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Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02/Oct/2017 to 14/Mar/2018
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
yes
Remarks:
On the first day of exposure period, the animals will be weight between 170 and 350 grams. Age: From 3-4 weeks old Clinical Biochemistry Organs weight: spleen and ovaries was included on weight
GLP compliance:
yes (incl. QA statement)
Limit test:
yes

Test material

Constituent 1
Reference substance name:
Reference substance 001
Cas Number:
62880-93-7
Test material form:
solid
Specific details on test material used for the study:
Purity : 99,72%

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Wistar Hannover strain, and albinos, with age between 5-6 weeks
Sex:
male/female
Details on test animals or test system and environmental conditions:
The animals used previously passed through at least 7 days in a quarantine period and 5 days an acclimation period, and considered healthy, without injuries or diseases.
The animals were housed in up to 1 individual per sex per box during acclimation and exposure periods. During the experimental period, the rats were housed in
polypropylene boxes with metal grids and lining material (autoclaved wood shavings). Cage cleaning were performed twice a week for all animals. The boxes
were distributed on the shelves to ensure uniform ventilation and brightness throughout the study. The animals were kept under controlled and monitored environmental conditions throughout the study conduction for: temperature, humidity, light cycles and ventilation. Temperature and humidity data were recorded in a validated computerized system. The temperature of the experimental rooms was maintained at 22 ± 3 ° C, with relative humidity of 30-70%. A 12-hour light / dark cyclewas maintained, and ambient air was renewed 10 to 20 times per hour.
The animals received a conventional diet for laboratory animals and filtered and autoclaved drinking water both ad libitum throughout the study.

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
water
Remarks:
The chosen vehicle, water, was made based on the information provided by the sponsor. Since, it has been described that the test substance is soluble in water, in volumes greater than 25% of the solution at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
After preparation of test solutions, an aliquot of dose evaluated was analyzed by chromatography to determine the effective concentration of the active ingredient (a.i.) in the solution.
Each aliquot was identified with a code (001/17) and 4 aliquots was analyzed (001/17-A, 001/17-B, 001/17-C and 001/17-D). The acceptance limit between the aliquots should be less than ± 20%.
Duration of treatment / exposure:
28 days
Frequency of treatment:
The test suspension or vehicle was administered to the animals daily (7 days a week) dermally at the same time of day (morning). Individual doses were calculated based on the most recent body weight.
Doses / concentrations
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
Based on the information provided, the test substance does not present a skin irritant potential. A pilot test at 1000 mg/kg was performed, using 20 animals (10 control and 10 treated) and did not present reaction during 7 days of administration. Because of this evaluation, the limit test was conducted with the same dose (1000 mg/kg).
No. of animals per sex per dose:
There were used 5 animals/sex/group, 20 animals (10 females and 10 males) on pilot test and 30 animals (15 females and 15 males) on definitive test, totalizing 50 animals
Control animals:
yes, concurrent vehicle
Details on study design:
Each experimental animal previously selected for the test was prepared by clipping the fur from the back at least 24 hours prior to the application of the test suspensions. Afterwards, the skin were examined for signs of injuries and only animals with healthy and intact skin will be used. The test substance should be applied uniformly over an area, which is approximately 10 per cent of the total body surface area and then covered with a gauze dressing, which were held to the test site by an adhesive and non-irritating tape. Removal and ingestion of the test item by the animal was prevented by placing a suitable adhesive tape (semiocclusive patch) around the trunk and test area. The test substance was in contact with the skin at least 6 hours.

