Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 Nov 2017 to 16 Mar 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to other study
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.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3500 (Preliminary Developmental Toxicity Screen)
GLP compliance:
yes (incl. QA statement)
Justification for study design:
In accordance with the ECHA guidance, the dermal route of exposure was selected because this is the most relevant route of human exposure during manufacture, handling or use of the test item.

Test material

Constituent 1
Chemical structure
Reference substance name:
3-(p-cumenyl)-2-methylpropionaldehyde
EC Number:
229-695-0
EC Name:
3-(p-cumenyl)-2-methylpropionaldehyde
Cas Number:
6658-48-6
Molecular formula:
C14H20O
IUPAC Name:
3-(4-isopropylphenyl)-2-methylpropanal
Specific details on test material used for the study:
Name (as stated in the report): Silvial
Batch No.: SC00021294
Expiration date: 25 March 2018
Chemical name (IUPAC): 2-METHYL-3-(4-(2-METHYLPROPYL)PHENYL)PROPANAL

Test animals

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. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source.
This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Environmental Acclimation:
- Test Facility toxicology accommodation for 7 days prior to start of the pretest period (females) or 8 days before the commencement of dosing (males).
- The females were acclimatized to the jackets 5 days prior to and during pretest and to the Tegaderm patches 3 days prior to start of exposure.
- The males were acclimatized to the jackets and Tegaderm patches according to standard procedures.

Animal Identification:
- Prior to start of the pretest period (females) or treatment period (males), each animal was identified using tattoo.
- Prior to the pretest period, reserve females were numbered R1 through R8 at random by indelible marker.
- A few reserve females replacing allocated females prior to treatment received identification by tattoo.
- Pups were identified on postnatal day (PND) 1. They were randomized per litter and individually identified by means of subcutaneous injection of Indian ink. When general hair growth blurred the identification, the pups were identified by tattoo on the feet.

Selection, Assignment, Replacement, and Disposition of Animals:
- A total of 40 females was selected at randomization before initiation of the pretest phase. Any selected female classified as not having regular estrous cycles during the pretest phase was replaced before initiation of dosing by one of the 8 additional females having regular estrous cycles. A total of 40 females with regular estrous cycles continued in the study. The supernumerary females were removed from the study, and their estrous cycle results were kept in the raw data but not reported.

Housing:
- On arrival and following the pre-test (females only) and pre-mating period, animals were single housed in polycarbonate cages (Macrolon type III, height 18 cm).
- During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm). Except for the exposure period. During the daily 6 hours of exposure, males returned to their home cage (Makrolon type III, height 18 cm).
- During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm). Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
- During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam.
The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles.

Environmental Conditions:
- Actual daily mean temperature: 19 to 21°C
- Actual daily mean relative humidity of: 546 to 69%
- Light cycle: 12-hour light/12-hour dark
- Air : Ten or greater air changes per hour with 100% fresh air (no air recirculation)

Food / water :
- Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study
- Municipal tap water was freely available to each animal via water bottles.

Veterinary Care:
- Veterinary care was available throughout the course of the study, and animals were examined by the veterinary staff as warranted by clinical signs or other changes.
- During the third and fourth week of exposure, Lomir Jackets were not applied for multiple males across dose groups due to wounds in the neck. For male nos. 14 and 18 (Group 2) the wounds were inspected by a veterinarian. At the time of inspection their wounds were slightly festering and inflamed but not bleeding. To prevent further inflammation and on advice of the veterinarian, the fur around the wound area was clipped; no further treatment was required. As the wounds were not located on the treated skin, the exposure of the males was not negatively impacted.

Administration / exposure

Route of administration:
dermal
Vehicle:
corn oil
Remarks:
Stability for at least 24 hours at room temperature, 8 days in a 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), Project 513140
Details on exposure:
At least 24 hours before first treatment, an area of approximately 6x10 cm on the back of the animals were 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 the skin. 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 the skin.

Males were treated for a minimum of 29 days, i.e. 2 weeks prior to mating, during mating and up to and including the day before scheduled necropsy. Females were treated for 45-50 days, i.e. 14 days prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of pregnancy up to Day 19 post-coitum and starting on Day 4 of lactation (according to the OECD 422 guideline), up to and including the day before scheduled necropsy.
The omission of exposure on Day 20 post-coitum up to PND 3 over a period of several weeks was not considered to affect the toxicological evaluation. Females which failed to deliver were treated for 35-38 days.

