3-(p-cumenyl)-2-methylpropionaldehyde

Endpoint summary

Description of key information

- A 28-Day Study of SILVIAL by dermal exposure of Wistar Rats study performed in 2017/2018 to determine the potential toxicity of SILVIAL, when given dermally for 28 days to Wistar rats and evaluate the No Observed Adverse Effect Level (NOAEL) .

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

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

a short-term toxicity study by the oral route does not need to be conducted because an appropriate dermal study is available and dermal is the most appropriate route of administration as based on the provided thorough and rigorous exposure assessment

Reason / purpose for cross-reference:

reference to other study

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

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

Study period:

10 Aug 2017 - 10 Nov 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The dermal route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.

Qualifier:

according to guideline

Guideline:

EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name (as stated in the report): Silvial
Batch No.: SC00020196
Expiration date: 09 November 2017
Chemical name (IUPAC): 2-METHYL-3-(4-(2-METHYLPROPYL)PHENYL)PROPANAL

Species:

rat

Strain:

other: Wistar Han rat

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Environmental Acclimation
- Test Facility toxicology accommodation for 15 days before the commencement of dosing
- Animals trained for the application routeduring acclimation period according to standard procedures

Animal Identification
- each animal was identified using tattoo

Environmental Conditions:
- Actual daily mean temperature: 21 to 22°C
- Actual daily mean relative humidity of: 50 to 73%
- Light cycle: 12-hour light/12-hour dark
- Air : Ten or greater air changes per hour with 100% fresh air (no air recirculation)

Housing:
- Animals were single housed (randomly) in polycarbonate cages containing appropriate bedding equipped with water bottles.

Food/Water:
- Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) ad libitum
- Municipal tap water freely available (via water bottles)

Veterinary Care:
- no examinations or treatments were required

Type of coverage:

semiocclusive

Vehicle:

corn oil

Remarks:

Stability for at least 24 hours at room temperature, 8 days in the refrigerator (2-8°C) 3 weeks in a freezer (≤-15°C) is confirmed over the concentration range 1.08 mg/g (low) to 212 mg/g (high).

Details on exposure:

At least 24 hours before first treatment, an area of approximately 6x10 cm on the back of the animals was clipped. Whenever necessary (during the course of the study) the skin-area was re-clipped at least 3 hours before a next application. Care was taken to avoid abrading of the skin.
The test item and vehicle were administered to the appropriate animals dermally once daily (for 6 hours ± 30 minutes) for 7 days a week for a minimum of 28 days. Application was performed approximately the same time each day. Animals were treated up to the day prior to necropsy. The dose volume for each animal was based on the most recent body weight measurement. The test substance formulation was applied onto a surgical gauze patch (Surgy 1D; less than or equal to 8 ply), with an area of approximately 10% of the total body surface (i.e. approx. 40 cm² for males and approx. 30 cm² for females). The gauze patch was mounted on Medical tape or Microfoam and held in contact with the skin with Tegaderm flexible bandage. To protect the application, Lomir jackets were used (Manufacturers: Laboratoires Stella s.a., Liege, Belgium (surgical gauze) and 3M, St. Paul, Minnesota, U.S.A. (Medical Tape and Microfoam) and Lomir Biomedical, Hull, UK (Jackets).
During the exposure period the dressing was checked regularly, at least every 2 hours (i.e. after application, and approximately 1, 3 and 5 hours after application). In case the application was found dislodged or improperly secured on the animal before or on the 3-hour bandage check time point, a new dressing with formulation was applied. The maximum time during which the animal was subsequently not exposed was estimated retrospectively.
The first day of dosing was designated as Day 1. The dosing formulations were stirred continuously during dose administration. A dose control system was used as additional check to verify the dosing procedure according to Standard Operating Procedures.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis:
- Interval : Week 1
- Concentration: All groups
- Homogeneity: Groups 2 and 4
- Stability: Performed previously in conjunction with the method development and validation study (Test Facility Study No. 513140) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in this study.
- Analytical method: Analyses were performed by using a validated analytical procedure (Test Facility Study No. 513140).
Instrument: Acquity UPLC system (Waters, Milford, MA, USA)
Detector: Acquity UPLC TUV detector (Waters)
Column: Acquity UPLC BEH C18, 100 mm x 2.1 mm i.d., dp =1.7 μm (Waters)
Column temperature: 40°C +/- 1°C
Injection volume: 10 μL
Mobile phase: 70/30 (v/v) acetonitrile/water
Flow: 0.5 mL/min
UV detection: 210 nm

