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Description of key information

The repeat dose toxicity of the substance were examined in 28-day and 90-day repeat dose oral studies in the dog and the rat. The 4-week oral study in rats was associated with gastrointestinal (portal of entry) effects, and systemic effects were attributed to chronic active inflammation and ulceration of the stomach.  The 4-week oral study in dogs found vomiting to be the dose-limiting effect, with no other signs of systemic toxicity.   Subsequent subchronic (90-day) studies in both rats and dogs supported these findings, and in the 90 day dog study a NOEL and NOAEL for systemic effects could not be established because of the vomiting that occurred in some dogs at all dose levels. Other toxicological findings were interpreted to be either secondary to the effects already noted or of limited toxicological significance. The NOAEL for systemic toxicity in rats was 50 mg/kg/day. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Mode of Action Analysis / Human Relevance Framework

Additional information

The repeat dose toxicity of CS-1246 has been examined in 28-day and 90-day repeat dose oral studies in the dog and the rat.

28-Day Studies

In the 28-day dog study four groups of male and female Beagle dogs (two male and two female animals per group) were dosed by gavage with CS-1246 diluted in corn oil giving doses of 0, 1.5, 5 or 15 mg/kg/day (Stebbins, K.E. and Brooks, K.J., 2006). With the exception of a slight decrease in absolute thymus weights, (which were not accompanied by histopthological changes), in high dose male dogs treatment did not produce any evidence of systemic toxicity. Animals in the high dose (15 mg/kg/day) group did however vomit periodically. While the vomiting was interpreted as treatment related the absence of any signs of histopathological changes in the stomach suggested it was due to nausea rather than gastric irritation.

 

For the 28-day rat study rats (5/sex/dose, 6-7 weeks of age at study start) were treated with CS-1246 (oral gavage) at dose levels of 0, 50, 100, 300, and 1000 mg/kg/day for 28 consecutive days (Stebbins, K.E. and Card, T.L., 2006). The animals were subject to the standard toxicological procedures and measurements including ophthalmology, body weights, food intake, daily cage side observations, haematology, clinical chemistry, organ weights (necropsy), and histopathology.

The only treatment-related clinical observation was the presence of salivation in males and females a treated at 100, 300, or 1000 mg/kg/day. Animals in the high dose, (1,000 mg/kg/day), showed statistically significant decreases in body weights and at the end of the study the mean body weight gains of males and females given 1000 mg/kg/day were 32.4 and 12.2% lower than controls, respectively. Feed consumption of males given 1000 mg/kg/day was also reduced and statistically identified for the first three weeks of the study.

Males given 1000 mg/kg/day showed evidence of hypochromic microcytic anemia, which, based on the reticulocytosis and polychromasia also seen at this dose level, was interpreted to be regenerative. Males given 300 or 1000 mg/kg/day, and females given 1000 mg/kg/day, had treatment-related increases in the mean white blood cell counts, considered to secondary to the gastric inflammation occurring in these animals corresponded to inflammation of the gastric mucosa at these dose levels. Additional treatment-related effects consisted of statistically-identified higher mean platelet counts in males and females given 300 or 1000 mg/kg/day, a slightly higher mean reticulocyte count in females given 1,000 mg/kg/day.

Treatment-related systemic hepatic effects consisting of increased liver weights and slight centrilobular hypertrophy of hepatocytes were noted in the livers of males given 1000 mg/kg/day. While there was no corresponding microscopic liver alteration in high-dose females both sexes dosed at 1,000 mg/kg/day showed increased AST, ALT, and triglyceride concentrations, and treatment-related statistically identified lower total protein, albumin, and glucose concentrations. Males and females given 300 mg/kg/day had statistically-identified higher mean AST activities that were interpreted to be related to treatment.  

Dose related gastric irritation was the most prominent effect related to treatment with one or more animals from all treatment groups showing hyperplasia of the limiting ridge of the stomach and multifocal or diffuse hyperplasia and hypertrophy of mucous cells in the glandular mucosa of the stomach. Males and females given 300 or 1000 mg/kg/day had treatment-related inflammatory and erosive/ulcerative effects of the gastric mucosa. Ulcers were also present in the glandular or nonglandular mucosa and pylorus of several males and females given 1000 mg/kg/day. In most instances the gastric inflammation was chronic active, with the most severe inflammation associated with sites of mucosal erosion or ulceration. The gastric irritation was interpreted to be caused by the presence of formaldehyde, which is a degradation product of CS-1246 in the acidic environment of the stomach. Some male rats in the 1,000 mg/kg/day treatment group also showed evidence of irritation in the duodenum characterized by slight diffuse mucosal hyperplasia and hypertrophy.

