Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Remarks:
Dermal application - OECD 422 study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Citrathal concentrate S TW
- Physical state: Yellow to brown yellow liquid
Specific details on test material used for the study:
Identification: Citrathal
Appearance: Clear yellow liquid (determined by Charles River Den Bosch)
Batch: SC00017573
Purity/Composition: UVCB
Test item storage: In refrigerator (2-8°C) protected from light
Stable under storage conditions until: 07 January 2018 (expiry date)

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Species: Rat.
Strain: Crl: WI(Han).
Condition: Outbred, SPF-Quality.
Source: Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L'Arbresle Cedex, France. Details will be documented in raw data and report.
Sex:
male/female
Details on test animals and environmental conditions:
Number of Males 50.
Number of Females 58. (nulliparous and non-pregnant)
Number of Pups Expected: Approximately 480 pups (40 litters x 12 pups).
Target Age at the Initiation of the Pretest Period: Females: approximately 12-14 weeks.
Target Age at the Initiation of Dosing: Males: approximately 10-12 weeks. Females: approximately 14-16 weeks.
Target Weight at the Initiation of Dosing: Males: 250 to 350 g. Females: 200 to 250 g.

The animals will be allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days prior to start of the pretest period (females) or at least 5 days before the commencement of dosing (males). The females will be acclimatized to the jackets at least 5 days prior to and during pretest and to the Tegaderm patches at least 3 days prior to start of exposure. The males will be acclimatized to the jackets and Tegaderm patches according to standard procedures.

The target conditions for animal room environment will be as follows:
Temperature: 18 to 24°C.
Humidity: 40 to 70%.
Light Cycle: 12-hours light and 12-hours dark (may be interrupted for designated procedures).
Ventilation: At least 10 air changes per hour.

Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) will be provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals will not have access to food for a maximum of 2 hours.

Municipal tap water will be freely available to each animal via water bottles. During motor activity measurements, animals will not have access to water for a maximum of 2 hours.

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

The dermal route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
The dose levels will be selected based on the results of a 14-day dose range finder with dermal administration of Citrathal in rats (Test Facility Study No. 516568, see section 8), and in an attempt to produce graded responses to the test item.
The high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

VEHICLE
Identification: Corn oil
Supplier: Fagron, Capelle aan de IJssel, The Netherlands.
Batch: Documented in raw data and included in the Final Report
Specific gravity 0.92
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 516573) 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. Stability data have been retained in the study records of Test Facility Study No. 516573.

Samples for 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).
Sample Volume : Approximately 500 mg accurately weighed.
Acceptance Criteria: For concentration, the criteria for acceptability will be mean sample concentration results within or equal to ± 10% for solutions. For homogeneity, the criteria for acceptability will be a coefficient of variation (CV) of concentrations of ≤ 10% for each group.
Duration of treatment / exposure:
The test item and vehicle will be administered to the appropriate animals dermally once daily (for 6 hours ± 30 minutes) for 7 days a week. Application will be performed approximately the same time each day. The dose volume for each animal will be based on the most recent body weight measurement.
Frequency of treatment:
Main males and Recovery males will be exposed for at least 28 days, including at least 2 weeks of exposure prior to mating and during the mating period for Main males. For both Main and Recovery males, treatment will end one day before scheduled necropsy of Main males.
Main females will be treated for at least 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, up to and including the day before scheduled necropsy. Recovery females will be treated until at least the first scheduled necropsy of Main females.
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels in this study were selected to be 0, 250, 500, 1000 mg/kg, based on the results of the dose range finder (Test Facility Study No. 516568).
Positive control:
No

