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Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Qualifier:
according to guideline
Guideline:
EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
435-030-7
EC Name:
-
Cas Number:
26452-81-3
Molecular formula:
C5H5N2OCl
IUPAC Name:
435-030-7
Test material form:
other: solid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: at least 6 weeks
- Weight at study initiation: 227 - 257 g (males) and 189 - 213 g (females)
- Fasting period before study: no
- Housing: Individually, in multiple rat racks suitable for animals of this strain and the weight range expected during the course of this study
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): ≥15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Type of coverage:
occlusive
Vehicle:
olive oil
Details on exposure:
TEST SITE
- Area of exposure: dorso-lumbar region
- % coverage: circa 10% or surface area of the rat
- Type of wrap: foil backed gauze patch, held in position by a cohesive bandage and blenderm tape. Six female animals showed signs of discomfort thought to be related to the bandage, therefore an attempt was made to hold the foil backed gauze patch in place by blenderm tape alone. This was found to be impossible to keep the patch in place and so animals 21, 23 - 27, 30 - 32 and 35 - 38 were given H shape bandages from days 14 - 19. This did not prevent animals chewing the bandage and as the overall condition of the animals had improved the original style of bandage was resumed and used until the end of the study.

REMOVAL OF TEST SUBSTANCE
- Washing: cleansed using clean cotton wool swabs soaked in 3% Teepol in warm water followed by warm water alone and then gently dried with clean tissue paper.
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amounts applied: 30, 75, 200 mg/kg/day
- Concentration: Nominal concentrations of 30, 75 and 200 mg/mL
- Constant volume or concentration used: No
- For solids, paste formed: no

USE OF RESTRAINERS FOR PREVENTING INGESTION: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were extracted with methanol, and resulting solutions diluted with methanol, as appropriate, to give sample solution concentrations within the range of the calibration standards used. Samples and standards were analysed by Gas Chromatography (GC).

CHEMICALS AND REAGENTS
Methanol, HPLC grade

CALIBRATION STANDARDS
CMP, with a purity of 99.5% w/w was used. Nominally 100 mg of CMP was accurately weighed into a 100 mL volumetric flask and diluted to volume with methanol (nominally 1.0 mg/mL). Further appropriate dilutions were made with methanol in volumetric flasks to give a range of solutions, nominally within 10 µg/mL to 100 g/mL. The purity of the test substance was not taken into account in the preparation of standard solutions.

PROCEDURE
Sample preparation: Appropriate portions of samples, 1 ± 0.5 g, were transferred to volumetric flasks, diluted to volume with methanol and shaken thoroughly for 5 minutes. The samples were further diluted, as required, with methanol to a known nominal concentration within the range of calibration standards and then filtered through 0.45 µm (PTFE type) syringe filters prior to analysis.

Gas Chromatography Conditions: Gas chromatograph - HP6890 (Agilent Technologies); Autosampler - HP7683 (Agilent Technologies); Column - 30 m x 320 µm DB624, 1.8 µm film (J&W); Oven temperature - 150 °C; Inlet type - Split; Liner - Straight glass with silanised glass wool; Split Ratio - 50:1 or 100:1; Injection temperature - 250 °C; Injection volume - 1 µL; Gas Saver - On after 4 minutes; Gas Saver Flow - 20 mL/min; Detector Nitrogen - Phosphorus detector (NPD); Detector temperature - 260 °C; Carrier gas - Helium, 4 mL/min; Auxiliary gas - Helium, 4 mL/min; Detector gasses - Air, 60 mL/min; Hydrogen 3 mL/min (all flow rates are nominal); Data Handling - Millennium 32 (Waters)

CALIBRATION
The analysis system was calibrated using a range of standards to determine the linearity of response. An appropriate standard of known concentration was interspersed at intervals throughout the analysis.

CALCULATION OF RESULTS
The analysed concentration was calculated using the equation below, after appropriate calibration and reprocessing.
Analysed Concentration (mg/mL) = (C x Df x D)/(W x 1000), Where
C = Calculated sample concentration from data system (µg/mL)
Df = Dilution factor
D = Density of dose preparation (g/mL) *
W = Weight of dose preparation taken (g)
* The density of the preparation was calculated from the known densities of the test substance and vehicle in the Central Dispensary computer system (ARTEMIS-CDY).