The test item and vehicle were administered to the appropriate animals dermally once daily (for at least 6 hours, with a maximum of 7 hours) for 7 days a week. Application was performed approximately the same time each day. 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, which were secured with Velcro tape when necessary.

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.


For most animals the total time of removal of the patch was less than 5% of the total exposure time period. Exceptions were male nos. 12, 14 (Group 2); 26 (Group 3); 31 and 39 (Group 4) and female nos. 43 (Control); 53 (Group 2); 65, 69 (Group 3) and 78 (Group 4) for which the total time of removal was 5-15%; for female nos. 71 and 76 (Group 4) the time of removal was 20% and 22%, respectively. In addition, Control female no. 44 had a removal time of 24% time of removal. As this concerned a control female, this could not be related to the test item and was not considered to affect the outcome of the study. Unscheduled removal of (part of) the patch from the treated skin area by the animals may relate to both the study procedure and a response of the animals to irritating substances in these types of studies. The incidence of bandage removal in this study was not related to the administered dose. Therefore, this occurrence was considered to be related to the study procedures. As the total time of removal of bandage per group was generally less than 5% of the total exposure time period, it was considered that the incidences of bandage removal did not adversely affect the outcome or the integrity of the study.

The first day of exposure was designated as Day 1. The dosing formulations were stirred continuously during dose administration. A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures.

Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.
Details on mating procedure:
After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. During the daily 6 hours of exposure, males returned to their home cage.
Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 postcoitum. Once mating had occurred, the males and females were separated.
From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration Analysis :
- Duplicate sets of samples (approximately 500 mg, accurately weighed) were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.

Homogeneity Analysis:
- Duplicate sets of samples (approximately 500 mg, accurately weighed) were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was  10%.

Stability Analysis:
- Stability analyses 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 the present 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
Duration of treatment / exposure:
Males were treated for a minimum of 29 days (2 weeks prior to mating, during mating and up to and including the day before scheduled necropsy).
Females were treated for 45-50 days (14 days prior to mating with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of pregnancy up to Day 19 post-coitum and starting on Day 4 of lactation (according to the OECD 422 guideline), up to and including the day before scheduled necropsy.
Frequency of treatment:
Daily (for at least 6 hours, with a maximum of 7 hours) for 7 days a week.
Details on study schedule:
see "details on exposure" field above
Doses / concentrationsopen allclose all
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:
10 males and 10 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
see "details on exposure" field above
Positive control:
Not applicable

Examinations

Parental animals: Observations and examinations:
The following parameters were evaluated in this study: mortality/moribundity, clinical signs, body weight (gain), food consumption, estrous cycle length and regularity, sperm parameters (count, motility, and morphology), serum level of thyroid hormones T4 and TSH (F0-males and females), gross necropsy findings, organ weights (thyroid and male reproductive organs) and histopathologic examination (skin, thyroid and reproductive organs).

In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, serum levels of thyroid hormones (T4 in PND 4 and PND 14-16 pups, TSH in PND 14-16 pups) and macroscopy).

Mortality/Moribundity Checks – F0-Generation:
- Observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day

Clinical Observations – F0-Generation:
- Performed twice daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy. During the dosing period, these observations were performed prior to exposure and after removal of the dressing (6 hours (± 30 minutes)). Nominal times were used for computer registration only (i.e. 06:00 and 12:00). During the treatment-free period between Day 20 post coitum and PND 4, females were observed once daily using the nominal time 06:00 for computer registration.

Body Weights – F0-Generation:
- weighed individually on the first day of treatment (prior to first dermal application), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. Terminal body weights were recorded on the day of necropsy (fasted for males, non-fasted for females).

Food Consumption – F0-Generation:
- measured weekly, except for males and females which were housed together for mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

Water Consumption – F0-Generation:
- Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was expected or noted at visual inspection of the water bottles.

Cohabitation/Mating Procedure – F0-Generation:
- After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. During the daily 6 hours of exposure, males returned to their home cage. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 postcoitum. Once mating had occurred, the males and females were separated.

General Reproduction Data – F0-Generation:
- From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day.

