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Description of key information

In a repeat dose dermal study, the NOAEL is considered to be > 800 mg/kg/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Principles of method if other than guideline:
DBD/RENACIT 10 was administered daily via gavage in Cremophor EL (2%)/tap water to 5 male and 5 female Wistar rats per dose group, in doses of 0, 100, 300 or 1000 mg/kg body weight for a period of 4 weeks.
The animals were regularly observed and weighed. Food and water intake was determined. Functional Observational Battery (FOB) as well as Motor and Locomotor Activity (MA/LMA) tests and clinical laboratory investigations on blood samples were performed. Organs and tissues were subjected to gross and histopathological investigations and selected organs were weighed.
GLP compliance:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
other: Cremophor EL (2%)/tap water
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
29 (males)/30 (females) days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 100, 300 or 1000 mg/kg bw for a period of 4 weeks
Basis:
actual ingested
No. of animals per sex per dose:
5 male and 5 female rats per dose group
Control animals:
yes, concurrent vehicle
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

The survival rate, clinical appearance, body weight gain as well as food and water intake of the rats were not affected up to 1000 mg/kg.

FOB measurements and MA/LMA tests did not indicate neurotoxicity.

There was no indication of test substance-related changes in blood parameters. Beginning at 100 mg/kg an increase in liver weights was seen in males, which was correlated with liver enlargement. In females increased relative liver weights were noted at 1 000 mg/kg.

One of five males each showed distinct liver lobulation beginning at 300 mg/kg.

Centrilobular liver hypertrophy was diagnosed at 1000 mg/kg in males.

Taken together there were indications of adaptive liver reaction beginning at 100 mg/kg in males and at 1000 mg/kg in females.

The serum concentrations of sodium, potassium, were not affected by the treatment.

In the renal tubules of males at 300 mg/kg and above degenerations with increased accumulation of hyaline droplets, indicating a rat specific and not human relevant alpha-globulin nephropathy, was diagnosed. P2 and P3 segments of the cortical tubules were characterized by slight tubular dilation, luminal debris or cast and epithelial degeneration. Moreover, basophilic cortical tubules were raised by severity at 300 mglkg and above in males. Slight transitional cell hyperplasia occurred in the renal pelvis of 1/5 males each at 300 and 1000 mg/kg. In females, basophilic cortical tubules were slighty increased at 1000 mg/kg.

Taken together in the rat toxicologically relevant changes were evident in them kidneys beginning at 300 mg/kg in males and at 1000 mg/kg in females, where the alpha-globulin nephropathy and tubular basophilia are not supposed to be relevant for humans.

There was no morphological correlate to elevated heart weight of 1000 mg/kg males.

Necropsy, organ weight measurements and histopathology revealed no toxicologically relevant alterations in the remaining organs investigated.

Conclusions:
Toxicologically relevant changes were evident in the kidneys beginning at 300 mg/kg in males and at 1000 mg/kg in females, where the alpha-globulin nephropathy and tubular basophilia are not supposed to be relevant for humans. Necropsy, organ weight measurements and histopathology revealed no toxicologically relevant alterations in the remaining organs investigated.
Therefore for humans a NOAEL of 1000 mg/kg bw is justified.
Executive summary:

DBD/RENACIT 10 was administered daily via gavage in Cremophor EL (2%)/tap water to 5 male and 5 female Wistar rats per dose group, in doses of 0, 100, 300 or 1000 mg/kg body weight for a period of 4 weeks.

The animals were regularly observed and weighed. Food and water intake was determined. Functional Observational Battery (FOB) as well as Motor and Locomotor Activity (MA/LMA) tests and clinical laboratory investigations on blood samples were performed. Organs and tissues were subjected to gross and histopathological investigations and selected organs were weighed.

The test substance was stable in the vehicle for the duration of use. Formulations given to the rats were prepared appropriately.

The survival rate, clinical appearance, body weight gain as well as food and water intake of the rats were not affected up to 1000 mg/kg.

FOB measurements and MA/LMA tests did not indicate neurotoxicity.

There was no indication of test substance-related changes in blood parameters. Beginning at 100 mg/kg an increase in liver weights was seen in males, which was correlated with liver enlargement. In females increased relative liver weights were noted at 1 000 mg/kg.

One of five males each showed distinct liver lobulation beginning at 300 mg/kg.

Centrilobular liver hypertrophy was diagnosed at 1000 mg/kg in males.

Taken together there were indications of adaptive liver reaction beginning at 100 mg/kg in males and at 1000 mg/kg in females.

The serum concentrations of sodium, potassium, were not affected by the treatment.

In the renal tubules of males at 300 mg/kg and above degenerations with increased accumulation of hyaline droplets, indicating a rat specific and not human relevant alpha-globulin nephropathy, was diagnosed. P2 and P3 segments of the cortical tubules were characterized by slight tubular dilation, luminal debris or cast and epithelial degeneration. Moreover, basophilic cortical tubules were raised by severity at 300 mglkg and above in males. Slight transitional cell hyperplasia occurred in the renal pelvis of 1/5 males each at 300 and 1000 mg/kg. In females, basophilic cortical tubules were slighty increased at 1000 mg/kg.

