Registration Dossier

Diss Factsheets

Administrative data

Description of key information

The repeated dose toxicity via dermal exposure of Aradur 1019 in the rabbit was tested.

Results show No Observed Effect Level of 20 mg/kg bw/day. Effects were seen on dermal irritation and corrosion at 100 mg/kg bw/day leading to a classification of STOT RE Cat. 2

Key value for chemical safety assessment

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 1980 to Jun 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
GLP compliance:
no
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animals KA 46 rabbits, New Zealand White strain equal numbers of males and females bred and raised at Tierfarm Sisseln
Initial body weight 2.3 to 3.0kg
Initial age 2 to 16 weeks
No. of animals per group 10 males, 10 females (5 males, 5 females (5 males, 5 females for a 14 day recovery period)
Individual identification By numbered ear tags and under cage number
Method of randomisation Animals were assigned to the different groups by means of random numbers generated by the IBM computer of CIBA-GEIGY Ltd., Basle, Switzerland
Husbandry T he experiment was carried out under Standard laboratory conditions.
"Temperature: 22 ± 2°C
Relative humidity: 55 ± 10%
15 - 17 air changes/hr."

Housing individually in stainless steel cages
"Neither insecticides nor chemicals were applied in the animal room, with the exception of a desinfectant:
Fungitext SB (Prod. No. 30071, CIBA-GEIGY AG; Switzerland)"
Food Pelleted standard diet, NAFAG No. 814/85 (NAFAG SA, Gossau/Switzerland) ad libitum
Food from the follwing batches was fed throughout the test:

February 25 to March 31, 1980: Tox. 52
April 1 to April 21, 1980: Tox. 57
April 22 to May 19, 1980: Tox. 12/80
May 19 to June 2, 1980: Tox. 21/80
June 3 to June 16, 1980: Tox. 28/80

Water Tap water from automatic drinking valves ad libitum (drinking water quality according to the specifications of the "Schweizerishes lebensmittelbuch"
Route and method
of application Dermal on the shaved skin of the back. Gauze patch 8.5 x 6.5 cm, covered with aluminium foil (10 x 8 cm). Cover with an occlusive dressing and fastened around the trunk with an adhesive elastic bandage. The area involved was equivalent to about 10% of the total body surface. The hairs were clipped from the dorsal surface, once per week throughout the study"
Preparation TK12271 was appllied as a 2% solution in physiological saline (20 mg/kg/day) and as a concentrate (100, 500 mg/kg/day).
Frequency of application Once/day (6 hrs) 5 times/week for 13 consecutive weeks.
Duration of application
period 90 days
Recovery period 14 days
Dose levels (mg/kg/day) 0 (Control)
20
100
500
Type of coverage:
occlusive
Vehicle:
physiological saline
Details on exposure:
TEST SITE
- Area of exposure: Skin of the back
- % coverage: Approx. 10%
- Type of wrap if used: 8.5 x 6.5 cm Gauze patch, covered with aluminium foil (10 x 8 cm), covered with an occlusive dressing
- Time intervals for shavings or clipplings: Weekly

REMOVAL OF TEST SUBSTANCE
- Washing (if done):
- Time after start of exposure:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):
- Constant volume or concentration used: yes/no
- For solids, paste formed: yes/no

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes/no
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once/day (6 hours) 5 times/week for 13 weeks
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control
Dose / conc.:
20 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
No. of animals per sex per dose:
10 males, 10 females per dose
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
Mortality Daily (a.m. and p.m.)
Symptoms: Daily
Food consumption Weekly Althought measured accurately, it is presented as approximate consumption only because of the unavoidable wastages by the rabbits.
Body weight Weekly
Local skin irritiation Daily, for the first 3 weeks of treatment, weekly thereafter, immediately prior to next application of the test substance according to the procedure described in the "Appraisal of the Safety of Chemicals in Food, Drugs and cosmetics" (1959), the US Association of Food and Druf Officials (AFDO)
Sacrifice and pathology:
At the end of the 3 month test period and after a recovery period of two weeks all control and test animals were bied under ntravenous anaesthesia with T 61 (HOECHST) and subjected to detailed autopsy. Adrenals, brain, gonads, heart, kidneys, liver, thyroid (with parathyroid) and pituitary were weighed.

