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Description of key information

The systemic NOAEL and a local NOAEL for repeated dermal dose toxicity was determined to be 1000 and 40 mg/kg/bw, respectively.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

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:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Qualifier:
according to guideline
Guideline:
EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Kleintierfarm Madoerin AG, CH 4414 Fuellinsdorf, Switzerland
- Age at study initiation: males: 10-12 weeks
- Weight at study initiation: males: 235-257g, females: 204 - 224 g
- Housing: Individually in Makrolon type-3 cages with wire mesh lids and standard softwood bedding ('Lignocel', Schill AG, 4132 Muttenz, Switzerland).
- Diet (e.g. ad libitum): Pelleted standard Kliba 343, Batch 28/85 rat maintenance diet ('Kliba'-Futter, Klingentalmuehle AG, 4303 Kaiseraugst, Switzerland), available ad libitum
- Water (e.g. ad libitum): Community water from Itingen, available ad libitum
- Acclimation period: One week under test conditions, after veterinary examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 10
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
occlusive
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
4%
Details on exposure:
Twenty-eight (28) dermal applications were administered to the shaved skin of the back. The skin area involved in the dermal application was equivalent to about 10 % of the total body surface. The hair was clipped from the dorsal surface once weekly throughout the study. The test article was applied evenly on the shaved skin and covered with an occlusive dressing. The dressing was wrapped around the abdomen and fixed with an elastic adhesive bandage, for 6 hours per day, 7 days per week for a total of 28 application days. The application volume was 2 mL/kg body weight. The animals of the control group were treated with the vehicle alone (2 mL/kg) under the same conditions as described above. Test article remainders were washed off with luke-warm tap water after termination of the daily treatment in animals of groups 2 to 4. The skin was dried with disposable paper towels.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 days, 6 hours per day
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
40, 200, 1000
Basis:
nominal per unit body weight
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels are based upon acute studies conducted at RCC.
Positive control:
No data
Observations and examinations performed and frequency:
- Symptoms (systemic): Twice daily.
- Symptoms (local): Daily prior to the following application according to Draize score.
- Viability/Mortality: Twice daily.
- Food consumption: Weekly
- Body weights: Twice weekly
- Ophthalmologic examinations: Observation by Ophthalmoscope, (Heine-Bifocal Ophthalmoscope, miroflex type) at termination of the application period.
- Clinical laboratory investigations: Blood samples for hematology and clinical biochemistry were collected from all animals under light ether anesthesia. The animals were fasted for 18 hours before blood sampling but water was provided. Blood samples were collected from each animal between the hours of 7.30 and 9.00 a.m. to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus. Urine was collected over an 18-hour period into a specimen vial using a metabolism cage. Parameters being measured for hematology: erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, reticulocyte count, nucleated erythrocytes normoblasts, total leukocyte count, differential leukocyte count, red cell morphology, thromboplastin time, partial thromboplastin time. Parameters being measured for clinical biochemistry: glucose, urea, creatinine, bilirubin, cholesterol, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, calcium, phosphorus, sodium, potassium, chloride, albumin, protein total. Parameters being measured for urinalysis: volume (18 hour), specific gravity, pH, protein, glucose, ketone, bilirubin, blood, urobilinogen, urine sediment.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- All animals were necropsied and descriptions of all macroscopic abnormalities were recorded. The following organ weights were recorded on the scheduled date of necropsy: adrenal glands, brain, heart, kidneys, liver, ovaries, spleen, testes.