Clinical Pathology (Hematology,Coagulation, Clinical Chemistry ) :
- Blood of F0-animals (except for females with total litter loss) was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retroorbital sinus under anesthesia using isoflurane in the animal facility. F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
Thyroid hormone:
- Blood samples were processed for serum, and serum was analyzed for Thyroxine (total T4) and Thyroid Stimulating Hormone (TSH)





























Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pre-test period), the first 14 days of treatment and during mating until evidence of copulation was observed. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus. This was done for all females.
Sperm parameters (parental animals):
Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility was assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal was recorded. Evaluation was performed for all samples. One epididymis (left) was removed, placed in labeled bags, and kept in the freezer at ≤-15°C. After thawing the left epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples.
Litter observations:
Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed.
Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or eficiencies in maternal care.
Postmortem examinations (parental animals):
Necropsy – F0-Generation:
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs and the (treated) skin.

Organ Weights – F0-Generation:
- Epididymis ; Gland, coagulation ; Gland, parathyroid ; Gland, prostate ; Gland, seminal vesicle ; Gland, thyroid ; Testes

Sperm Analysis – F0-Generation:
- See "Sperm parameters (parental animals" field above

Tissue Collection and Preservation – F0-Generation:
- Representative samples of the tissues ( Cervix; Right Epididymis; Gland, coagulation; Gland, mammary; Gland, parathyroid; Gland, pituitary; Gland, prostate; Gland, seminal vesicle; Gland, thyroid; Gross lesions/masses; Ovaries; Skin (treated); Testes; Uterus; Vagina) were collected from all animals and preserved in 10% neutral buffered formalin

Histology – F0-Generation:
- The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin:
All animals > Epididymis, skin (treated), thyroid gland, gross lesions/masses, ovaries and testes.
Males that failed to sire (except for males which were selected), females that failed to deliver pups and females with total litter loss > Cervix, coagulation gland, prostate gland, seminal vesicles, uterus and vagina
Female with total litter loss > Mammary gland

Histopathology – F0-Generation:
- All tissues as defined under Histology were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. Target tissues identified by the study pathologist during microscopic evaluation were communicated to the Study Director; tissues were evaluated and reported. For the testes of all males detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings were noted.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Reproductive indices:
Fertility index, gestation index and duration
Offspring viability indices:
Post-Implantation Survival Index, Live birth index, Viability index, Lactation index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No parental toxicity was observed up to the highest dose level tested (1000 mg/kg).
Dermal irritation (if dermal study):
effects observed, treatment-related
Description (incidence and severity):
Hyperkeratosis of the epidermis of the treated skin occurred at increased incidence and/or severity in 300 mg/kg males (minimal) and in 1000 mg/kg males (up to slight) and females (minimal). This correlated with the scales noted at necropsy and was considered to be test item related. The hyperkeratosis (with associated scale formation) was regarded as nonadverse based on its nature and slight severity. Clinically, scales were noted at all dose levels in a dose-related manner, and focal erythema of treated skin (generally slight) was noted in about half of the 1000 mg/kg animals (both sexes) and several females at 100 and 300 mg/kg.
Mortality:
no mortality observed
Description (incidence):
Survival, general condition, body weight (gain) and food consumption were not affected by treatment.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Survival, general condition, body weight (gain) and food consumption were not affected by treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Survival, general condition, body weight (gain) and food consumption were not affected by treatment.
Food efficiency:
no effects observed
Description (incidence and severity):
Survival, general condition, body weight (gain) and food consumption were not affected by treatment.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was expected or noted at visual inspection of the water bottles.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum levels of total T4 and TSH were measured in parents. However, possible adversity of this effect could not be assessed within this type of screening study and was therefore not taken into account when determining the NOAEL.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Hyperkeratosis of the epidermis of the treated skin was observed at an increased incidence and/or severity in males at 300 mg/kg and in rats of both sexes at 1000 mg/kg. No other significant effects were noted.
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No effects noted
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Epididymal sperm count, sperm motility and sperm morphology were considered not to be affected by treatment.
Reproductive performance:
no effects observed
Description (incidence and severity):
There was 1/10 couple of the control group (male no. 10 and female no. 50) and 1/10 couple treated at 100 mg/kg/day (male no. 19 and female no. 59) that failed to deliver pups.
Furthermore 1/10 females of the 1000 mg/kg/day group (female no. 75) showed total litter loss on post-natal day 2. Histopathology did not reveal any changes in the reproductive
organs that could explain this lack of healthy offspring.

