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Description of key information

Data from two oral sub-chronic toxicity studies in rats, from the read-across substances Guanidinium Hydrochloride and Ammonium Thiocyanate were used to assess repeated dose toxicity of Guanidine Thiocyanate.

Reflecting differences in molecular weight between the source substance Ammonium thiocyanate and the target substance Guanidine Thiocyanate a factor of 1.55 was applied to the NOAEL 20 mg/kg bw/d obtained from a sub-chronic toxicity study with Ammonium Thiocyanate resulting in a NOAEL of 31 mg/kg bw/d, which is used for derivation of Guanidine Thiocyanate DNELs.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Generally it is assumed that Guanidinium Thiocyanate dissociates in aqueous media in the corresponding ions Guanidinium and Thiocyanate. Therefore both ions, independently of their source, can be considered relevant for assessment of repeated dose toxicity potential.
Thus theoretically salts of both ions with a relatively nontoxic counter ion are suitable to assess the toxicological potential of Guanidinium Thiocyanate after sub-chronic exposure.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Refer to attached document.

3. ANALOGUE APPROACH JUSTIFICATION
Refer to attached document.

4. DATA MATRIX
Refer to attached document.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Dose descriptor:
NOAEL
Effect level:
ca. 20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see remark
Remarks on result:
other: the lowest value obtained form the study with the read-across substance Ammonium thiocyanate is used for assessment
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 31 mg/kg bw/day (nominal)
Based on:
other: Calculation from Ammonium Thiocyanate to Guanidinium Thiocyanate Factor 1.55
Sex:
male/female
Basis for effect level:
other: Result was obtained from read across substance
Key result
Critical effects observed:
not specified
Conclusions:
The lowest value obtained form the study with the read-across substance Ammonium thiocyanate is used for assessment.
Executive summary:

As a conservative approach the lowest NOAEL of 20 mg/kg bw/day from Ammonium Thiocyanate study was used for assessment of Guanidine Thiocyanate.

Reflecting differences in molecular weight between the source substance Ammonium thiocyanate and the target substance Guanidine Thiocyanate a factor of 1.55 was applied to the NOAEL 20 mg/kg bw/d obtained from a sub-chronic toxicity study with Ammonium Thiocyanate resulting in a NOAEL of 31 mg/kg bw/d, which is used for derivation of Guanidine Thiocyanate DNELs.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
31 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Data from a GLP compliant guideline studies with reliability 1.
Organ:
not specified

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

Sub-acute toxicity:

According to column 2 of Annex VIII, section 8.6.1, testing on short term toxicity (28 days) does not need to be conducted if data from a reliable sub-chronic (90 days) are available. Data from a sub-chronic toxicity study are available. Thus, conducting of a short term toxicity (28 days) study is not necessary.

 

Sub-chronic toxicity:

Generally it is assumed that Guanidinium Thiocyanate dissociates in aqueous media in the corresponding ions Guanidinium and Thiocyanate. Therefore both ions, independently of its source, can be considered relevant for assessment of repeated dose toxicity potential.

Thus theoretically salts of both ions with a relatively nontoxic counter ion are suitable to assess the toxicological potential of Guanidine Thiocyanate after sub-chronic exposure.

 

Data from two oral sub-chronic toxicity studies in rats, one with Guanidine Hydrochloride and one with Ammonium Thiocyanate were used to assess repeated dose toxicity.

A detailed justification document for the read-across is attached in the respective target records of IUCID.

 

Guanidine Hydrochloride 

In a sub-chronic toxicity study according to OEDC Guideline 408 (adopted 21 September 1998), the read-across substance 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.

 

Based on a nephropathy at a dose level of 300 mg/kg body weight/day the No Observed Adverse Effect Level (NOAEL) of Guanidine Hydrochloride was 100 mg/kg body weight/day. A (NOEL) could not be established, due to the local irritant effects of Guanidine Hydrochloride in the glandular stomach of all dose groups. The Low Observed Effect level (LOEL) was considered to be 25 mg/kg body weight/day.

 

Ammonium Thiocyanate

In the second sub-chronic toxicity study according to OECD Guideline 408 (adopted 21 September 1998), the read-across substance Ammonium Thiocyanate was administered by daily gavage in the rat. Based on a 14-day range finding study, the dose levels for this 90-day oral gavage study were selected to be 0, 20, 100 and 500 mg/kg bw/day.

The test substance was administered daily for 92 or 93 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females.

From the results of this sub-chronic to toxicity study in rats with Ammonium Thiocyanate a no observed adverse effect level (NOAEL) of 20 mg/kg/day was concluded.

At the high dose of 500 mg/kg bw/day there were treatment related deaths. Other organs which showed treatment-related pathological lesions in the 500 mg/kg dose group included the forestomach (squamous hyperplasia), liver (hepatocellular hypertrophy), testes (seminiferous epithelial degeneration), spleen (lymphoid hyperplasia and increased 0 hemopoiesis), thymus (atrophy) and bone marrow (erythroid hyperplasia), 100 mg/kg bw/day some minimal clinical signs and changes in haematology and clinical biochemistry parameters were observed. Histopathoiogical examination revealed squamous hyperplasia of the forestomach and hepatocellular hypertrophy in some animals. Increased kidney weights in males were not supported by any histopathological evidence of renal dysfunction. 20 mg/kg bw/day there were no changes noted that were considered to be an effect of treatment.