Examinations

Observations and examinations performed and frequency:
Daily Observation: During the treatment period, all animals were observed daily, two times a day (in the beginning and at the end of each day), for mortality and morbidity.
Clinical exam: All animals were submitted a detailed clinical examination, out of the box, prior to initiation of treatment and weekly during the treatment period. Signs to be observed included changes in the skin, hair, eyes, mucous membranes, occurrences of secretions and excretions, and autonomic activity (lacrimation, piloerection, pupil size and altered respiratory pattern). Changes in locomotion, posture and reaction to manipulation, as well as presence of clonic or tonic
movements, stereotyped (excessive self-cleaning, repetitive circular movements) or bizarre behavior (self-mutilation, walking backwards) were recorded.
Body Weight: Animals were weighed on the first day of dosing, weekly thereafter, and at termination.
Feed consumption: Feed consumption was evaluated weekly and the average feed consumption was calculated per animal per day.
Hematological Alysis and Clinical Biochemistry : The following examinations were performed on all animals:
- Hematology, including hematocrit, hemoglobin concentration, erythrocyte count, total leucocyte count and differential, and platelet count.
- Clinical biochemistry determination on blood were performed at the end of the test period: calcium, phosphorus, chloride, sodium, potassium, glucose, serum alanine aminotransferase, serum aspartate aminotransferase, gamma glutamyl
transpeptidase, cholesterol, triglycerides, urea nitrogen, albumen, total serum protein, blood creatinine.
Sacrifice and pathology:
All animals were euthanized at the end of the study.
At the end of the study, all animals were examined macroscopically for any structural abnormalities or pathological changes. The gross necropsy included examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents.
During necropsy the following organs of the male and female animals were collected and preserved in 10% formalin: normal and treated skin, liver, kidney, spleen and lung. Macroscopic alterations were also collected and preserved for histopathological evaluation.
The liver, kidneys, adrenals, spleen, testes and ovaries were dissected and weighed wet. Peer organs were weighed together.
Statistics:
Quantitative variables such as body weight, food consumption, organs weight, hematological, clinical biochemistry, coagulation tests was analyzed by Two way ANOVA followed by Bonferroni’s Test. White Blood Count (WBC) was evaluated by T-Test. The values of AST and ALT was analyzed by Two way ANOVA followed by Tukey’s Test. The level of significance will be set at 5%.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
During one week the animals of pilot study was observed and only test system AM 1/03 presented nasal porphyria for one day, and it was a control animal. This result confirmed the choice of dose in definitive test.
During the exposition of definitive test the only reported abnormality was the presence of nasal porphyria for one day in the test systems AM 1/13, AM 1/17, AM 2/11, AM 2/17, AM 2/19, AM 3/07 and AM 3/08. In addition, test system AM 3/04 presented nasal porphyria for 3 days on the first week.
Another exception was the test system AM 1/18 that died on the 14th day of administration, no presenting signals before the death. Thus, it was replaced by the animal AM 1/22, which showed no clinical signs or abnormalities during the 28 days of administration.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
The use of the semi-occlusive pet during the test substance administrations causes a physical aggression to the epidermis evidenced by the changes observed in the microscopic examination of the cutaneous tissue of the test systems in the three groups of this study. However, these lesions had a greater extent (distribution and frequency) and degree of involvement (spiny and corneous stratum) of the epidermis in the test systems of the 1000 mg/Kg group, suggesting that the test substance might have contributed to the aggravation damage to the epidermis. After the aggressive stimulus to the epidermis, as well as administration of the test substance, there was a slight reversibility of the adaptive alterations suffered by the epidermis due to aggression (physical and/or chemical). In conclusion, these alterations was local on the superficial of the skin, and there was no evidence of systemic alterations.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Another exception was the test system AM 1/18 that died on the 14th day of administration, no presenting signals before the death. Thus, it was replaced by the animal AM 1/22, which showed no clinical signs or abnormalities during the 28 days of administration.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
When evaluating body weight in both males and females, there was no statistical difference (Two Way ANOVA followed by Bonferroni’s Test) between the control and treated group, as shown in Figure 01. The individual values of each test system of the control and treated group are described in Tables 02 to 07. Thus, there was no evaluation of the satellite group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