All testes of all males had a normal spermatogenic staging profile.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
dermal irritation

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
1/10 females of the 1000 mg/kg/day group (female no. 75) showed total litter loss on post-natal day 2. Histopathology did not reveal any changes in the reproductive
organs that could explain this lack of healthy offspring. This was considered unrelated to the test item.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
A trend for decrease in body weight of the pups at top dose was observed however all means remained within the historical control data. A comparison of the results within the study
showed significant reduction for female pup body weights on days 1, 7 and 13 however no statistical significance was achieved for the male/combined pup weight on any of the days in all dose
groups.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Serum levels of total T4 and TSH were measured in pups. However, possible adversity of this effect could not be assessed within this type of screening study and was therefore not taken into account when determining the NOAEL.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 300 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Dose Formulation Analyses:

Accuracy: The concentrations analyzed in the formulations of Groups 2, 3 and 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 ( Control group)

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

Applicant's summary and conclusion

Conclusions:
No evidence of reproductive toxicity was shown in the study.
Executive summary:

The objectives of this study were to determine the potential toxic effects of Silvial when given dermally for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development.

In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.

The dose levels in this study were selected to be 0, 100, 300 and 1000 mg/kg, based on the results of a 28-day repeated dose toxicity study with dermal administration of Silvial in rats (Test Facility Study No. 513139).

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

The following parameters were evaluated in this study: mortality/moribundity, clinical signs, body weight (gain), food consumption, estrous cycle length and regularity, sperm parameters (count, motility, and morphology), serum level of thyroid hormones T4 and TSH (F0-males and females), gross necropsy findings, organ weights (thyroid and male reproductive organs) and histopathologic examination (skin, thyroid and reproductive organs).

In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and

duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, serum levels of thyroid hormones (T4 in PND 4 and PND 14-16 pups, TSH in PND 14-16 pups) and macroscopy).

Formulation analysis showed that formulations were prepared accurately and homogeneously.

Parental results

No parental toxicity was observed up to the highest dose level tested (1000 mg/kg).

Hyperkeratosis of the epidermis of the treated skin occurred at increased incidence and/or severity in 300 mg/kg males (minimal) and in 1000 mg/kg males (up to slight) and females (minimal). This correlated with the scales noted at necropsy and was considered to be test item related. The hyperkeratosis (with associated scale formation) was regarded as nonadverse based on its nature and slight severity. Clinically, scales were noted at all dose levels in a dose-related manner, and focal erythema of treated skin (generally slight) was noted in about half of the 1000 mg/kg animals (both sexes) and several females at 100 and 300 mg/kg.

An increased incidence and severity (up to slight degree) of follicular cell hypertrophy was observed in 100, 300 and 1000 mg/kg treated females. Since there was not a clear dose response, and this finding can be observed as a spontaneous finding at similar degrees, the relation with the test item was not established. However, in females treated at 1000 mg/kg, the thyroid gland weight was statistically significant increased (absolute and relative to body weight) compared to the control group. Therefore, a relation with treatment with the test item for 1000 mg/kg treated females is possible. Based on the low severity, the follicular cell hypertrophy itself was considered non-adverse.

Mean serum levels of T4 and TSH values at all dose levels were measured. However, possible adversity of this effect could not be assessed within this type of screening study and was therefore not taken into account when determining the parental NOAEL.

Survival, general condition, body weight (gain) and food consumption were not affected by treatment.

Findings related to the treatment procedure (use of Tegaderm tape and protective jacket) were noted in untreated skin adjacent to treated area (neck) and, less frequently, at the flank(s), back or shoulders. These findings occurred in all groups, the control group included, without a dose-related pattern. Clinical findings included scales (also noted at necropsy), scabs, scars, wounds and focal erythema. Microscopic changes consisted of erosion/ulceration (up to marked), inflammatory cell infiltrate of the dermis (up to moderate), and slight degrees of hyperkeratosis or hyperplasia of the epidermis, pustule(s), hemorrhage, and/or perifollicular inflammatory cell infiltrate.

Reproductive results

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg).

Developmental results:

A decrease in body weight of the pups at top dose was observed.

A trend for decrease in body weight of the pups at top dose was observed however all means remained within the historical control data. A comparison of the results within the study showed significant reduction for female pup body weights on days 1, 7 and 13 however no statistical significance was achieved for the male/combined pup weight on any of the days in all dose groups.

Mean serum levels of T4 and TSH values at all dose levels were measured. However, possible adversity of this effect could not be assessed within this type of screening study and was therefore not taken into account when determining the developmental NOAEL.

In conclusion, based on the results of this reproduction/developmental toxicity screening test, the No Observed Adverse Effect Level (NOAELs) of Silvial was established at 300mg/kg.