scabs on the neck or shoulder. This is occasionally seen and since these animals were not exposed to the test substance, this finding was not considered toxicologically relevant.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two males treated with 800 mg/kg guanidine phosphate (1:1) were found dead during the study on day 3 and day 13. No further mortality occurred.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced body weight gain was noted in males at 800 mg/kg, predominantly in the first two weeks. No other deviation from expected body weight gain was found.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption before or after allowance for body weight was slightly lower in males and females at 800 mg/kg compared to control animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Lower and higher white blood cells counts were found in males and females resp. In females, also lower eosinophil levels and lower platelet counts were detected.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At 800 mg/kg bw/d, increased creatinine level (males), increased inorganic phosphate level (males and females), decreased chloride and glucose level (females) and increased alanine aminotransferase, calcium, potassium and bile acids levels (females) were detected.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Higher absolute and relative kidney and liver weights were noted in males and females, except for the absolute liver weight in males which was comparable to controls. The absolute and relative adrenal weights were higher in males only.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There were treatment-related macroscopic findings present at the highest dose males and females only which comprised enlarged kidneys and thickened small intestine (duodenum, jejunum, ileum). In females at 800 mg/kg a firm mass was noted in the stomach.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related morphologic alterations consisted of a variety of changes in the kidneys, consisting of presence of tubular basophilia, concomitant with tubules showing desquamation of tubular epithelial cells, as well as foci of degeneration and necrosis of tubular epithelial cells. All these changes were predominantly present within the outer medulla and/or medullary rays (within the cortex). As reaction to the degenerative changes, peritubular fibrosis and lymphoid cell infiltrates was noted among animals in the 800 mg/kg treated group.
Outspoken mineralization was noted at the corticomedullary junction in a single female of the 800 mg/kg treated group.
Further, tubular dilation was observed in most animals of this group. This change is mainly thought responsible for the enlargement of the kidneys of animals in this group, grossly.

Although generally mild, duodenal hypertrophy was noted among animals in the 800 mg/kg treated group. Grossly, the duodenal hypertrophy resulted in thickening of the small intestine in two animals. Mild hypertrophy of the adrenal cortex was present in the males of the 800 mg/kg treated group.
Histopathological findings: neoplastic:
not examined
Dose descriptor:
NOAEL
Effect level:
>= 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Mortality and macroscopic and histopathologic effects on kidneys, liver, duodenum and adrenals at 800 mg/kg bw/day.
Critical effects observed:
yes
Lowest effective dose / conc.:
800 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
Based on the results of a sub-acute study (oral; 28 days exposure) performed according to OECD/EC guidelines and GLP principles, a No Observed Adverse Effect Level (NOAEL) for guanidine phosphate (1:1) of 200 mg/kg was established.

Executive summary:

A sub-acute study was performed according to OECD/EC guidelines and GLP principles. Male and female Wistar rats were exposed via oral gavage to 0, 50, 200 or 800 mg/kg bw/ day. Two males treated at 800 mg/kg were found dead on Days 3 and 13. No further mortality occurred. In the highest dose group, animals showed lethargy (with lower motor activity in females), hunched posture, swelling of the abdomen, piloerection, pale faeces, lean appearance and salivation. Reduced body weight gain was noted in males at 800 mg/kg, paralleling slightly lower food consumption in rats of the highest dose group. No other deviations in expected body weights and body weight gain were noted. Higher relative kidney and liver weights were noted in males and females. There were treatment-related macroscopic findings present at 800 mg/kg only (enlarged kidneys and thickened small intestine (duodenum, jejunum, ileum)). This coincided with changes in clinical biochemistry parameters at 800 mg/kg bw/ d such as higher creatinine level in males, higher inorganic phosphate level (both sexes), and in females lower chloride, glucose, alanine aminotransferase, calcium, potassium and bile acids levels.

Based on these data, a No Observed Adverse Effect Level (NOAEL) for guanidine phosphate (1:1) of 200 mg/kg was established. Since the effects were not found to be significantly toxic (not significantly different from the effects seen at the acute testing), the test substance is not classified according to Regulation (EC) No 1272/2008.