When evaluating food consumption in both males and females, there was no statistical difference between the control and test group. As in the satellite group there was no difference in consumption between the 4 weeks of treatment and the 2 subsequent weeks when the test substance was discontinued.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
When evaluating hematological analysis, red blood, in both males and females, there was no statistical difference between the control and test group. Only exception are white blood count WBC in males.
Analyzing only the WBC of control and satellite group, it was observed that there was no statistically significant difference. The white blood series only presented statistically difference in quantity of lymphocytes of males, a result already expected due to the increase of WBC previously mentioned. Furthermore, there is no statistically significance difference between control and satellite group of males.
The last point evaluated was coagulation through the fibrinogen concentration, time of thrombin and prothrombin, where no statistically significant difference was found between treated and control groups, in both males and females.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
When evaluating clinical biochemistry in females, there was no statistical difference between the control and test group. However, the males presented a statistically significant difference for the serum levels of AST and ALT.
The AST and ALT values were evaluated together with satellite group for Two-Way ANOVA followed Tukey’s Test. No statistical difference was observed on AST values and on ALT values a statistical difference was observed when compared treated and satellite groups with control group.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Both males and females test systems did not present statistically significant difference were observed in relative values of the organs weight when compared to the treated group with the control group (Two-way ANOVA followed by Bonferroni’s post-test).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The macroscopic and histopathological evaluation did not present any evidence of toxicity from the test substance; the only evidence was some alterations on the skin. In the 3 groups studied was observed epidermal hyperplasia with less intensity in the control group and the treated group was also observed orthokeratotic hyperkeratosis. In the satellite group was observed epidermal hyperplasia as the same as control group and orthokeratotic hyperkeratosis as the same as treated group, however less intensity. There was no inflammatory response and no dermal alterations.
In the histopathological evaluation of cutaneous tissue of all test systems of the control group, multifocal areas with discrete thickening of the epidermis were observed. The occurrence of another form of distribution occurred in some of the test systems: uniform distribution (AM 1/15); multifocal to coalescing distribution (AM 1/17 and AM 1/19). Epidermal hyperplasia was associated with mild keratinocyte hypertrophy, sometimes accompanied by caryomegalalia, without altering the tintorial capacity of the nucleus. Also irregular keratinocyte stratification was observed; however, keratinocytes maintained their normal polarity, were not atypical and did not invade the basement membrane.
In the histopathological evaluation of cutaneous tissue of all the test systems of the 1000 mg/Kg group, greater evidence of epidermal thickening was observed when compared to that observed in the control group. In the 1000 mg/Kg group, the distribution varied from multifocal to coalescing (AM 2/11 and AM 2/18) or was regular / continuous (AM 2/12, AM 2/13, AM 2/14, AM 2/15, AM 2/16, AM 2/17, AM 2/19 and AM 2/20) and intensity ranging from mild to moderate. In these areas, irregular keratinocyte stratification, cellular hyperplasia and/or cellular histoptrophy were observed, with nuclei with increased size and from norm to hypochromic. Moreover, all keratinocytes maintained their normal polarity, were not atypical and did not invade the basement membrane. In addition, in this same group of test systems, discrete foci of orthokeratotic hyperkeratosis predominantly laminated (AM 2/11, AM 2/12, AM 2/13, AM 2/14, AM 2/15, AM 2/16, AM 2/17, AM 2/18 , AM 2/19 and AM 2/20), sometimes associated with focal thickening of the adjacent granular layer (AM 2/13, AM 2/15, AM 2/17 and AM 2/20) or interspersed with foci of compact hyperkeratosis (AM 2/14, AM 2/15, AM 2/17, AM 2/18, AM 2/19 and AM 2/20).
In the histopathological evaluation of all the test systems of the satellite group, there were areas with discrete thickening of the epidermis, associated with a discrete thickening of the granular stratum, characterized by hyperplasia and / or keratinocyte hypertrophy, with multifocal distribution (AM 3/01, AM 3/02, AM 3/05, AM 3/07, AM 3/08 and AM 3/09), or rare (AM 3/04, AM 3/06 and AM 3/10). In the present study, multifocal discrete orthokeratotic hyperkeratosis of laminated type (AM 3/01, AM 3/02, AM 3/05, AM 3/06, AM 3/07, AM 3/08 and AM 3/09) or compact (AM 3/04, AM 3/05, AM 3/07, AM 3/08 and AM 3/09) associated with epidermal hyperplasia. Test systems AM 3/05 and AM 3/09 also presented discrete foci of thickening of the granular stratum.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