HISTOPATHOLOGY: Yes
- Sections of the following organs were examined microscopically from all rats of of the 0 and 1000 mg/kg bw dose groups (number of sections per organ): Skin (untreated, treated; 1), liver (1), kidneys (2), and all gross lesions. From all other rats, only the treated skin and all gross lesions were examined.
Statistics:
Univariate one-may analysis of variance was used to assess the significance of intergroup differences. If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups. The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution. For the overall spontaneous mortality data, the Fisher's exact test for 2x2 tables was applied. Group means were calculated for continuous data and medians were calculated for discrete data (scores).
Details on results:
- Mortality: No death occurred during the study.
- Symptoms: Slight to moderate crust formations were observed in one male of the 40 mg/kg bw, four males and five females of the 200 mg/kg bw group and five males and five females of the 1000 mg/kg bw group. No other local or systemic symptoms were observed.
- Food consumption: No differences in food consumption were observed between the animals of the test article-treated and control group during the test period.
- Body weights: No differences in body weight gain were observed between the animals of the test article-treated and control group during the test period.
- Ophthalmoscopic examinations: One female of the high dose group showed an opalescent and slight bluish discolored cornea at termination of treatment. No other treatment-related findings were observed in any animal of the test article-treated or control group.
- Clinical laboratory investigations: The assessment of hematological, biochemical and urinalysis data indicated no changes of toxicological significance at termination of the treatment. All statistical differences in the results of the hematological, biochemical and urinalysis parameters were considered to be incidental and of normal biological variation.
- Organ weight: No significant differences in absolute and relative organ weights and ratios were observed between the animals of the treated and control groups.
- Macroscopic findings: Small focal or multifocal sores and eschars were observed at the application site of three rats of the low dose group, eight rats of the intermediate dose group, and 10 rats of the high dose group. Moreover, a few spontaneous gross lesions were encountered in both control and treated rats. These lesions were correlated with their histologic diagnoses.
- Microscopic findings: Skin lesions were observed at the application site of three rats of the low dose group, eight rats of the intermediate dose group, and nine rats of the high dose group. They were characterized by focal or multifocal inflammation associated with multifocal or diffuse acanthosis. In the more severe cases, ulcerative dermatitis was observed. The severity of the lesions was similar in the rats of the intermediate and high dose groups.
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Effect level based on clinical symptoms, body weight, food consumption, ophthalmoscopic examination, clinical laboratory investigations, and pathology
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
40 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Effects observed on the treated skin.
Critical effects observed:
not specified
Conclusions:
The systemic NOAEL and a local NOAEL for repeated dermal dose toxicity was determined to be 1000 and 40 mg/kg/bw, respectively.
Executive summary:

In a GLP compliant repeated dose toxicity study, performed according to OECD guideline 410, Wistar rats (5/sex/dose) were treated with the test substance (0, 40, 200, and 1000 mg/kg bw) by repeated dermal application, for a period of 28 days, 6 hours per day. No treatment related effects were observed on mortality, clinical signs, food consumption, body weight, ophthalmoscopic examinations, clinical laboratory investigations, and organ weights.Treatment-related gross and microscopic findings were observed at the application site at dose-dependent incidence and severity. Macroscopically, the lesions consisted in sores and scabs of varying size, microscopically, minimal to severe inflammation with slight to marked acanthosis was observed. Since only treatment-related dose depended alterations were observed macroscopically and microscopically in the treated skin of different animals of the test article treated groups, the NOAEL for systemic effects was determined to be 1000 mg/kg/bw and the NOAEL for local effects was determined to be 40 mg/kg bw.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP compliant guideline study, klimisch 1

