1-Propanaminium, 2-hydroxy-N,N,N-trimethyl-3-[(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)thio]-, chloride (1:1)

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Feb 2018 - 4 Jun 2018

Reliability:

1 (reliable without restriction)

Data source

Reference

Title:

Unnamed

Year:

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:

The Test Suspensions (Group 2 and 2 - S) or vehicle (Group 1) will be administered daily, by topic route on the skin, approximately at the same period of day (in the morning) on a 7-day-a-week during 21 days

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

{2-hydroxy-3-[(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)sulfanyl]propyl}trimethylazanium chloride

EC Number:

811-523-6

Cas Number:

88992-45-4

Molecular formula:

C14 H19 F13 N O S . Cl

IUPAC Name:

{2-hydroxy-3-[(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)sulfanyl]propyl}trimethylazanium chloride

Test material form:

solid: particulate/powder

Details on test material:

Batch nº: 2007-0817-15
Analyzed concentration: 99.56% (w/w)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System
This study was conducted using rats (Rattus norvegicus), Wistar Hannover strain, and albinos,
with age between 4-6 weeks, (Batches 08/2017 - Pilot and 004/18 - Definitive). There were used 5
animals/sex/group, 10 animals (5 females and 5 males) on pilot test 1. In the pilot test 2 were used 5 animals/sex/group, 20 animals (10 females and 10 males). In addition, in the definitive test were used 5 animals/sex/group, 60 animals (30 females and 30 males) on definitive test, totalizing 90 animals.
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 supplier was CEMIB - Centro Multidisciplinar para Investigação Biológica na Área de Ciências de Animais de Laboratório.
Environmental conditions
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 g
rids 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 cycle was maintained, and ambient air was renewed 10 to 20 times per hour.

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

water

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 (003/17) and 3 of each dose aliquots was analyzed for each gender, totalizing 9 samples (003/18- A175, 003/18 A350, 003/18 A700, 003/18- B175, 003/18 - B350, 003/18 - B700, 003/18- C175, 003/18 – C350 and 003/18- C700). The acceptance limit between the aliquots should be less than ± 20%

Duration of treatment / exposure:

21 d

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)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

175 mg/kg bw/day

Remarks:

lower dose

Doses / concentrations 2

Dose / conc.:

350 mg/kg bw/day

Remarks:

Intermediate dose

Doses / concentrations 3

Dose / conc.:

700 mg/kg bw/day

Remarks:

higher dose

No. of animals per sex per dose:

There were used 5 animals/sex/group, 10 animals (5 females and 5 males) on pilot test 1.
In the pilot test 2 were used 5 animals/sex/group, 20 animals (10 females and 10 males).
In addition, in the definitive test were used 5 animals/sex/group, 60 animals (30 females and 30 males) on definitive test, totalizing 90 animals.

Control animals:

yes, concurrent vehicle

Details on study design:

Selection of the doses
Due to the absence of literature information on the toxicity of the test substance, a pilot test was perfor med at the dose of 1000 mg.Kg-1. The pilot test used 10 animals (5 males and 5 females) and it presented severe skin lesions since the 4th day of administration, except for 3 of 5 females that has presented servere skin lesions since the 3rd day of administration. Therefore, a second pilot was performed, based on the sponsor’s information that the test substance is used at a concentration of 30% in the final product, and the LD50 of the active ingredient is higher than 600 mg.Kg-1. Thus, the second pilot aims using 10 animals for dose (5 males and 5 females), totaling 20 animals. The objective was to establish a higher dose than LD50, which causes clinical signs, but does not cause severe skin injuries.
The second pilot was performed at doses 700 and 800 mg.Kg-1 and there was presence of injuries in the skin at both doses, however, at dose 800 mg.Kg-1 the erythema was multifocal (3 of 5) and moderated (2 of 5). After the results, the higher dose selected for definitive test was 700, and using a factor 2, the intermediate and lowest dose were 350 and 175 mg.Kg-1, respectively.
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.
Experimental procedure
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. Some animals were trichotomized 2 times in the period of 21 days and others only once. The criterion for repeating or not the trichotomy was the absence of hair and animal welfare, at the intermediate doses and
higher doses that presented skin injuries.

Examinations

Observations and examinations performed and frequency:

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 tr
eatment 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 day before 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

Sacrifice and pathology:

Euthanasia
All animals were euthanized at the end of the study.
Necropsy and Pathology
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.
Weight of organs
The liver, kidneys, adrenals and testes were dissected and weighed wet. Peer organs were weighed together.

Other examinations:

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. Moreover, the clotting parameters were evaluated (Prot
hrombin time, fibrinogen concentration and thromboplastic time);
- 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, ornithine decarboxylase, bilirubin total, direct and indirect. The value of indirect bilirubin was obtained was obtained by subtracting direct bilirubin from the total bilirubin value.