Examinations

Observations and examinations performed and frequency:
During treatment, Main and Recovery animals will be observed at least twice daily, up to the day prior to necropsy or start of the recovery period, respectively. These clinical observations will at least be conducted prior to exposure and after removal of the dressing (6 hours (± 30 minutes)).
During the recovery period, Recovery animals will be observed at least once daily, up to the day prior to necropsy.
Animals will be observed for specific clinical signs. The time of onset, grade and duration of any observed signs will be recorded. Signs will be graded for severity and the maximum grade will be predefined at 3 or 4. Grades will be coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) will be scored. In the data tables, the scored grades will be reported, as well as the percentage of animals affected in summary tables.
Other examinations:
Arena Observations – F0-Generation
Frequency: Once before the first administration of the test item and at weekly intervals during the treatment period.
Procedure: Animals will be observed for specific clinical signs outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs will be recorded.
discrete data (scores) in the summary tables. Test statistics will be calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may be rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Statistics:
All statistical tests will be conducted at the 5% significance level. All pairwise comparisons will be conducted using two sided tests and will be reported at the 1% and 5% levels.
Numerical data collected on scheduled occasions will be analyzed according to sex and occasion. Descriptive statistics number, mean and standard deviation will be reported whenever possible. Values may also be expressed as a percentage of predose or control values when deemed appropriate. Inferential statistics will be performed according to the matrix below when possible, but will exclude semi-quantitative data, and any group with less than 2 observations.
Datasets with at least 3 groups (the designated control group and 2 other groups) will be compared using Dunnett-test (many-to-one-t-test).
Datasets with at least 3 groups will be compared using a Steel-test (many-to-one rank test).
The motor activity data set (at least 3 groups) will be compared using an overall Kruskal-Wallis. Whenever, the overall test is significant, the Wilcoxon Rank-Sum test will be applied to compare the treated groups to the control group.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No clinical signs of toxicity were noted in the males during the observation period.
In the females, several clinical signs were observed related to the dermal route of exposure.
In the control group, 2/15 females had brown discolouration/ brown staining on the treated skin. Of the affected females, one female had discolouration or staining on Days 59 to 61 of treatment and the other female had this finding on Days 39 to 50 and the first four days of the recovery period.
At 250 mg/kg, 4/10 females had scales (primarily between Days 6 to 10 of treatment), 3/10 females had general erythema, and 2/10 females had scabs on the treated skin on one or multiple occasions. Additionally, brown discolouration/ brown staining on the treated skin was observed in 3/10 females, primarily between Days 39 to 55 of treatment.
At 500 mg/kg, 2/10 females had scales on the treated skin on Days 9 and 10 of treatment, and one of these females also had general erythema on the treated skin on Days 7 to 9 of
treatment.
At 1000 mg/kg, 9/15 females had scales on the treated skin, primarily between Days 5 and 10 and Days 33 and 50 of treatment. For one of these females, scales were also observed on Day 1 and Days 7 to 10 of the recovery phase. General or maculate erythema was observed in two females on one or more occasions during treatment, one female also presented fissures on one single day. Additionally, brown discolouration/ brown staining on the treated skin was observed in 8/15 females, primarily between Days 39 and 55 of treatment.

Chromodacryorrhoea was noted in 3/10 males and 1/10 females at 500 mg/kg and 3/15 males and 7/15 females at 1000 mg/kg. For most of the affected males and females,
chromodacryorrhoea was observed on one or two occasions during the first two weeks of treatment. Chromodacryorrhoea was also observed two females during the sixth week of
treatment. The incidence of this finding was slightly higher than normally observed in oral gavage or diet studies, however as this finding was also observed on two consecutive days in
one control male it was considered unrelated to treatment. It could be related to the exposure method, but as the chromodacryorrhoea was scored the lowest grade, for most animals on one
occasion only and without other signs of stress (e.g. body weight loss or other clinical signs) there is no indication that the stress of the exposure method would impact the study results.