LIMIT OF DETECTION
The limit of detection was calculated to be approximately 2 µg/mL test substance in the analysed solution, corresponding to a formulation concentration of 1 mg/mL
Duration of treatment / exposure:
20 applications of 6 hours over a 28 day period on days 1 - 3, 6 - 10, 13 - 17, 20 - 24, 27 and 28 (replicates 1 - 5) and days 1, 2 ,5 - 9, 12 - 16, 19 - 23 and 26 - 28 (replicates 6 - 10)
Frequency of treatment:
Once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
30, 75, 200 mg/kg/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels selected were based on the results of a preliminary dermal toxicity study in rats
- Rationale for animal assignment: The animals were distributed amongst experimental groups by a method designed to ensure that any unhealthy rats or rats at the extremes of the weight range were excluded. The weights were recorded and sorted using a computer sort feature for each sex separately. All the stock animals were weighed and the data recorded directly electronically. The weights were then sorted electronically into order with the highest weight listed first, followed by the next highest etc. Animals at extremes of the weight range and any unhealthy animals were discarded.
The sexes were randomised separately. A Latin Square was generated and was used for the allocation of animals to the experimental groups. The heaviest animal was allocated the lowest number in the group of replicate 1 denoted by the number on the Latin Square and ear­ punched with the lowest available experimental number for that cage shown on the rack plan. This procedure was carried out until all cages in each replicate contained one rat.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily, prior to dose application and after decontamination, or at a similar time of day on the days when the animals were not dosed.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily, prior to dose application and after decontamination, or at a similar time of day on the days when the animals were not dosed.

DERMAL IRRITATION: Yes
- Time schedule: daily, prior to dose application and after decontamination, or at a similar time of day on the days when the animals were not dosed.

BODY WEIGHT: Yes
- Time schedule for examinations: daily, immediately before dose application, at a similar time on days when the animals were not dosed and prior to termination on day 29.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, food consumption has been measured as g/rat/day

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at scheduled termination, day 29
- Anaesthetic used for blood collection: Yes, halothane Ph. Eur. Vapour
- Animals fasted: No
- How many animals: all
- Parameters examined: red blood cell count, haemoglobin, haematocrit, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, total white blood cell count (WBC), differential WBC, platelet count, prothrombin time and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at scheduled termination, day 29
- Animals fasted: No
- How many animals: all
- Parameters examined: urea, creatinine, glucose, albumin, total protein, albumin/globulin ratio, cholesterol, triglycerides, sodium, potassium, chloride, calcium, total bilirubin, phosphorus (as phosphate), gamma-glutamyl transferase activity, creatine kinase activity, alkaline phosphatase activity, aspartate aminotransferase activity, alanine aminotransferase activity

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes, all tissues processed from the control and top dose (200 mg/kg/day) were examined by light microscopy: abnormal tissue, adrenal gland, kidney, liver, skin (treated), skin (untreated), testis, epididymis, spleen, heart, ovary
Other examinations:
organ weights: adrenal glands, kidneys, liver, testes/ovaries
Statistics:
- Body weights were considered by analysis of covariance on initial (day 1) body weight, separately for males and females.
- Weekly food consumption was considered by analysis of variance, separately for males and females.
- Haematology and blood clinical chemistry were considered by analysis of variance. Male and female data were analysed together and the results examined to determine whether any differences between the control and the treated group were consistent between the sexes.
- Organ weights were considered by analysis of variance and analysis of covariance on final body weight, separately for males and females. Summary data are presented for organ to body weight ratios but these were not analysed statistically as the analysis of covariance provides a better method of allowing for differences in terminal body weights.
- With the exception of body weights, analysis of variance and covariance allowed for the replicate structure of the study design and were carried out using the MIXED procedure in SAS (1996). Least-squares means for each group were calculated using the LSMEAN option in SAS PROC MIXED. Unbiased estimates of differences from control were provided by the difference between each treatment group least-squares mean and the control group least­ squares mean. Differences from control were tested statistically by comparing each treatment group least-squares mean with the control group least-squares mean using a two-sided Student's t-test, based on the error mean square in the analysis.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no mortalities. Treatment related clinical changes e.g. lachrymation, signs of salivation and salivation were seen in 1 or more top dose animals on day one of dosing. These changes did not persist and are considered to be of no toxicological significance. A number of clinical observations e.g. thickening, desquamation, oedema, small scattered scabs and scabs at the edge of the application area, consistent with those commonly seen in dermal toxicity studies as a consequence of bandaging, were seen in control animals at a similar incidence and/or showed no evidence of a dose-response and are considered to be unrelated to administration of test substance. There was a slight increase in incidence and duration of erythema in all treated groups although there was not a clear dose-response relationship.