Concentration and Homogeneity Analysis:
- Duplicate middle samples for Groups 1 and 3 (concentration analysis only) and duplicate top, middle, and bottom samples for Groups 2 and 4 (concentration and homogeneity analysis) (approximately 500 mg) for each sampling time point were sent to the analytical laboratory.
- Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10%. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10%.

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals dermally for a minimum of 28 days.

Frequency of treatment:

daily (for 6 hours ± 30 minutes) for 7 days a week

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 > Corn oil only (control group)

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 2 > Dose concentration = 20 mg/mL

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Group 3 > Dose concentration = 60 mg/mL

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 4 > Dose concentration = 200 mg/mL

No. of animals per sex per dose:

5 males and 5 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

see "details on exposure" field above

Positive control:

Not applicable

Observations and examinations performed and frequency:

The following parameters and end points were evaluated in this study: clinical signs, body weights, food consumption, clinical pathology parameters (hematology, coagulation, clinical chemistry), gross necropsy findings, organ weights, and histopathologic examinations.

Mortality/Moribundity Checks:
- Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.

Clinical Observations:
- Performed twice daily (prior to dermal exposure and after removal of the application), beginning during the first day of exposure and lasting throughout the dosing period. During the dosing period, these observations were performed directly after removal of the application.

Arena Observations:
- Animals were removed from the cage and placed in a standard arena, and a detailed clinical observation was performed weekly, beginning before the first administration of the test item. These observations were conducted after dosing.

Body Weights:
- Animals were weighed individually weekly, starting on Day 1. A fasted weight was recorded on the day of necropsy.

Food Consumption:
- Quantitatively measured weekly starting on Day 1 and continuing weekly throughout the dosing period.

Water Consumption:
- Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

Clinical Pathology (Hematology,Coagulation, Clinical Chemistry ) :
- Blood was collected between 7.00 and 10.30 a.m. from the retro-orbital sinus under anesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) of fasted animals.Animals were fasted overnight (with a maximum of 24 hours) before their scheduled necropsy.

Necropsy :
- All animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their
associated organs and tissues.

Organ Weights:
- Gland adrenal, kidney weighed at necropsy for all scheduled euthanasia animals.

Tissue Collection and Preservation:
- Representative samples of the tissues were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands)

Histology:
- The adrenal gland, heart, kidney, liver, skin, spinal cord, testis and gross lesions were embedded in paraffin (Klinipath, Duiven, The Netherlands), sectioned, mounted on glass slides, and stained with hematoxylin and eosin (Klinipath, Duiven, The Netherlands).

Histopathology:
- Histopathological evaluation was performed by a board-certified toxicological pathologist with training and experience in laboratory animal pathology.

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of systemic toxicity were noted during daily detailed clinical observations or during weekly arena observations.

Dermal irritation:

effects observed, non-treatment-related

Description (incidence and severity):

Slight erythema and/or scales were noted on the treated skin in males and females predominantly at 1000 mg/kg during the treatment period.
Other skin effects outside the treated area (including erythema, scabs, scales and/or wounds) were noted at all dose levels, including controls. These were considered to have occurred due to the application method. As these findings were slight, they were considered not to
toxicologically significant. Incidental findings of chromodacryorrhoea during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Slightly lower body weight gain was noted in males at 1000 mg/kg throughout the study period.
Body weights and body weight gain of males at 100 and 300 mg/kg and females up to 1000 mg/kg remained in the same range as controls over the study period.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption after correction for body weight was slightly higher for females at 1000 mg/kg during the study period, reaching statistical significance in Weeks 3 and 4.
Food consumption before or after correction for body weight in females up to 300 mg/kg and males up to 1000 mg/kg was similar to the control level over the study period.