Additional scabs, wounds, and scales on the skin were observed, likely resulting from the use of tegaderm tape and/or the jacket used for the application, and unrelated to treatment. All
other clinical signs remained within normal limits.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
In the control group, 2/15 females had brown discolouration/ brown staining on the treated skin. Of the affected females, one female had discolouration or staining on Days 59 to 61 of treatment and the other female had this finding on Days 39 to 50 and the first four days of the recovery period.
At 250 mg/kg, 4/10 females had scales (primarily between Days 6 to 10 of treatment), 3/10 females had general erythema, and 2/10 females had scabs on the treated skin on one or multiple occasions. Additionally, brown discolouration/ brown staining on the treated skin was observed in 3/10 females, primarily between Days 39 to 55 of treatment.
At 500 mg/kg, 2/10 females had scales on the treated skin on Days 9 and 10 of treatment, and one of these females also had general erythema on the treated skin on Days 7 to 9 of treatment.
At 1000 mg/kg, 9/15 females had scales on the treated skin, primarily between Days 5 and 10 and Days 33 and 50 of treatment. For one of these females, scales were also observed on Day 1 and Days 7 to 10 of the recovery phase. General or maculate erythema was observed in two females on one or more occasions during treatment, one female also presented fissures on one single day. Additionally, brown discolouration/ brown staining on the treated skin was observed in 8/15 females, primarily between Days 39 and 55 of treatment.
Additional scabs, wounds, and scales on the skin were observed, likely resulting from the use of tegaderm tape and/or the jacket used for the application, and unrelated to treatment.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically relevant changes in body weights and body weight gain in males or females during the treatment or recovery periods. Body weights and body weight
gain of treated animals remained in the same range as controls over the treatment period.
During the recovery period of the males, males at 1000 mg/kg had a slightly higher body weight and body weight gain at each weighing interval compared to the control males. The
lower terminal body weight of the main males at 1000 mg/kg compared with the higher terminal body weights of the recovery males at 1000 mg/kg were considered incidental and
unrelated to Citrathal.
At the end of the post coitum period, body weight and body weight gain of treated females were slightly higher at 250, 500, and 1000 mg/kg when compared to controls, but the
differences were not increased in a dose dependent manner. The increase in body weight and body weight gain of treated females at 250, 500, and 1000 mg/kg was therefore considered to
be unrelated to Citrathal.
From Day 15 of treatment onwards, the body weight of non-mated recovery females at 1000 mg/kg was slightly lower and the corresponding body weight gain in these females was
statistically significant lower on Days 15 and 29 when compared to concurrent controls. As the decrease was minimal it was considered non adverse.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before correction for body weight was similar between treated and control males.
A slight, statistically significant, increase (about 10%) was noted in the relative food consumption of recovery females treated at 1000 mg/kg during the last two weeks of
treatment and the first week of the recovery period. This increase was caused by the observed decrease in body weight, without an effect on the absolute food consumption during the same
time period. In addition, given the direction and magnitude of the change, the observed increase was not considered adverse.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Chromodacryorrhoea was noted in 3/10 males and 1/10 females at 500 mg/kg and 3/15 males and 7/15 females at 1000 mg/kg. For most of the affected males and females,
chromodacryorrhoea was observed on one or two occasions during the first two weeks of treatment. Chromodacryorrhoea was also observed two females during the sixth week of
treatment. The incidence of this finding was slightly higher than normally observed in oral gavage or diet studies, however as this finding was also observed on two consecutive days in
one control male it was considered unrelated to treatment. It could be related to the exposure method, but as the chromodacryorrhoea was scored the lowest grade, for most animals on one
occasion only and without other signs of stress (e.g. body weight loss or other clinical signs) there is no indication that the stress of the exposure method would impact the study results.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no toxicologically relevant changes noted in haematological parameters in males or females at the end of the treatment period.
At the end of recovery period, there was a minimal, but statistically significant decrease in red blood cell distribution width (RDW) in males at 1000 mg/kg (7% lower than controls). This
difference was not considered related to Citrathal because of the minimal magnitude of the change and it occurred during the post treatment period.
In recovery females, there was a minimal, but statistically significant decrease in haemoglobin levels at 1000 mg/kg at end of the treatment period. This difference in
haemoglobin levels occurred in the absence of changes in red blood cells and haematocrit. At the end of the recovery period, there was a minimal, non-statistically significant decrease in
red blood cells, haemoglobin, and haematocrit (9%, 9% and 10% lower compared to controls, respectively) and a minimal increase in platelets (6% higher compared to controls) in females
in the 1000 mg/kg dose group. These differences in haematological parameters were not considered related to Citrathal because of the minimal magnitude of the change.
The statistically significant increase in red blood cell distribution width (RDW) in main females at 250 mg/kg noted at the end of the treatment period was considered unrelated to
Citrathal because it was not dose dependent.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant changes noted in clinical biochemistry parameters in males or females at the end of the treatment or recovery periods.
A statistically significant increase was noted in sodium levels at the end of treatment in males at 500 and 1000 mg/kg. As the difference between treated males and control males was
minimal (about 1%) and in the absence of a dose response, the increase was considered unrelated to treatment.
Other statistically significant alterations in clinical biochemistry parameters were considered unrelated to administration of the test item due to the minimal magnitude of the change,
absence of a dose response, and/or absence of biological relevance.