As hyperplasia of the gastric mucosa occurred at all dose levels, a no-observed-effect level (NOEL) could not be determined.

90-Day Studies

In a 90-day repeat dose oral dog study four groups of four male and four female Beagledogswere administeredCS-1246by gavage at targeted dose levels of 0, 1.5, 5, or 15 mg/kg body weight/day (Stebbins, K. E. and Brooks, K. J., 2007). One male and one female dog given 1.5 mg/kg/day vomited once during the study (days 33 and 71, respectively), one female dog given 5 mg/kg/day vomited on day 63, and three male and two female dogs given 15 mg/kg/day vomited at least once during the study. The vomiting was considered to be treatment related, and was most likely due to nausea rather than direct gastric irritation, since there was no microscopic evidence of gastric irritation at any dose level. Therefore, the vomiting was interpreted to be a protective, non-adverse effect. Females given 15 mg/kg/day had slightly higher urine pH and in some animals phosphate crystals were present in the urine. There were however no associated histopathologic alterations of the urinary tract. Because of the vomiting that occurred in some dogs from all treatment groups, it was not possible to establish a NOEL. 

 

In the 90-day repeat dose study in the rat groups of ten male and ten female rats were administered CS-1246 in corn oil by gavage at dose levels of 0, 10, 50, or 250 mg/kg /day (Stebbins, K.E.et al, 2007). The animalswere subject to the standard toxicological procedures and measurements including ophthalmology, body weights, food intake, daily cage side observations, haematology, clinical chemistry, organ weights (necropsy), and histopathology.Detailed clinical observations (DCO) were conducted on all animals pre-exposure and once per week throughout the study; the examination included cage-side, hand-held and open-field observations, which are recorded categorically or using explicitly defined scales (ranks). The functional tests (sensory evaluation, rectal temperature, grip performance and motor activity) were conducted pre-exposure and during the last week of treatment.

There were no treatment-related effects in functional tests, body weights, feed consumption, ophthalmic observations, prothrombin time, clinical chemistry parameters, or organ weights. Males and females given 250 mg/kg/day had a treatment-related increase in the incidence of salivation immediately following treatment. Rats in this treatment group also had higher mean reticulocyte counts. While the increase was considered to be related to treatment the absence of any other hematologic effects and the normal microscopic appearance of the bone marrow in all animals indicated the response was of minimal toxicological significance. 

Females given 250 mg/kg/day had an increase in the incidence and severity of blood in the urine, but as there was no evidence of histopathologic alterations in the urinary tract the effect was considered to be of minimal toxicological significance. Males given 250 mg/kg/day exhibited slight increased eosinophilia of the centrilobular hepatocytes. AS this minor effect was not accompanied by clinical chemistry or liver weight changes, the alteration was interpreted to be a non-adverse effect of minimal toxicological significance.

The major toxicological consequence of treatment was the gross and microsopic pathological changes in the stomach of exposed animals. All animals in the high dose group together with all females and 8/10 males in the 50 mg/kg/day treatment group exhibited gross pathological changes of a thickened limiting ridge of the stomach. In addition, one male given 250 mg/kg/day had a treatment-related ulcer of the glandular mucosa of the stomach. There were no treatment-related gross observations in males or females given 10 mg/kg/day. Microscopically, all males and 6/10 females given 50 mg/kg/day, and all males and females given 250 mg/kg/day had slight hyperplasia of the squamous epithelium lining the limiting ridge of the stomach. Very slight or slight hypertrophy of the surface epithelial cells lining the glandular mucosa of the stomach was present in 2/10 males given 50 mg/kg/day, and in all males and females given 250 mg/kg/day. Subacute to chronic inflammation of the glandular mucosa and submucosa occurred in a few males and females given 50 mg/kg/day and in the majority of males and females given 250 mg/kg/day. Five males given 250 mg/kg/day had increased numbers of mitotic figures in the mucous cells of the glandular mucosa. Additional stomach effects consisted of multifocal erosions in the glandular mucosa of one male given 250 mg/kg/day, a focal ulcer in the glandular mucosa of another male given 250 mg/kg/day, and focal necrosis of the glandular mucosa of one female given 250 mg/kg/day. All of the above effects were interpreted to be the result of direct irritation to the gastric mucosa by CS-1246. There were no treatment-related effects in the stomachs of males and females given 10 mg/kg/day.