Taken together in the rat toxicologically relevant changes were evident in them kidneys beginning at 300 mg/kg in males and at 1000 mg/kg in females, where the alpha-globulin nephropathy and tubular basophilia are not supposed to be relevant for humans.

There was no morphological correlate to elevated heart weight of 1000 mg/kg males.

Necropsy, organ weight measurements and histopathology revealed no toxicologically relevant alterations in the remaining organs investigated.

Under the conditions described a no-observed-effect-level (NOEL) can not be determined for males due to adaptative changes in the liver considered to be not adverse. Deduced from species-specific kjdney alterations, the no-observed-adverse-effect-level (NOAEL) is assigned at 100 mg/kg for male and at 300 mg/kg (=NOEL) for female rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
OECD Guideline for Testing of Chemicals No. 410. Repeated Dose Dermal Toxicity:21/28-Day,1-8. May. 1981 State Environmental Protection Administration : The Guidelines for the Testing of Chemicals Ministry of Health,2005: Technical standards for testing of chemicals .
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: State Environmental Protection Administration : The Guidelines for the Testing of Chemicals
Deviations:
no
Qualifier:
according to
Guideline:
other: Ministry of Health,2005: Technical standards for testing of chemicals .
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Test species: Rats.
Strain: SD.
Supplier: Guangdong Medical Laboratory Animal Center [certified animal number SCXK (Guangdong) 2008-0002].
Number of animals: 25 males and 25 females.
Number of groups: 5(control group, Low-dose group, Intermediate-dose group, High-dose group and Additional group of high-dose).
Number of animal/group: 5 males and 5 females per group.
Body weight at the start of experiment: 200 ~ 274g .
Identification of animals: Tags marked with animal group number and treatment details were attached to cages. Each animal was given an unique number.
Acclimatization: Five days prior to the experiment in the test room.
Randomization: Animals were assigned to five groups randomly.


Environmental conditions
Test Room: In the Center
Animal house conditions: The test facility was an air-conditioned room with 12h artificial fluorescent light and 12h dark.
Temperature range: 20~25oC.
Humidity range: 40~70%.

Husbandry Practices
Caging: Stainless steel cages were used. Autoclaved clean dry corncob was used as the bedding material. Animals were housed in one group according to sex in cages.
Water bottle: Each cage was supplied with a polypropylene water bottle with a stainless steel nozzle.
Sanitation: Bedding material was changed daily.
Food and water: Standard pellet feed supplied by Guangdong Medical Laboratory Animal Center and ultra-pure filtered sterilized water were provided to the animals freely.
Frequency of providing feed and drinking water: Both drinking water and feed were provided ad libitum.
Type of coverage:
open
Vehicle:
water
Details on exposure:
Healthy young adult animals are acclimatized to the laboratory conditions for 5 days prior to the test. Before the test, animals are randomized and assigned to the treatment and control groups. An area of about 5×8 cm2 on the back of the animal was clipped free of hair. Repeat clipping or shaving is usually needed at approximately weekly intervals. Pepton 22 was administered on the hair-free site of the rats for 6 hours per day on a 5-day per week basis, for a period of 28 days. The rats were fasten in the stainless steel shelves for 6h, then the test site were cleaned with warm water every time. Animals of the Low-dose group were conducted in the 40 mg/kg dose level, the Intermediate-dose group were conducted in the 200 mg/kg dose level, the High-dose group were conducted in the 800 mg/kg dose level, the Additional group of high-dose were conducted in the 800 mg/kg dose level too, the control group were given distilled water instead of tested compounds, with the same procedure as all the test groups.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
28 days
Frequency of treatment:
Pepton 22 was administered on the hair-free site of the rats for 6 hours per day on a 5-day per week basis, for a period of 28 days.
Remarks:
Doses / Concentrations:
Low-dose group: 40 mg/kg
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
Medium-dose group: 200 mg/kg
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
High-dose group: 800 mg/kg
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
High-dose group (additional): 800 mg/kg
Basis:
nominal per unit body weight
No. of animals per sex per dose:
5 males and 5 females per group
25 males and 25 females in total
Control animals:
yes, concurrent vehicle
Details on study design:
Healthy young adult animals were acclimatized to the laboratory conditions for 5 days prior to the test. Before the test, animals are randomized and assigned to the treatment and control groups. An area of about 5×8 cm2 on the back of the animal was clipped free of hair. Repeat clipping or shaving were usually needed at approximately weekly intervals. Pepton 22 was administered on the hair-free site of the rats for 6 hours per day on a 5-day per week basis, for a period of 28 days. The rats were fasten in the stainless steel shelves for 6h, then the test site were cleaned with warm water every time. Animals of the Low-dose group were conducted at the 40 mg/kg dose level, the Medium-dose group were conducted at the 200 mg/kg dose level, the High-dose group were conducted at the 800 mg/kg dose level, the Additional high-dose group were conducted at the 800 mg/kg dose level too, the Control group were given distilled water instead of tested compounds, with the same procedure as all the test groups.
Positive control:
The Control group was given distilled water instead of tested compounds, using the same procedure as all the test groups.
Observations and examinations performed and frequency:
Observation period: All animals were observed signs of toxicity, including the time of onset the degree and duration, everyday for a period of 28 days, but animals of Additional high-dose group scheduled for follow-up observations were kept for a further 14 days without treatment to detect recovery from, or persistence of, toxic effects.

