Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From November 8, 2011
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Product: CarboNeXT
Batch: 08/07/11

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female

Administration / exposure

Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SAMPLE PREPARATION
The test product was neat.
Sodium chloride injection was used as control.
EXPERIMENTAL DESIGN
To perform the assay were used 30 rats, 15 male and 15 female.
10 animals (5 per sex) were treated with the neat test substance at a dose level of 2000 mg/kg, 10 animals (5 per sex) were treated with sodium chloride injection at a dose level of 2000 mg/kg and were used as controls and 10 animals were used as satellite group, so were treated in the same way of the group treated with the test sample, but observed for 14 more days from the end of the treatement period to determine the reversibility, persistence, o delayed occurrence of toxic effects.

The limit test was conducted at a dose level of 2000 mg/kg since the sample has already proven to be non irritant and not toxic after a test conducted according to OECD 402 and a dose range finding test of repeated exposure.
It was decide to perform the test at a dose double respect the limit test considering the risk for human exposure.
Duration of treatment / exposure:
TREATMENT
Skin preparation
24 hours before the beginning of the test the back and the sides of the animals was shaved on an area of about 30cm2. This procedure was repeated periodically when necessary.
Application
The test sample and the control solvent were given, at a ratio of 2000 mg/kg, with a square of gauze directly on animals skin after humidification with sodium chloride injection and fixed with a skin patch in an area of about 10% of the total body surface.
The gauze was put on the back of animals and further fixed with hypo-allergenic occlusive tape.
The whole trunk of the animal was protected with an elastic bandage.
6 hours after application bandage and adhesive tape were removed.

The same treatment was repeated daily (5 days/week) for 28 days.

Removal
6 hours after application bandage and adhesive tape were removed.
Skin was cleaned form sample excess by a tampon soaked with sodium chloride injection.
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
2000 mg/kg
Basis:

No. of animals per sex per dose:
30 rats (15 male and 15 females) divided in :
- treated group (5 male and 5 females)
- control group (5 male and 5 females)
- satellite group ((5 male and 5 females)
Details on study design:
- Dose selection rationale: during the Dose Range Finging the test product has been daily administered with a dose of2000, 1000 and 500 mg/kg by topical application to 3 groups of 5 female rats for 14 days by topical application.
In all groups no mortality has been detected; neither symptoms nor signs of toxicity were recorded. At the autopsy no abnormalities were detected.
On the basis of the results the dose of 2000 mg/kg was selected for the Repeated Dose Dermal Toxicity, OECD 410.

Examinations

Observations and examinations performed and frequency:
OBSERVATIONS
Body weight
Every week, before the first treatment, each animal was weighted.

Clinical observations
A clinical examination was made every day and any clinical symptoms were recorded.
Clinical observations have include:
- evaluation of organic bodies functions
- evaluation of tegumentary apparatus
- evaluation of mucus conditions
- evaluation of somatomotor activity and sensorial conditions.

Clinical pathology
24 hours after the last treatment, to animals of treated and control group was made a blood levy with the purpose to analyze all hematologic and chemistry parameters.
Analysed hematologic parameters were: leukocytes, erythrocytes, plateletes, haemoglobin concentration, haematocrit, mean corpuscular value.
Analysed chemistry parameters were: CPT, GOT, gamma GT, creatinine, glucose, total protein, Azotaemia, Sodium, Potassium and Albumin.
These parameter weren't executed on satellite group because no abnormalities were detected in analysed blood sample of the other two group.

Gross pathology
24 hours after the last application the rats were sacrificed and a full necroscopy was performed.
The following organs were collected and weighted: treated skin, spleen, liver, kidneys, ovaries, testicles, heart, lungs and thyms.
This operation wasn't executed on satellite group because no abnormalities were detected in organs sample of the other two group.

Hystopathology
An histological evaluation was performed on:
- treated skin, kidney, liver, gonads, thyms.
Organs were put in formalin solution 10% and sent to Accelera test site.
The specimens of treated skin, kidney, liver, gonads and thymus were trimmed, embedded in paraffin, sectioned, stained with hematoxylin and erythorin and examined histologically, according to the methods described in the "Standard Operating Procedures" adopted at Accelera pathology facilities.
Residual tissues were preserved in fixative.
To improve the input of the data, macroscopic changes recorded on the necropsy forms at the Test Facility were trasferred into the Pristima (R) System.
Hystological evalutation data were directly entered into the Pristima (R) System.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Haematological findings:
no effects observed
Details on results:
During the study no mortality case or toxic symptoms has been detected in male and female rats.
The weight increase is complied to the standards of species and race. No abnormalities have been detected comparing treated and control groups.
At the autopsy no injuries has been detected, all collected organs haven't shown any abnormalities.
No abnormalities in hematologic and chemistry parameters analysed in male and female rats.
No treatment-related lesions were observed in the skin. On the whole, cutaneous changes mainly characterized by squamous cell hyperplasia and hyperkeratosis were noted in control and treated groups with a siilar incidence and severity.
Similarly, findings noted in the examined internal organs were not considered related to the treatment as they occurred with similar incidence and severity in control and treated animals and/or they are known to occur in laboratory experience as background pathology in this species.

Effect levels

Dose descriptor:
NOAEL
Effect level:
> 2 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
No toxic symptoms have been observed after 28 day repeated exposure at the dose of 2000 mg/kg of the test substance CarboNeXT.