Statistics:

GraphPad Prism 7 evaluated all parameters according to described below:
- Two Way Variance (ANOVA) and post-test of Tukey’s: body weight, feed consumption;
- One Way Variance (ANOVA) and post-test of Bonferroni’s Test: hematological (erithrocytes, pl
atelets, HCT, HBG, VCM, HCM and CHCM), biochemistry and coagulation parameters (PT, APTT
and fibrinogen), organs weight and White Blood Cells (segmented, lymphocytes, monocytes and
leukocytes);
- T Parametric Test: biochemistry and clotting parameters of satellite groups, organs weight of
satellite groups clinical pathology parameters (CBC, WBC, coagulation and biochemistry).
All tests was conducted of significance level was 5%

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During the exposition the only reported abnormality was the absence of grooming in the test systems
AN 3/06, AN 4/02, AN 4/10. On the other hand, evaluating the skin lesions was observed erythema on 1 of 5 males and 4 of 5 females at dose of 175 mg.Kg-1; erythema on 4 of 5 males and 5 of 5 females at dose of 350 mg.Kg-1; and erythema on 4 of 5 males and 5 of 5 females of higher dose.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

Males: In the skin, the groups at doses of 350 mg.Kg-1 (4 of 5 animals) and 700 mg.Kg-1 (all animals ) presented crust formations. On the other hand, only in one animal of control group and 175 mg.Kg-1 was observed presence of crust.
Females: In the skin the control group did not present alterations, however in all animals of treated groups were observed abnormalities as below:
- 175 mg.Kg-1: presence of discrete crusts in 04 animals and in one test system was observed
moderated crusts on the administration site;
- 350 mg.Kg-1: in 4 animals was observed multifocal crustal lesions and only one with discreet crusts on the administration site;
- 700 mg.Kg-1: presence of multifocal crustal lesions in all animals and presence of alopecic areas in 3 of 5 animals.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

When evaluating body weight in both males and females, there was statistical difference (Two Way ANOVA followed by Tukey’s Test) between the control versus intermediate and higher dose. Similarly, there was a significant difference between the lowest dose versus the intermediate dose and the higher dose.
The data of satellite groups there was statically difference during the period of exposition of test substance between control and dose of 700 mg.Kg-1, including after the interruption of administration

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

When evaluating food consumption in males, there was statistical difference between the control
and higher dose of 700 mg.Kg-1. However, there was difference between intermediate and higher
dose with lower dose, for males, and it was observed statistical difference between the lower and intermediate doses with control group. When evaluating the data of satellite groups, it is possible to observe that no difference between control and higher dose on females. On the other hand, in feed consumption of males rats there was statically different on the second and fourth week

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

When evaluating hematological analysis, red blood, in both males and females, there was no statistical difference or biological relevance between the control and treated groups. Due to the satellite groups were not evaluated statistically.
In the evaluation of the white blood cells also no statistical difference was found between control
group and treated groups at doses of 175, 350 and 700 mg.Kg-1. Due to the satellite groups were not evaluated statistically.
The last point evaluated was coagulation through the fibrinogen concentration, time of thrombin and prothrombin, where statistically significant difference was found:
- Fibrinogen in males rats between control group and dose of 700 mg.Kg-1;
- Time of Prothrombin in males rats at dose 350 mg.Kg-1 when compared to control group;
- Time of Prothrombin in females rats at dose 350 and 700 mg.Kg-1 when compared to control group;
- Time of Thromboplastic time in females at dose 700 mg.Kg-1;
When evaluating the satellite values of control and higher dose groups, it was observed that the
concentration of fibrinogen and thromboplastic time persist different of control values. Although, the values of satellite group was lower than control, in opposite the values of main test

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Evaluating clinical biochemistry in males there was no statistical difference or biologic significance between control and treated groups. However, the females presented a statistically significant difference for the serum levels of Phosphorous, ALT, glucose and urea. These values area evaluating in satellite group for unpaired test T. No statistical difference was observed on values when treated satellite group with control satellite group.
There was no valor of normality of ornithine decarboxylase and there was statistical difference when comparing dose of 700 mg.kg-1 with control group. Although, there was no statistical difference between satellite groups.
The similarly results was found on males when comparing treated groups with control group, however in satellite groups the statistical difference remained.