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Qualifier:
according to guideline
Guideline:
EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Kleintierfarm Madoerin AG, CH 4414 Fuellinsdorf, Switzerland
- Age at study initiation: males: 10-12 weeks
- Weight at study initiation: males: 235-257g, females: 204 - 224 g
- Housing: Individually in Makrolon type-3 cages with wire mesh lids and standard softwood bedding ('Lignocel', Schill AG, 4132 Muttenz, Switzerland).
- Diet (e.g. ad libitum): Pelleted standard Kliba 343, Batch 28/85 rat maintenance diet ('Kliba'-Futter, Klingentalmuehle AG, 4303 Kaiseraugst, Switzerland), available ad libitum
- Water (e.g. ad libitum): Community water from Itingen, available ad libitum
- Acclimation period: One week under test conditions, after veterinary examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 10
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
occlusive
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
4%
Details on exposure:
Twenty-eight (28) dermal applications were administered to the shaved skin of the back. The skin area involved in the dermal application was equivalent to about 10 % of the total body surface. The hair was clipped from the dorsal surface once weekly throughout the study. The test article was applied evenly on the shaved skin and covered with an occlusive dressing. The dressing was wrapped around the abdomen and fixed with an elastic adhesive bandage, for 6 hours per day, 7 days per week for a total of 28 application days. The application volume was 2 mL/kg body weight. The animals of the control group were treated with the vehicle alone (2 mL/kg) under the same conditions as described above. Test article remainders were washed off with luke-warm tap water after termination of the daily treatment in animals of groups 2 to 4. The skin was dried with disposable paper towels.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 days, 6 hours per day
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
40, 200, 1000
Basis:
nominal per unit body weight
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels are based upon acute studies conducted at RCC.
Positive control:
No data
Observations and examinations performed and frequency:
- Symptoms (systemic): Twice daily.
- Symptoms (local): Daily prior to the following application according to Draize score.
- Viability/Mortality: Twice daily.
- Food consumption: Weekly
- Body weights: Twice weekly
- Ophthalmologic examinations: Observation by Ophthalmoscope, (Heine-Bifocal Ophthalmoscope, miroflex type) at termination of the application period.
- Clinical laboratory investigations: Blood samples for hematology and clinical biochemistry were collected from all animals under light ether anesthesia. The animals were fasted for 18 hours before blood sampling but water was provided. Blood samples were collected from each animal between the hours of 7.30 and 9.00 a.m. to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus. Urine was collected over an 18-hour period into a specimen vial using a metabolism cage. Parameters being measured for hematology: erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, reticulocyte count, nucleated erythrocytes normoblasts, total leukocyte count, differential leukocyte count, red cell morphology, thromboplastin time, partial thromboplastin time. Parameters being measured for clinical biochemistry: glucose, urea, creatinine, bilirubin, cholesterol, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, calcium, phosphorus, sodium, potassium, chloride, albumin, protein total. Parameters being measured for urinalysis: volume (18 hour), specific gravity, pH, protein, glucose, ketone, bilirubin, blood, urobilinogen, urine sediment.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- All animals were necropsied and descriptions of all macroscopic abnormalities were recorded. The following organ weights were recorded on the scheduled date of necropsy: adrenal glands, brain, heart, kidneys, liver, ovaries, spleen, testes.