Thyroid hormone analyses:
There were no toxicologically relevant changes in serum levels of T4 in F0 males.
The statistically significant changes in serum levels of T4 levels in in F0 males at 250 mg/kg was considered unrelated to administration of the test item due to the absence of a dose response.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
At the end of the treatment period, grip strength in the hind limbs was 21% lower in 1000 mg/kg treated males compared to the concurrent controls. This difference was not
statistically significant when compared to the concurrent controls. The average value (373 gram) was below the historical control range of the Testing Facility1. At the end of the
recovery phase, no differences in grip strength of the hind limbs were observed between control and males at 1000 mg/kg.
During motor activity testing, the mean number of total movements and mean number of ambulations was lower in males at 1000 mg/kg at the end of the treatment period (12% and
11% of controls, respectively). The average values for total movements (5764) and ambulations (1055) did not reach statistical significance when compared to the concurrent
controls. All groups demonstrated a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
In females, motor activity (total number of movements and ambulations) was similar across the groups, with all groups demonstrating a similar motor activity habituation profile with a
decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically relevant alterations in organ weights.
At end of treatment a decrease of spleen weight (absolute and relative) was noted in males treated at 1000 mg/kg. Spleen weight was statistically significant different for absolute
weight only; which was 20% decreased when compared to control. No difference in spleen weight was noted at end of recovery and the mean absolute and relative weight remained
within historical control data2, therefore the decrease was considered not toxicologically relevant.
A slight increase of testes and seminal vesicle weight (absolute and relative) was noted at the end of treatment in males dosed at 1000 mg/kg (8% and 12%, respectively in relative weights
compared to control). A similar increase was noted in males treated at 500 mg/kg (not statistically significant, 9% increase compared to control). No differences were noted in
testes or seminal vesicles weight at end of recovery. Furthermore, the mean weight of both organs remained well within historical control data3 and the increases were therefore
considered not toxicologically relevant.
The observed decrease in absolute and relative weights of the ovaries at the end of treatment were considered to have arisen from the relatively high control mean weight. In combination
with the lack of a dose-response, the decreases were found unrelated to treatment.
Slight (statistically significant) changes at the end of recovery were considered unrelated to Citrathal because it occurred during the post treatment period.
Other organ weights and organ to body weight ratios among the dose groups were similar to control levels or were considered unrelated to treatment, based on the absence of a dose
relationship.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no Citrathal-related microscopic observations.
There were some microscopic findings in the treated skin consisting of orthokeratotic hyperkeratosis of the epidermis, the presence of pustule(s) and/or (peri)follicular infiltrate.
These findings were noted at minimal degree in all dose groups including controls and lacked a dose response. Therefore they were considered as unrelated to the treatment with the test
item. Furthermore, there were some microscopic findings in the skin adjacent to the treated area (back of the neck, shoulder and thoraco-dorsal region) consisting of erosion/ulceration (up to
marked), orthokeratotic hyperkeratosis (slight) and/or hyperplasia of the epidermis (minimal) and the presence of pustule(s) (up to slight). In absence of a clear dose response, these
lesions which were sometimes present in controls as well, were considered to be procedure related (trauma to the wearing of the protective jacket).
The recovery was complete for all skin lesions.
Lymphoid atrophy of the thymus was recorded in 1/5 females of the 250 mg/kg group (slight), in 1/5 females of the 500 mg/kg group (minimal) and in 3/5 females of the 1000 mg/kg group (minimal). Since there was no clear dose response, no statistically significant lower thymus weights and since mild lymphoid atrophy sometimes can be observed in control females of this study type (Ref. 1), this finding was considered as unrelated to treatment with the test item.
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.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
dermal irritation

Target system / organ toxicity

Key result
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
other: Whole animal

Applicant's summary and conclusion

Conclusions:
Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the parental, reproduction, and developmental no-observed-adverse-effect levels (NOAEL) of Citrathal of at least 1000 mg/kg were established.
Executive summary:

(NOAEL) of Citrathal of at least 1000 mg/kg were established.