Food efficiency:

not examined

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

not examined

Description (incidence and severity):

Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical pathology investigations revealed changes predominantly at 1000 mg/kg. Lower
platelet counts were noted in females at 300 and 1000 mg/kg accompanied by a longer
protrombine time (PT) and Activated Partial Thromboplastin Time (APTT) in females at
1000 mg/kg. Based on the magnitude of the affected coagulation parameters the findings at
1000 mg/kg are considered to be adverse.
Other findings included lower total protein (in females accompanied by decreased albumin
levels), cholesterol and calcium levels in both sexes at 1000 mg/kg. In addition, higher total
bilirubin and urea levels were noted in females at 1000 mg/kg. These changes were only
slight and therefore considered to be not adverse.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

A test item-related macroscopic finding was present in the liver of all females treated at 1000 mg/kg and consisted of pale discoloration. The microscopic correlate was microvesicular vacuolation and hepatocellular hypertrophy.
In one male treated at 1000 mg/kg, pale discoloration of the liver was noted as well but without a microscopic correlate.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

An increased incidence and severity of diffuse hepatocellular hypertrophy and microvesicular
vacuolation was observed in the livers of females starting at 300 mg/kg. Minimal single cell necrosis and centrilobular pigmented cells (up to slight degree) were observed in livers of female rats treated at 1000 mg/kg.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical biochemistry

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Dose Formulation Analyses:

- The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test item was detected in the Group 1 formulation.

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Conclusions:

In conclusion, daily dermal SILVIAL exposure was well tolerated in rats at levels up to 300 mg/kg. Adverse test item-related effects were observed at dose levels of 1000 mg/kg and consisted of liver findings and changes in coagulation parameters. Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 300 mg/kg.

Executive summary:

The objective of this study was to determine the potential toxicity of SILVIAL, when given dermally for 28 days to Wistar rats. In addition, a No Observed Adverse Effect Level (NOAEL) was evaluated.

The study design was as follows (dose level): Group 1 (5 males 5 females ) : 0 mg/kg/day; Group 2 (5 males 5 females ) : 100 mg/kg/day; Group 3 (5 males 5 females ) : 300 mg/kg/day; Group 4 (5 males 5 females ) : 1000 mg/kg/day.

Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity.

The following parameters and end points were evaluated in this study: clinical signs, body weights, food consumption, clinical pathology parameters (hematology, coagulation, clinical chemistry), gross necropsy findings, organ weights, and histopathologic examinations.

Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

No mortality occurred in this study and no signs of systemic toxicity were noted at dose levels up to 1000 mg/kg. The treated skin showed slight erythema and/or scales during mainly the second half of the treatment period in males and females predominantly at 1000 mg/kg.

Slightly lower bodyweight gain was noted for males at 1000 mg/kg, without correlating changes in food consumption. Relative food consumption was slightly higher for females at 1000 mg/kg. In the absence of correlating changes in body weight or other parameters, this slight change was considered not toxicologically relevant.

At 1000 mg/kg, the livers of all females showed pale discoloration. This was accompanied by higher liver weights, microscopically correlating with microvesicular vacuolation and hepatocellular hypertrophy. The increased incidence and severity of diffuse hepatocellular hypertrophy and microvesicular vacuolation was also observed in the livers of females at 300 mg/kg. Additional findings in livers of females at 1000 mg/kg included minimal single cell necrosis and centrilobular pigmented cells (up to slight degree). Based on the presence of single cell necrosis, which is an indication for cell damage, the liver findings at 1000 mg/kg/ are considered to be adverse.

Clinical pathology investigations revealed changes predominantly at 1000 mg/kg. Lower platelet counts were noted in females at 300 and 1000 mg/kg accompanied by a longer protrombine time (PT) and Activated Partial Thromboplastin Time (APTT) in females at 1000 mg/kg. Based on the magnitude of the affected coagulation parameters the findings at 1000 mg/kg are considered to be adverse.

Other findings included lower total protein (in females accompanied by decreased albumin levels), cholesterol and calcium levels in both sexes at 1000 mg/kg. In addition, higher total bilirubin and urea levels were noted in females at 1000 mg/kg. These changes were only slight and therefore considered to be not adverse.