HISTOPATHOLOGY: Yes
- Sections of the following organs were examined microscopically from all rats of of the 0 and 1000 mg/kg bw dose groups (number of sections per organ): Skin (untreated, treated; 1), liver (1), kidneys (2), and all gross lesions. From all other rats, only the treated skin and all gross lesions were examined.
Statistics:
Univariate one-may analysis of variance was used to assess the significance of intergroup differences. If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups. The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution. For the overall spontaneous mortality data, the Fisher's exact test for 2x2 tables was applied. Group means were calculated for continuous data and medians were calculated for discrete data (scores).
Details on results:
- Mortality: No death occurred during the study.
- Symptoms: Slight to moderate crust formations were observed in one male of the 40 mg/kg bw, four males and five females of the 200 mg/kg bw group and five males and five females of the 1000 mg/kg bw group. No other local or systemic symptoms were observed.
- Food consumption: No differences in food consumption were observed between the animals of the test article-treated and control group during the test period.
- Body weights: No differences in body weight gain were observed between the animals of the test article-treated and control group during the test period.
- Ophthalmoscopic examinations: One female of the high dose group showed an opalescent and slight bluish discolored cornea at termination of treatment. No other treatment-related findings were observed in any animal of the test article-treated or control group.
- Clinical laboratory investigations: The assessment of hematological, biochemical and urinalysis data indicated no changes of toxicological significance at termination of the treatment. All statistical differences in the results of the hematological, biochemical and urinalysis parameters were considered to be incidental and of normal biological variation.
- Organ weight: No significant differences in absolute and relative organ weights and ratios were observed between the animals of the treated and control groups.
- Macroscopic findings: Small focal or multifocal sores and eschars were observed at the application site of three rats of the low dose group, eight rats of the intermediate dose group, and 10 rats of the high dose group. Moreover, a few spontaneous gross lesions were encountered in both control and treated rats. These lesions were correlated with their histologic diagnoses.
- Microscopic findings: Skin lesions were observed at the application site of three rats of the low dose group, eight rats of the intermediate dose group, and nine rats of the high dose group. They were characterized by focal or multifocal inflammation associated with multifocal or diffuse acanthosis. In the more severe cases, ulcerative dermatitis was observed. The severity of the lesions was similar in the rats of the intermediate and high dose groups.
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Effect level based on clinical symptoms, body weight, food consumption, ophthalmoscopic examination, clinical laboratory investigations, and pathology
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
40 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Effects observed on the treated skin.
Critical effects observed:
not specified
Conclusions:
The systemic NOAEL and a local NOAEL for repeated dermal dose toxicity was determined to be 1000 and 40 mg/kg/bw, respectively.
Executive summary:

In a GLP compliant repeated dose toxicity study, performed according to OECD guideline 410, Wistar rats (5/sex/dose) were treated with the test substance (0, 40, 200, and 1000 mg/kg bw) by repeated dermal application, for a period of 28 days, 6 hours per day. No treatment related effects were observed on mortality, clinical signs, food consumption, body weight, ophthalmoscopic examinations, clinical laboratory investigations, and organ weights.Treatment-related gross and microscopic findings were observed at the application site at dose-dependent incidence and severity. Macroscopically, the lesions consisted in sores and scabs of varying size, microscopically, minimal to severe inflammation with slight to marked acanthosis was observed. Since only treatment-related dose depended alterations were observed macroscopically and microscopically in the treated skin of different animals of the test article treated groups, the NOAEL for systemic effects was determined to be 1000 mg/kg/bw and the NOAEL for local effects was determined to be 40 mg/kg bw.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP compliant guideline study, klimisch 1

Additional information

A 5-day range finding study was performed to select the dosages for a 28-day dermal toxicity study (RCC 1985).

In a GLP compliant repeated dose toxicity study, performed according to OECD guideline 410, Wistar rats (5/sex/dose) were treated with the test substance (0, 40, 200, and 1000 mg/kg bw/day) by repeated dermal application, for a period of 28 days, 6 hours per day. No treatment related effects were observed on mortality, clinical signs, food consumption, body weight, ophthalmoscopic examinations, clinical laboratory investigations, and organ weights.Treatment-related gross and microscopic findings were observed at the application site at dose-dependent incidence and severity. Macroscopically, the lesions consisted in sores and scabs of varying size, microscopically, minimal to severe inflammation with slight to marked acanthosis was observed. Since only treatment-related dose depended alterations were observed macroscopically and microscopically in the treated skin of different animals of the test article treated groups, the NOAEL for systemic effects was determined to be 1000 mg/kg/bw/day and a marginal NOAEL for local effects was determined to be 40 mg/kg bw.


Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Only 28-day study available

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Only 28-day study available

Justification for classification or non-classification

Based on the findings of the repeated dose toxicity study, the substance does not need to be classified according to Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

The study results of the 28 -day repeated dose dermal toxicity study in rats (RCC Study 052648) do not warrant classification of FAT 40181 according to the CLP (Reg. 1272/2008) or DSD (Dir. 67/548/EEC) regulations. The justification for this conclusion of non-classification regarding specific target organ toxicity (STOT) or skin irritation/corrosion has been detailed in the attached expert statement (dated 13thMay 2014).