The following organs and tissues were preserved in 10 % neutral formalin: brain (cerebrum, cerebellum, brainstem), spinal cord, sciatic nerve, eye, pituitary, heart, thymus, thyroid (with parathyroid if present in plane section), trachea, lungs (with mainstem bronchi), spleen, bone marrow, lymph nodes (cervical, mesenteric), salivary gland, oesophagus, stomach, small and large intestine, pancreas, liver, adrenal glands, kidneys, urinary bladder, testes, epididymis and prostate or ovaries and uterus, skeletal muscle, skin (sections from treated and untreated areas) and appropriate tissue of any animals with signs of gross lesions. The fixed tissue samples were embedded in paraplast, sectioned at 3-5 um, stained with haematoxylin and eosin and subjected to microscopical examination.
Statistics:
For each time point and parameter a uni-variate statistical analysis was conducted. Due to the routine manner of the analysis system parameter free methods were applied. Each treated group was compared to the control group in respect of dispersion and displacement*. In addition a trend test** was applied considering all groups.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The control and 20 mg/kg/day groups failed to show general symptoms of treatment.
For the 100 mg/kg/day group, starting with day 7 of the test period the rabbits showed slight sedation, ruffled fur, ataxia and partially dispnoe and tremor. Ruffled fur and sedation were still seen in the animals which remained for recovery.
For the 500 mg/kg/day group, Starting with day 4 of the test all rabits showed ataxia, dyspnea, sedation, curved position, ruffled fur and later tremor.

At the end of the recovery period curved position, ruffled fur and sedation were seen in the rabbits which remained for the recovery period. No death occured during the test and recovery period.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
The mean skin irritations at the application sites were

Application period Recovery period
control group none none
20 mg/kg/day minimal (Index 0.1) none
100 mg/kg/day extreme (index 6.3) marked (index 5.4)
500 mg/kg/day extreme (index 7.1) extreme (index 6.5)
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
With the exeption of a significant (Sign. L. = 0.01) negative trend in the bodyweights of the male rabbits during weeks 6 to 13 of the test period, all the other values were comparable to those of the controls. Additionally the male rabbits of group 4 showed a significant difference in bodyweights. ( Sign. L. = 0.01 ). during weeks 9 to 11 and 13. The decrease of all bodyweights during weeks 4 to 6 was due to the change in water quality as described above. During weeks 14 and 15 of the recovery period a negative trend (Sign. L = 0.01) from the control to the highest dose group in the male rabbits was still present.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
For each time point and parameter a uni-variate statistical analysis was conducted. Due to the routine manner of the analysis system parameter free methods were applied. Each treated group was compared to the control group in respect of dispersion and displacement*. In addition a trend test** was applied considering all groups.
Food efficiency:
no effects observed
Description (incidence and severity):
With the exeption of small intergroup differences the food utilization of the rabbits of the test groups was comparable to that of the controls during the test period.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Thrombocyte count:
The males of the 500 mg/kg/day groups at 6 and 12 weeks of treatment and the females of the 500 mg/kg/day group at 12 weeks of treatrment had increased thrombocyte counts and a positive trend from the control to the highest dosage group. Total Leucocyte count
In the males, a decrease was observed in the 20 mg/kg/day group at week 6. Increases were seen in both sexes of the 500 mg/kg/day group at week 6; positive trends from the control to the highest dosage group were observed at 6 and 12 weeks in the males and at week 12 in the females.
Differential Leucocyte count
Band Neutrophils; Decreased counts were seen in the males of the 100 and 500 mg/kg/day group at week 6.
Segmented Neutrophilst Increased counts were found in the males f the 100 mg/kg/day group at week 12 and in the 500 mg/kg/day group at 6 and 12 weeks. A positive trend from the control to the highest dosage group was also observed at 6 and 12 weeks. In the females increases were found in the 100 mg/kg/day group at weeks 6 and 12 and in the 500 mg/kg/day group at week 6. At week 6 a positive trend from the control to the highest dosage group was also seen.
Eosinophils: The females of the 100 mg/kg/day group had lówer values at week 12. 7.
Basophils: At week 12 a decrease was observed in the females ofthe 100 mg/kg/day group.
Lymphocytes: Decreased counts were seen in the male 100 mg/kg group at week 12 as well as in the 500 mg/kg group at both terms, on which also a negative trend from the control to the highest dosage group was apparent. In the females a decrease was found only in the 500 mg/kg group at week 6 with a simultaneous negative trend from the control to the highest dosage group. The haematological changes can be summarized as slight thrombocytosis, moderate leucocytosis with an absolute
neutrophilia, effects seen mainly in the 500 mg/kg/day groups and somewhat less pronounced in the 100 mg/kg/day groups. In view of the marked dermal irritations these findings can be considered as normal reactions to the local injuries. They are therefore rather consequences of the injury caused by TK 12271 than systemic effects attributable to the compound.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Glucose-concentration
Lower values were observed in the males of the 20 and 100 mg/kg/day group at week 6 and at 6 and 12 weeks in the 500 mg/kg/day group. A negative trend from the control to the highest dosage group was found at both test dates. At week 12 lower values were seen in the females of all treatment groups. These decreases are small and almost situated around the lower physiological limits.