In conclusion, daily dermal SILVIAL exposure was well tolerated in rats at levels up to 300 mg/kg. Adverse test item-related effects were observed at dose levels of 1000 mg/kg and consisted of liver findings and changes in coagulation parameters. Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 300 mg/kg.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

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

Study period:

10 Aug 2017 - 10 Nov 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The dermal route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.

Qualifier:

according to guideline

Guideline:

EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name (as stated in the report): Silvial
Batch No.: SC00020196
Expiration date: 09 November 2017
Chemical name (IUPAC): 2-METHYL-3-(4-(2-METHYLPROPYL)PHENYL)PROPANAL

Species:

rat

Strain:

other: Wistar Han rat

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Environmental Acclimation
- Test Facility toxicology accommodation for 15 days before the commencement of dosing
- Animals trained for the application routeduring acclimation period according to standard procedures

Animal Identification
- each animal was identified using tattoo

Environmental Conditions:
- Actual daily mean temperature: 21 to 22°C
- Actual daily mean relative humidity of: 50 to 73%
- Light cycle: 12-hour light/12-hour dark
- Air : Ten or greater air changes per hour with 100% fresh air (no air recirculation)

Housing:
- Animals were single housed (randomly) in polycarbonate cages containing appropriate bedding equipped with water bottles.

Food/Water:
- Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) ad libitum
- Municipal tap water freely available (via water bottles)

Veterinary Care:
- no examinations or treatments were required

Type of coverage:

semiocclusive

Vehicle:

corn oil

Remarks:

Stability for at least 24 hours at room temperature, 8 days in the refrigerator (2-8°C) 3 weeks in a freezer (≤-15°C) is confirmed over the concentration range 1.08 mg/g (low) to 212 mg/g (high).

Details on exposure:

At least 24 hours before first treatment, an area of approximately 6x10 cm on the back of the animals was clipped. Whenever necessary (during the course of the study) the skin-area was re-clipped at least 3 hours before a next application. Care was taken to avoid abrading of the skin.
The test item and vehicle were administered to the appropriate animals dermally once daily (for 6 hours ± 30 minutes) for 7 days a week for a minimum of 28 days. Application was performed approximately the same time each day. Animals were treated up to the day prior to necropsy. The dose volume for each animal was based on the most recent body weight measurement. The test substance formulation was applied onto a surgical gauze patch (Surgy 1D; less than or equal to 8 ply), with an area of approximately 10% of the total body surface (i.e. approx. 40 cm² for males and approx. 30 cm² for females). The gauze patch was mounted on Medical tape or Microfoam and held in contact with the skin with Tegaderm flexible bandage. To protect the application, Lomir jackets were used (Manufacturers: Laboratoires Stella s.a., Liege, Belgium (surgical gauze) and 3M, St. Paul, Minnesota, U.S.A. (Medical Tape and Microfoam) and Lomir Biomedical, Hull, UK (Jackets).
During the exposure period the dressing was checked regularly, at least every 2 hours (i.e. after application, and approximately 1, 3 and 5 hours after application). In case the application was found dislodged or improperly secured on the animal before or on the 3-hour bandage check time point, a new dressing with formulation was applied. The maximum time during which the animal was subsequently not exposed was estimated retrospectively.
The first day of dosing was designated as Day 1. The dosing formulations were stirred continuously during dose administration. A dose control system was used as additional check to verify the dosing procedure according to Standard Operating Procedures.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis:
- Interval : Week 1
- Concentration: All groups
- Homogeneity: Groups 2 and 4
- Stability: Performed previously in conjunction with the method development and validation study (Test Facility Study No. 513140) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in this study.
- Analytical method: Analyses were performed by using a validated analytical procedure (Test Facility Study No. 513140).
Instrument: Acquity UPLC system (Waters, Milford, MA, USA)
Detector: Acquity UPLC TUV detector (Waters)
Column: Acquity UPLC BEH C18, 100 mm x 2.1 mm i.d., dp =1.7 μm (Waters)
Column temperature: 40°C +/- 1°C
Injection volume: 10 μL
Mobile phase: 70/30 (v/v) acetonitrile/water
Flow: 0.5 mL/min
UV detection: 210 nm

Concentration and Homogeneity Analysis:
- Duplicate middle samples for Groups 1 and 3 (concentration analysis only) and duplicate top, middle, and bottom samples for Groups 2 and 4 (concentration and homogeneity analysis) (approximately 500 mg) for each sampling time point were sent to the analytical laboratory.
- Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10%. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10%.