According to OECD Guidance Notes No. 32 (2000), not all clinical signs observed may be related to pathological or morphological changes in organs or tissues. In addition, it should be considered that statistical significance does not necessarily imply biological significance, just as an effect with biological significance does not necessarily present statistical significance. Thus, the results found were evaluated together when possible, besides severity and persistence, in order to define which of the alterations found were considered adverse effects.
In order to evaluate the hematological parameters, the animals of control group present leucopenia (low amount of leukocytes) due to lymphopenia (low amount of lymphocytes) compared to normal values ranging from 5,000 to 12,000 mm3 (QUINTON, 2005). In addition, when the value of treated group was compared with the normal values of literature (QUINTON, 2005), it was in accordance with the normal range. Furthermore, when the values obtained for the males of the control and treated groups were compared, a statistically significant difference was observed. Therefore, the statistical significance found does not reflect a biological significance, since the treated group is in accordance with the normal parameters for species. And this change was not considered as an adverse effect to the test substance.
Regarding the biochemical evaluation, a statistically significant reduction in the ALT enzyme values was observed when the control group was compared with the dose of 1000 mg/Kg and the satellite dose group. However, this enzyme is not hepato-specific because ALT is also found in renal cells in lesser amounts. Considering the range of normality described in the literature of 23 to 50 U/L (BOEHM, OLAF et al., 2007), it is noted that the treated group presents ALT values close to this range. Although the control group presented high ALT values at the biochemical examination, the liver and renal histopathological analysis did not present results to justify this exacerbation. In both the liver and kidney, no cellular or tissue changes such as fibrosis and / or necrosis were found to warrant the intracytoplasmic release of ALT into the blood. Thus, due to the absence of other alterations, the result found was not considered biologically significant and therefore was not considered an adverse effect in the present study.
According to the second statistical evaluation, the AST enzyme values did not present a significant statistical difference and did not require further evaluation.
Regarding the integumentary system, it is known that in the normal epidermis there is an established equilibrium between the rate of basal cell proliferation and the rate of loss of differentiated surface cells, resulting in the constant thickness of the epidermis and of each of its layers. However, epidermal responses lead to changes in epidermal growth or differentiation such as keratinization disorders (hyperkeratosis, for example) and epidermal hyperplasia (increase in the number of cells within the epidermis, more frequently in the epidermal thorny stratum). Furthermore, changes in the fluid balance and cellular adhesion of the epidermis may occur; inflammatory lesions of the epidermis; alterations in epidermal pigmentation, which were not observed in the test systems of the present study.
Histopathological changes in superficial or deep dermis were also not observed.
The use of the semi-occlusive pet during the test substance administrations causes a physical aggression to the epidermis evidenced by the changes observed in the microscopic examination of the cutaneous tissue of the test systems in the three groups of this study. However, these lesions had a greater extent (distribution and frequency) and degree of involvement (spiny and corneous stratum) of the epidermis in the test systems of the 1000 mg/Kg group, suggesting that the test substance might have contributed to the aggravation damage to the epidermis. After the aggressive stimulus to the epidermis, as well as administration of the test substance, there was a slight reversibility of the adaptive alterations suffered by the epidermis due to aggression (physical and/or chemical). In conclusion, these alterations was local on the superficial of the skin, and there was no evidence of systemic alterations.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
clinical signs
dermal irritation
gross pathology
haematology
organ weights and organ / body weight ratios

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The results obtained in the present study indicated that dermal administration of test substance 2-methyl-2[(1-oxo-3-[(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)thio]propyl)amino]-1-propanesulfonic acid, sodium salt at dose of 1000 mg/Kg/day in Wistar rats (Rattus novergicus) during 28 days produced the following findings related to the test substance:
- No adverse systemic effects related to the test substance;
- Local response on the skin (epidermal).
Based on these results, the NOAEL (No-Observed-Adverse-Effect-Level) for rats males and females was equal to or greater than 1000 mg/Kg/day.
Executive summary:

The NOAEL (No-Observed-Adverse-Effect-Level) for rats males and females was equal to or greater than 1000 mg/Kg/day.

The substance 2-methyl-2[(1-oxo-3-[(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)thio]propyl)amino]-1-propanesulfonic acid, sodium salt induced a local response on the skin in combination with the semi-occlusive system.