 

An effect on the thyroid gland, a known target organ of Ammonium Thiocyanate, was not evident in this study; particularly total serum cholesterol and triglycerides are decreased in the 100 mg/kg dose group when compared to the control group (respectively -6.9% and -32% in males and -22.8%and -1 9% in females), while these parameters are usually increased in situations of hypothyroidism. This is consistent with the lack of pathotogical lesions and the unchanged weight of the thyroid gland in any of the dose groups, which are the most sensitive parameters for evaluating the thyroid function. Consequently, evaluations of levels of T3 and T4 have not been performed.

Conclusion

Reflecting differences in molecular weight between the source substance Ammonium thiocyanate and the target substance Guanidine Thiocyanate a factor of 1.55 was applied to the NOAEL 20 mg/kg bw/d obtained from a sub-chronic toxicity study with Ammonium Thiocyanate resulting in a NOAEL of 31 mg/kg bw/d, which is used for derivation of Guanidine Thiocyanate DNELs.

Justification for classification or non-classification

Repeated dose toxicity of Guanidine Thiocyanate was assessed based on data from two oral sub-chronic toxicity studies in rats, one with Guanidine Hydrochloride and one with Ammonium Thiocyanate.

Generally it is assumed that Guanidine Thiocyanate dissociates in aqueous media in the corresponding ions Guanidinium and Thiocyanate. Therefore data for both ions, independently of its source, can be considered relevant for assessment of repeated dose toxicity potential.

 

In an oral sub-chronic toxicity study in rats with Guanidine Hydrochloride a nephropathy was observed at the high dose of 300 mg/kg bw/day. Based on these findings, the no observed adverse effect level (NOAEL) in this study was considered to be 100 mg/kg body weight/day.

The histopathologically observed nephropathy in high dose animals from both sexes consisted mainly of an interstitial inflammation with related peritubular (interstitial) fibrosis at a minor severity. All findings in the two lower dose groups (35 and 100 mg/kg bw/day) were within the range of normal renal background alterations and hence not related to treatment.

 

From the results of a sub-chronic to toxicity study in rats with Ammonium Thiocyanate a no observed adverse effect level (NOAEL) of 20 mg/kg/day was concluded.

At the high dose of 500 mg/kg bw/day there were treatment related deaths. Other organs which showed treatment-related pathological lesions in the 500 mg/kg dose group included the forestomach (squamous hyperplasia), liver (hepatocellular hypertrophy), testes (seminiferous epithelial degeneration), spleen (lymphoid hyperplasia and increased 0 hemopoiesis), thymus (atrophy) and bone marrow (erythroid hyperplasia), 100 mg/kg bw/day some minimal clinical signs and changes in haematology and clinical biochemistry parameters were observed. Histopathological examination revealed squamous hyperplasia of the forestomach and hepatocellular hypertrophy in some animals. Increased kidney weights in males were not supported by any histopathological evidence of renal dysfunction. 20 mg/kg bw/day there were no changes noted that were considered to be an effect of treatment.

 

By definition of CLP Regulation, classification with STOT-RE Category 2 is relevant for ”substances that, on the basis of evidence from studies in experimental animals can be presumed to have the potential to be harmful to human health following repeated exposure. Substances are classified in category 2 for target organ toxicity (repeat exposure) on the basis of observations from appropriate studies in experimental animals in which significant toxic effects, of relevance to human health, were produced at generally moderate exposure concentrations.”

 

According to guidance on CLP moderate exposure concentrations relevant for STOT-RE –Category 2 classification are 10 to ≤ 100 mg/kg bw/day for the oral route for data obtained from a sub-chronic toxicity study.

In the study with Guanidine nitrate the NOAEL was 100 mg/kg bw/day and thus there were no treatment related alterations at 35 and 100 mg/kg bw/day.

In the study with Ammonium Thiocyanate the NOAEL was 20 mg/kg bw/d, at a dose level of 100 mg/kg bw/d some minimal clinical signs and changes in haematology and clinical biochemistry parameters were observed. Histopathoiogical examination revealed squamous hyperplasia of the forestomach and hepatocellular hypertrophy in some animals. Increased kidney weights in males were not supported by any histopathological evidence of renal dysfunction. Therefore the observed effects at 100 mg/kg bw/d were not considered “significant/severe” effects and thus classification as STOT RE is not warrant.

 

In conclusion considering the evaluated effects from the sub-chronic toxicity studies a classification with STOT-RE according to GHS Regulation EC No 1272/2008 for Guanidine Thiocyanate was considered not warrant, as no clear target organ toxicity could be identified below the harmful cut-off values for classification Cat 2 (10-100 mg/kg bw) in the CLP regulation.