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals dermally for a minimum of 28 days.

Frequency of treatment:

daily (for 6 hours ± 30 minutes) for 7 days a week

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 > Corn oil only (control group)

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 2 > Dose concentration = 20 mg/mL

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Group 3 > Dose concentration = 60 mg/mL

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 4 > Dose concentration = 200 mg/mL

No. of animals per sex per dose:

5 males and 5 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

see "details on exposure" field above

Positive control:

Not applicable

Observations and examinations performed and frequency:

The following parameters and end points were evaluated in this study: clinical signs, body weights, food consumption, clinical pathology parameters (hematology, coagulation, clinical chemistry), gross necropsy findings, organ weights, and histopathologic examinations.

Mortality/Moribundity Checks:
- Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.

Clinical Observations:
- Performed twice daily (prior to dermal exposure and after removal of the application), beginning during the first day of exposure and lasting throughout the dosing period. During the dosing period, these observations were performed directly after removal of the application.

Arena Observations:
- Animals were removed from the cage and placed in a standard arena, and a detailed clinical observation was performed weekly, beginning before the first administration of the test item. These observations were conducted after dosing.

Body Weights:
- Animals were weighed individually weekly, starting on Day 1. A fasted weight was recorded on the day of necropsy.

Food Consumption:
- Quantitatively measured weekly starting on Day 1 and continuing weekly throughout the dosing period.

Water Consumption:
- Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

Clinical Pathology (Hematology,Coagulation, Clinical Chemistry ) :
- Blood was collected between 7.00 and 10.30 a.m. from the retro-orbital sinus under anesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) of fasted animals.Animals were fasted overnight (with a maximum of 24 hours) before their scheduled necropsy.

Necropsy :
- All animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their
associated organs and tissues.

Organ Weights:
- Gland adrenal, kidney weighed at necropsy for all scheduled euthanasia animals.

Tissue Collection and Preservation:
- Representative samples of the tissues were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands)

Histology:
- The adrenal gland, heart, kidney, liver, skin, spinal cord, testis and gross lesions were embedded in paraffin (Klinipath, Duiven, The Netherlands), sectioned, mounted on glass slides, and stained with hematoxylin and eosin (Klinipath, Duiven, The Netherlands).

Histopathology:
- Histopathological evaluation was performed by a board-certified toxicological pathologist with training and experience in laboratory animal pathology.

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of systemic toxicity were noted during daily detailed clinical observations or during weekly arena observations.

Dermal irritation:

effects observed, non-treatment-related

Description (incidence and severity):

Slight erythema and/or scales were noted on the treated skin in males and females predominantly at 1000 mg/kg during the treatment period.
Other skin effects outside the treated area (including erythema, scabs, scales and/or wounds) were noted at all dose levels, including controls. These were considered to have occurred due to the application method. As these findings were slight, they were considered not to
toxicologically significant. Incidental findings of chromodacryorrhoea during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Slightly lower body weight gain was noted in males at 1000 mg/kg throughout the study period.
Body weights and body weight gain of males at 100 and 300 mg/kg and females up to 1000 mg/kg remained in the same range as controls over the study period.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption after correction for body weight was slightly higher for females at 1000 mg/kg during the study period, reaching statistical significance in Weeks 3 and 4.
Food consumption before or after correction for body weight in females up to 300 mg/kg and males up to 1000 mg/kg was similar to the control level over the study period.