Cholesterol-concentration
The Cholesterol-concentration was increased in the males of the 100 mg/kg/day group at week 12, and in the 500 mg/kg/day group at weeks 6 and 12; a positive trend from the control to the highest dosage group was observed at weeks 6 and 12. The changes in the 500 mg/kg/day group are obvious and must be considered as effects of the application of TK 12271.

Total Protein Concentration
At week 12 in the male 500 mg/kg/day group an increase as well as a positive trend from the control to the highest dosage group was seen.

Electrophoresis relative
Whereas albumin was decreased in the males of the 100 mg/kg/day group at week 12 and in all animals of the 500 mg/kg/day group at the end of the test with negative trends from the controls to the highest dosage groups, the A1 and B Globulines were increased. For the A1 Globulines, this was seen in both checks of the 100 and 500 mg/kg/day group with the exception of the female 100 mg/kg/day group at week 12. The B-Globulins were increased in the 100 mg/kg/day groups at week 12 in the males and at week 6 in the females and at both terms in all 500 mg/kg/day animals (500 mg/kg/day males week 12 not significant). Positives trends from the control to the highest dosage groups were also present in all cases. The changes in the Protein-Electrophoresis in the 100 and 500 mg/kg/day groups can be summarized as follows: Normoprotëinaemia, Hypalbuminaemia and Hyperglobulinaemia A and SB. A similar phenomenon is described in H.H. Marki in W. Siegenthaler, Klinische Pathophysiologie, G. Thieme Verlag, 1970, p 91 f for inflammatory processus. Therefore the changes observed in the 100 and 500 mg/kg/day groups are consequences of the irritation set by the test compound and not of systemic toxicity of TK 12271.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were slight differences in the individual weights of some organs among the treated and control animals as well. The analysis of the organ weights and organ to body weight ratios however revealed no consistent compound related trends oralterations. Organ weights and ratios for the compound treated rabbits were generally comparable to those of the control animals.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
All details regarding the gross anatomical observations are listed within the macroscopical findings in individual rabbits. In the skin at the site of dermal application of the tested compound severe local reaction with ulceration of the skin was seen in all rabbits from the 100 and 500 mg/kg/day dosage groups. After an additional recovery period of 14 days skin ulcers were still present in all (5/5) male and in 4/5 female rabbits from the 500 mg/kg/day and in 1/5 male and 1/5 female animals from the 100 mg/kg/day dosage groups. In the remaining rabbits of both these groups thickened skin at the site of dermal application of the tested compound was noted. No gross skin lesion were observed in rabbits treated with 20 mg/kg/day and in control animals. All other gross changes seen in a few control and treated rabbits were incidental in nature and not related to the treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the skin at the site of local administration of the tested compound in all male and female rabbits from the 500 mg/kg/day group there were severe local reactive changes showing large ulcers with necrosis, haemorrhages and suppuration at the base of the ulcers and proliferation of granulation tissue and initial fibrosis in the vicinity. Similar changes, however in some animals little less pronounced, were found also in all male and female rabbits from the 100 mg/kg/day group.