BODY WEIGHT AND WEIGHT GAIN
Group mean body weight in males and females given 200 or 75 mg/kg/day was slightly lower than concurrent controls during the first few days of the study with the difference being more persistent in males than females. Body weights in males given 200 mg/kg/day remained lower than controls throughout the study (maximum differences 6 - 7%) although the difference was not statistically significant after day 11. Males given 75 and females given 200 or 75 mg/kg/day were similar to or above control values for the remainder of the study.

FOOD CONSUMPTION
Food consumption in males and females given 200 mg/kg/day was slightly lower than control values during the first week of the study but returned to control values thereafter.

HAEMATOLOGY
At a dose level of 200 mg/kg/day in males, lymphocyte count was slightly lower and large unstained cell count was slightly higher than controls but, in the absence of any effects in total white blood cell count or any of the other differential counts, these effects are considered to be of no toxicological significance. At a dose level of 200 or 75 mg/kg/day in females, mean cell volume and mean cell haemoglobin were slightly lower than control values. Mean cell volume and mean cell haemoglobin are derived from measured parameters (red blood cell count, haematocrit and haemoglobin concentration) which themselves were similar to control values, therefore these changes are considered to be of no toxicological significance. There was an apparent statistically significant reduction in activated partial thromboplastin times in males given 200 or 30 mg/kg/day when compared to control values. The observed changes were small, attributable to a very low value for control male 2, showed no dose-response relationship and are considered to be unrelated to administration of test substance.

CLINICAL CHEMISTRY
Plasma cholesterol and plasma potassium levels showed differences from control values. However these differences were relatively minor, only seen in males and, in the absence of any changes in liver or kidney weights or in microscopic finding, these changes are considered to be of no toxicological significance. Plasma aspartate aminotransferase activity in females given 200 or 75 mg/kg/day was lower than controls. This was attributed to a very high activity for control female 21 and is considered to be unrelated to administration of test substance. There were a number of other statistically significant differences from control values but these were confined to intermediate dose groups and a single sex only and were considered to be unrelated to administration of test substance.

ORGAN WEIGHTS
Organ weights in all groups were similar to controls.

GROSS PATHOLOGY
There were no compound-related effects.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no compound-related effects. A number of changes were observed in skin in control and treated rats. It is considered that these changes were related to the application of the dressing rather than to application of the compound .

Effect levels

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicologically significant effects were observed at the highest dose tested.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of the test, the major adverse effect observed was a reduction in body weight (maximum difference was 6 - 7% and the difference was not statistically significant after day 11) in the highest dose tested (200 mg/kg bw).
Executive summary:

In a GLP compliant repeated dose 28-day dermal toxicity study, performed according to OECD guideline 410, groups of five male and five female Alpk:APfSD (Wistar-derived) rats were administered 30, 75 or 200 mg test substance/kg/day (in olive oil) by dermal application over a 28 day period (20 applications). A concurrent control group was similarly treated, but olive oil only was applied. Clinical observations, body weights and food consumption were recorded throughout the study and, at the end of the scheduled period, the animals were killed and subjected to an examination post mortem. Cardiac blood samples were taken for clinical pathology, selected organs were weighed and specified tissues were taken for subsequent histopathology examination. The achieved concentration and stability of the dosing solutions was satisfactory throughout the study. There were no mortalities. At a dose level of 200 mg/kg/day, male body weights were reduced and remained lower than controls throughout the study. However, the maximum difference was 6 - 7% and the difference was not statistically significant after day 11. Food consumption in both sexes and female body weights were slightly lower than controls during the first week of the study. There were no compound-related changes in organ weights, macro- or micro-pathology. At a dose level of 75 mg/kg/day there were transient differences in body weights in both sexes but these did not persist beyond the first week of the study. At a dose level of 30 mg/kg/day there were no compound-related effects.