Food efficiency:

not examined

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

not examined

Description (incidence and severity):

Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical pathology investigations revealed changes predominantly at 1000 mg/kg. Lower
platelet counts were noted in females at 300 and 1000 mg/kg accompanied by a longer
protrombine time (PT) and Activated Partial Thromboplastin Time (APTT) in females at
1000 mg/kg. Based on the magnitude of the affected coagulation parameters the findings at
1000 mg/kg are considered to be adverse.
Other findings included lower total protein (in females accompanied by decreased albumin
levels), cholesterol and calcium levels in both sexes at 1000 mg/kg. In addition, higher total
bilirubin and urea levels were noted in females at 1000 mg/kg. These changes were only
slight and therefore considered to be not adverse.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

A test item-related macroscopic finding was present in the liver of all females treated at 1000 mg/kg and consisted of pale discoloration. The microscopic correlate was microvesicular vacuolation and hepatocellular hypertrophy.
In one male treated at 1000 mg/kg, pale discoloration of the liver was noted as well but without a microscopic correlate.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

An increased incidence and severity of diffuse hepatocellular hypertrophy and microvesicular
vacuolation was observed in the livers of females starting at 300 mg/kg. Minimal single cell necrosis and centrilobular pigmented cells (up to slight degree) were observed in livers of female rats treated at 1000 mg/kg.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical biochemistry

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Dose Formulation Analyses:

- The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test item was detected in the Group 1 formulation.

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Conclusions:

In conclusion, daily dermal SILVIAL exposure was well tolerated in rats at levels up to 300 mg/kg. Adverse test item-related effects were observed at dose levels of 1000 mg/kg and consisted of liver findings and changes in coagulation parameters. Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 300 mg/kg.

Executive summary:

The objective of this study was to determine the potential toxicity of SILVIAL, when given dermally for 28 days to Wistar rats. In addition, a No Observed Adverse Effect Level (NOAEL) was evaluated.

The study design was as follows (dose level): Group 1 (5 males 5 females ) : 0 mg/kg/day; Group 2 (5 males 5 females ) : 100 mg/kg/day; Group 3 (5 males 5 females ) : 300 mg/kg/day; Group 4 (5 males 5 females ) : 1000 mg/kg/day.

Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity.

The following parameters and end points were evaluated in this study: clinical signs, body weights, food consumption, clinical pathology parameters (hematology, coagulation, clinical chemistry), gross necropsy findings, organ weights, and histopathologic examinations.

Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

No mortality occurred in this study and no signs of systemic toxicity were noted at dose levels up to 1000 mg/kg. The treated skin showed slight erythema and/or scales during mainly the second half of the treatment period in males and females predominantly at 1000 mg/kg.

Slightly lower bodyweight gain was noted for males at 1000 mg/kg, without correlating changes in food consumption. Relative food consumption was slightly higher for females at 1000 mg/kg. In the absence of correlating changes in body weight or other parameters, this slight change was considered not toxicologically relevant.

At 1000 mg/kg, the livers of all females showed pale discoloration. This was accompanied by higher liver weights, microscopically correlating with microvesicular vacuolation and hepatocellular hypertrophy. The increased incidence and severity of diffuse hepatocellular hypertrophy and microvesicular vacuolation was also observed in the livers of females at 300 mg/kg. Additional findings in livers of females at 1000 mg/kg included minimal single cell necrosis and centrilobular pigmented cells (up to slight degree). Based on the presence of single cell necrosis, which is an indication for cell damage, the liver findings at 1000 mg/kg/ are considered to be adverse.

Clinical pathology investigations revealed changes predominantly at 1000 mg/kg. Lower platelet counts were noted in females at 300 and 1000 mg/kg accompanied by a longer protrombine time (PT) and Activated Partial Thromboplastin Time (APTT) in females at 1000 mg/kg. Based on the magnitude of the affected coagulation parameters the findings at 1000 mg/kg are considered to be adverse.

Other findings included lower total protein (in females accompanied by decreased albumin levels), cholesterol and calcium levels in both sexes at 1000 mg/kg. In addition, higher total bilirubin and urea levels were noted in females at 1000 mg/kg. These changes were only slight and therefore considered to be not adverse.

In conclusion, daily dermal SILVIAL exposure was well tolerated in rats at levels up to 300 mg/kg. Adverse test item-related effects were observed at dose levels of 1000 mg/kg and consisted of liver findings and changes in coagulation parameters. Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 300 mg/kg.

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Does not justify a classification for repeat dose toxicity.