There was partial recovery of the inflammation of the stomach, with treatment-related chronic inflammation of the glandular submucosa still present in a few males and females given 50 mg/kg/day, and in the majority of males and females given 250 mg/kg/day, following the 28-day recovery period. There was complete recovery of all other treatment-related effects. 

The no-observed-effect level (NOEL), for point of contact localized gastric irritation was 10 mg/kg/day CS-1246. The NOEL for the minor systemic toxicological effects was identified as 50 mg/kg/day for both sexes.

 

Overall Assessment

Based on the results from the repeat dose toxicity studies it appears that high dose exposures to CS-1246 in the rat can produce a slight increase in reticulocyte counts, (without change to other hematological parameters), together with mild changes in liver histopathology. Such effects occur at doses of 250 mg/kg/day and above. The major effect seen in the rat was local gastric irritation characterized by multifocal erosions andhyperplasia of the squamous epithelium lining the limiting ridge of the stomach. Oral treatment can be characterized as producing two types of toxicological responses. The first and most severe is the local irritation seen in the stomach at point of contact of treatment.In the 90-day repeat dose study in the rat the NOEL for this point of contact gastric irritation/local effect was 10 mg/kg/day. The toxicological effects associated with systemic exposures were relatively mild in nature and included some minor hepatic and a slight increase in reticulocyte counts without change to other hematological parameters. The NOEL for systemic effects, (i.e. mild hepatic and haematological effects) is 50 mg/kg/day.

As treatment with CS-1246 produced nausea and vomiting in the dog the data obtained from the repeat dose studies in this species are really of little help in identifying and quantifying the health risk posed by exposure to CS-1246.

While the neurotoxic potential of CS-1246 has not been evaluated directly there are no indications from acute or repeated-dose testing that CS-1246 is a potential neurotoxicant. There have been no in-life observations indicative of neurotoxicity in acute studies by the oral, dermal, and inhalation routes, nor have there been any systemic effects noted in repeated-dose studies that would suggest a potential for neurotoxicity. Gross pathological examinations and histopathology on experimental animals, (rats and dogs), treated orally for 28 to 90 days have shown no evidence of changes in the brain, spinal cord, or peripheral nerves. Additionally, there are no structural/chemical properties that would suggest a neurotoxic potential or cholinesterase inhibition. Thus based on the results of available toxicology studies, in two species, there is no evidence that exposure to CS-1246 will produce neurotoxicity.  

There is no information available for systemic or local toxicity after repeated dermal exposures.

DNEL Calculations

The NOEL from the 90-day oral rat study will be used to calculate DNEL. For DNEL local gastric irritation the NOEL of 10 mg/kg/day will be used and for systemic DNEL a NOEL of 50 mg/kg/day will be used.

 

References:

Stebbins, K.E. and Card, T.L. (2006) Bioban CS-1246: 28-Day Oral Toxicity Study in CRL:CD(SD) Rats. The Dow Chemical Company Report No: DR-0365-7827-002 GLP, Unpublished

 

Stebbins, K.E. and Brooks, K.J. (2006) CS-1246: 28-Day Oral Gavage Toxicity Study in Beagle Dogs. The Dow Chemical Company Report No: DR-0365-7827-003 GLP, Unpublished

 

Stebbins, K.E., Card, T.L., and Andrus, A.K. (2007) CS-1246: 90-Day Oral Gavage Toxicity Study in CRL:CD(SD) Rats. The Dow Chemical Company Report No: DR-0365-7827-004. GLP, Unpublished

 

Stebbins, K. E. and Brooks, K. J. (2007). CS-1246: 90-Day Oral Gavage Toxicity Study in Beagle Dogs. The Dow Chemical Company Report No: DR-0365-7827-007 GLP, Unpublished

Justification for classification or non-classification

Based the available data regarding effects on target organs following repeated exposure, classification is not warranted according to EU Directive 67/548/EEC and CLP Regulation (EC) No. 1272/2008.