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The justification to read across the data is attached in section 13.
Reason / purpose:
read-across source
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
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.
Mortality:
mortality observed, treatment-related
Description (incidence):
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.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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):
no effects observed
Description (incidence and severity):
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
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalmologic findings were observed in any of the animals of this study.
Haematological findings:
no effects observed
Description (incidence and severity):
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 biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
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 findings:
effects observed, treatment-related
Description (incidence and severity):
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.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
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.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
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 pathological findings:
effects observed, treatment-related
Description (incidence and severity):
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.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
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.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Examination of Fertility Parameters:
Guanidinium 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. Guanidinium 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.
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: 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:
test mat.
Sex:
male/female
Basis for effect level:
other: Local irritant effects of guanidinium hydrochloride in the glandular stomach of all dose groups.
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

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%.

Conclusions:
Based on a 90-day repeated dose study performed according to OECD guideline and GLP principles, the no observed adverse effect level (NOAEL) of guanidinium hydrochloride is considered to be 100 mg/kg body weight/day. This value is read across to Guanidinium phosphate.
Executive summary:

In a subchronic toxicity study performed according to OEDC Guideline 408 (adopted 21 September 1998) and GLP principles, guanidinium 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. Guanidinium 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.

This value is read across to Guanidinium phosphate.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The study was performed according to OECD/EC guidelines and GLP principles.
System:
urinary
Organ:
kidney

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A sub-acute repeated dose oral toxicity study was performed with guanidine phosphate (1:1) according to OECD/EC guidelines and GLP principles. Male and female Wistar rats were exposed via oral gavage to 0, 50, 200 or 800 mg/kg bw/ day. Two males treated at 800 mg/kg were found dead on resp. days 3 and 13. No further mortality occurred. In the highest dose group, animals showed lethargy (with lower motor activity in females), hunched posture, swelling of the abdomen, piloerection, pale faeces, lean appearance and salivation. Reduced body weight gain was noted in males at 800 mg/kg bw, slightly lower food consumption in rats of the highest dose group was observed. No other deviations in expected body weights and body weight gain were noted. Higher relative kidney and liver weights were noted in males and females. There were treatment-related macroscopic findings present at 800 mg/kg bw/day only (enlarged kidneys and thickened small intestine (duodenum, jejunum, ileum)). This coincided with changes in clinical biochemistry parameters at 800 mg/kg bw/ d such as higher creatinine level in males, higher inorganic phosphate level (both sexes), and in females lower chloride, glucose, alanine aminotransferase, calcium, potassium and bile acids levels. Based on these data, a No Observed Adverse Effect Level (NOAEL) for guanidine phosphate (1:1) of 200 mg/kg was established.

A subchronic toxicity study with substance analogue guanidine hydrochloride was performed according to OEDC Guideline 408 and GLP principles. In this study, the test item was administered to 10 Wistar rats/sex/dose 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 high dose (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. 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.

Based on these data (nephropathy at a dose level of 300 mg/kg body weight/day), the NOAEL of guanidine hydrochloride was established to be 100 mg/kg bw/day. Following the rationale attached in section 13, this data is read across to guanidine phosphate (1:1). In short, comparing the observations in the repeated dose studies performed with the target and the source chemical, it becomes clear that for both guanidine phosphate and guanidine hydrochloride, the main target organ is the kidney. The nephropathy was manifested as tubular basophilia, tubular cell necrosis and peritubular fibrosis at a minor severity. This toxicity is expected to be related to the guanidine ions present in both the target and the source substance. The results of two studies suggest that both substances have comparable toxicological profiles, with kidney being the main target organ. Therefore it is considered acceptable to read-across the results from a 90-day toxicity study from the source chemical guanidine hydrochloride to the target chemical guanidine phosphate (1:1). Furthermore, for both substances hypertrophy of the adrenal glands was observed, for guanidine phosphate at 800 mg/kg bw/day, for guanidine hydrochloride at 300 mg/kg bw/ day (although in this study this was considered to be related to stress rather than substance-related). The similarities of the effects observed confirm that both substance have a comparable toxicological profile, which is most likely related to the presence of the guanidine ion for both substances.

Since the effects were not found to be significantly toxic (not significantly different from the effects seen at the acute testing), guanidine phosphate (1:1) is not classified for repeated dose exposure according to Regulation (EC) No 1272/2008.

Justification for classification or non-classification

Based on the available data, the substance is not classified for repeated dose exposure according to Regulation (EC) No 1272/2008.