Observation: A careful clinical examination was made once each day. Additional observations was made daily with appropriate actions taken to minimize loss of animals to the study. Necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals.

Body weight: Body weight of each animal was recorded weekly and before necropsy.

Hematology test: Including erythrocyte count, hematocrit, hemoglobin concentration, platelet count, total and differential leucocyte count, thromboplastin time and partial thromboplastin time were investigated at the end of the test period.

Biochemistry test: Clinical biochemistry determination on blood was carried out at the end of the test period. Blood parameters of liver and kidney function are appropriate. The selection of specific tests was influenced by observations on the mode of action of the substance.
Include serum glutamic-pyruvic transaminase(ALT), serum glutamic-oxaloacetic transaminase(AST), gamma glutamyl transpeptidase(GGT), total bilirubin(TB), total serum protein(TP), albumen(ALB), blood creatinine(CRE) and urea nitrogen(BUN) measurements.

Gross necropsy: All animals in the study were subjected to a full gross necropsy which included examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. The liver, kidneys, adrenals and testes were weighed wet as soon as possible after dissection to avoid drying.
The following organs and tissues were preserved in a suitable medium for possible future histopathological examination: normal and treated skin, brain, heart, liver, spleen, lungs, kidney, paranephroi, testis, epididymides, ovaries and uteruses.

Histopathology: Histological examination was performed with microscopy on H.E. stained slides of the preserved organs and tissues of all the tested groups and the control group.
Sacrifice and pathology:
At the end of the experiment, all the animals were sacrificed to test some relevant indexes.
Clinical signs:
no effects observed
Dermal irritation:
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
platelet count PLT increased (P <0.05) in males, thromboplastin time (PT) prolonged (P <0.05) in males.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Biochemistry test: Compared to the result of the indexes of the Control group, there were no significant differences both in females and males in Low-, Medium-, and High-dose group. But the result of the indexes of Additional high-dose group compared with
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Compared to the result of the indexes of the Control group, there were no significant differences in females in Low-, Medium-, and High-dose group. Compared with the High-dose group, the liver coefficients and Ovaries of the Additional high-dose group wer
Gross pathological findings:
no effects observed
Description (incidence and severity):
Despite some pathological changes of the brain, cerebellum, pituitary, kidney, lung, testis and uterus of each dose group, the regularity, frequency and the degree of injury showed no significant dose-effect relationship in different dose levels. Consider
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
dermal irritation
Dose descriptor:
NOEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
dermal irritation
Dose descriptor:
NOAEL
Effect level:
> 800 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
dermal irritation
Critical effects observed:
not specified

For tables and other data, please view the full report.

Conclusions:
NOAEL determined by the report assessor
>200 mg/kg/d in males
>200 mg/kg/d in females
Executive summary:

Although the Chinese test report suggests a NOAEL of 40 mg/kg/day, there is no evidence for this in the test report and minor blood parameter changes are conisdered adaptive. A NOEL of 200 mg/kg/day is suggested by the reviewer, based on adaptive changes and findings of low statistical significance. It is considered that there were no adverse effects at the highest treated group of 800 mg/kg/day.

The NOAEL is therefore estimated to be > 800 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
800 mg/kg bw/day
Study duration:
subacute
Species:
rat

Additional information

The substance appears to be absorbed through dermal contact.

Changes reported in the subacute dermal study are conisdered to be adaptive. A group dosed at 800 mg/kg/day for 6 weeks showed no clinical signs or reduction in weight gain compared with controls and is considered to be the no adverse effect level.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

Key study - reliable

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:

Key study - reliable

Justification for classification or non-classification

The 28 day oral study showed minimal effects with a NOAEL considered to be 1000 mg/kg/day.

Toxicologically relevant changes were evident in the kidneys beginning at 300 mg/kg in males and at 1000 mg/kg in females, where the alpha-globulin nephropathy and tubular basophilia are not supposed to be relevant for humans. Necropsy, organ weight measurements and histopathology revealed no toxicologically relevant alterations in the remaining organs investigated.

Although the Chinese dermal test report suggests a NOEAL of 40 mg/kg/day, there is no evidence for this in the test report and minor blood parameter changes are conisdered adaptive. A NOEAL of 400 mg/kg/day is suggested by the reviewer, but it is likely that there were no adverse effects at the highest treated group of 800 mg/kg/day.