Endocrine findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males test systems present statistically significant difference in relative values of the kidneys and
testicles weight when compared to the doses with the control group (One-way ANOVA followed by Bonferroni’s post-test). Moreover, females present statistically significant difference in relative values of kidneys and adrenals weight when compared the dose of 700 mg.Kg-1 with control group. On the satellites groups, only the adrenals weight in females rats reversed the effects. The others organs have presented statistical difference when compared treated group with control group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The macroscopic and histopathological evaluation demonstrate evidences of alterations on liver, kidneys and skin. Macroscopically in liver males, 02 of 05 animals of control group presented moderated lattice pattern; on the lower dose 02 of 05 animals presented moderated lattice pattern and 02 of 05 presented accentuated lattice
pattern; at dose of 350 mg.Kg-1 03 of 05 animals presented moderated lattice pattern and 01 of 05 presented accentuated lattice pattern; on the higher dose 05 of 05 presented moderated lattice pattern and only one presented accentuated lattice pattern. Furthermore, the alterations persisted on the satellite groups.
Evaluating kidneys, it is possible to observed alterations only in treated groups: 02 animals of lower dose presented alterations (AN2/27 – discreet dilatation and AN2/29 – hydronephrosis); 03 of 05 animals at dose 350 mg.Kg-1 with pelvis dilatation and 01 of 05 with medullary cortical congestion; at dose of 700 mg.Kg-1 all males presented alterations, 03 of 05 with congestion and 02 of 05 with pelvis dilatation. Furthermore, the alterations persisted on the satellite groups.
In the skin, the groups at doses of 350 mg.Kg-1 (4 of 5 animals) and 700 mg.Kg-1 (all animals) presented crust formations. On the other hand, only in one animal of control group and 175 mg.Kg-1 was observed presence of crust.
Therefore, it is possible to observe abnormalities on kidneys (pelvis dilatation on kidneys in two test systems and congestion in another two animals) and liver satellite control group, however the skin did not present any alteration. In the treated satellite group was observed abnormalities, mainly on liver (discreet lattice pattern on 4 animals and one with moderated lattice pattern) and on the skin (diffuse growth of hairs).
In addition, females rats presented the same macroscopic findings as males. On the liver, in only one animal was found moderated lattice pattern in control group.
In the treated groups, 3 of 5 animals at doses of 175 and 350 mg.Kg-1 presented discreet lattice pattern, with exception of test system AN3/06 and AN 2/32 that presented changed lattice pattern. In the higher dose, in all animals was observed lattice pattern, moderated in 2 animals, discreet on another 2 animals and accentuated in only one animal.
Evaluating kidneys of females no alterations was observed on control group and only one test system at lower and intermediate dose presented discreet depression focus with discreet pelvis dilatation (AN2/28); and discreet congestion (AN3/08). On the other hand, at dose of 700 mg.Kg-1 in 4 of 5 test systems was observed alterations: AN4/02 with external surface with a discrete focal area of grayish coloration, AN4/06 with external surface with a multifocal area of grayish coloration and accentuated cut red wine, AN4/08 with moderated and larger focus of pelvis dilatation; and AN4/10 with surface cut diffusely red wine.
In the skin the control group did not present alterations, however in all animals of treated groups were observed abnormalities as below:
- 175 mg.Kg-1: presence of discrete crusts in 04 animals and in one test system was observed moderated crusts on the administration site;
- 350 mg.Kg-1: in 4 animals was observed multifocal crustal lesions and only one with discreet crusts on the administration site;
- 700 mg.Kg-1: presence of multifocal crustal lesions in all animals and presence of alopecic areas in 3 of 5 animals.
On the satellite control group no abnormalities was observed and the treated satellite group presented moderated pelvis dilatation on wright kidney (AN4S/06), moderately pale liver (AN4S/02 and AN4S/10) and crusts formations in all test systems.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histological findings was observed both males and females on liver, kidneys and skin. Moreover, it is
possible to note that the lesions was more severe at dose of 700 mg.kg-1.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

175 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

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The results obtained in the present study indicated that dermal administration of test substance with
2-hydroxy-N,N,N-trimethyl-3-[(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)thio]-1-propanimium chloride in Wistar rats (Rattus novergicus) during 21 days produced the following findings related to the test substance:
- 175 mg/Kg/day
No adverse systemic effects related to the test substance.
- 350 mg/Kg/day
Local response on the skin (epidermal);
Reduction of body weight gains in males;
Reduction of feed consumption in males;
Increase of time for conversion of Prothrombin in males and females;
Increase of serum values of ALT and glucose in females;
Macroscopic findings on kidneys, liver and skin;
Increase of kidneys weight in females;
Histological findings on liver and skin.
- 700 mg/Kg/day
Local response on the skin (epidermal);
Reduction of body weight gains in males and females;
Reduction of feed consumption in males;
Increase of fibrinogen concentration in males and time for conversion of Prothrombin in females;
Reduction of time for conversion of Thromboplastic (APTT) in females;
Increase of serum values of Glucose, Urea, Phosphorous and ALT in females;
Increase of serum value of Ornithine decarboxylase in males and females;
Increase of relative weight of kidneys and adrenals in females;
Increase of relative weight of kidneys and testicles in males;
Macroscopic findings on kidneys, liver and skin;
Histological findings on kidneys, liver and skin.
Based on these results, the NOAEL (No-Observed-Adverse-Effect-Level) for rats males and females was 175 mg/Kg/day.