After an additional recovery period of 14 days histopathological signs of healing of the skin ulcers were obvious. There were reepithelialization under the crusts, fibrosis and chronic inflammatory cell infiltration in the dermis and subcutis and slight or moderate acanthosis and parakeratosis in the adjacent epidermis. Residual skin ulceration was found histologically in all (5/5) male and in 4/5 female rabbits from the 500 mg/kg/day and in 1/5 male and 1/5 female animals from the 100 mg/kg/day dosage groups.

No other microscopical findings which could be related to the dermal administration of the tested compound were noted in treated animals. With exception of the local dermal lesions all histopathological changes seen in some treated and control rabbits were incidental in nature and are attributed to spontaneously occurring diseases in rabbits.
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOEL
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
dermal irritation
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
20 mg/kg bw/day (actual dose received)
System:
other: Skin corrosion
Organ:
skin
Treatment related:
yes
Executive summary:

TK 12271 was administered dermally to rabbits 5 times per week over a period of 90 days at dosages of 0 (control), 20, 100 and 500 mg/kg/day. The test compound was applied under occlusive dressing for 6 hours per application. The animals were observed for an additional 14 days without treatment. The control and 20 mg/kg/day groups failed to show general symptoms of treatment. In the 100 and 500 mg/kg/day groups sedation, ruffled fur, ataxia and occasionally dispnoe and tremor was observed. The body weights of the males of the 100 and 500 mg/kg/day groups were slightly reduced in the second half of the application period. The mean skin irritations at the application sites were application period recovery period control group 20 mg/kg/day 100 mg/kg/day 500 mg/kg/day none minimal (index 0.1) extreme (index 6.3) extreme (index 7.1) non none marked (index 5.4) extreme (index 6.5) During the recovery period, a healing process was observable in the 100 mg/kg/day group, but not in the 500 mg/kg/day group.

The haematological examination revealed in the 500 mg/kg/day and partially in the 100 mg/kg/day groups a slight Thrombocytosis and a moderate Leucocytosis with an absolute neutrophilia. In the blood chemistry investigation, the same groups showed Normoprotein-aemia, Hyperalbuminaemia, Hyperglobulinamia A and SB. These observations are common to inflammatory processus and are therefore not to be considered as specific effect of the test compound. In addition to these observations the cholesterol concentration was increased in the males of the higher dosage groups.

Post mortem findings

At the site of dermal application of the test compound severe local changes such as ulceration of the skin with necrosis, haemorrhages and local suppuration were seen in all male and female rabbits treated with 500 mg/kg/day of TK 12271. Similar changes were observed also in all male and female animals of the 100 mg/kg/day group. After a recovery period of 14 days, a healing processus with fibrosis and reepitalisation was in progress. Residual skin ulcers were found in all male ( 5/5 ) and in 4/5 female rabbits of the 500 mg/kg/day group and in one male ( 1/5 ) and in one female of the 100 mg/kg/day group. No other macroscopical or histopathological changes which could be related to the dermal application of TK 12271 were observed.

No effect level

No differences in all observed parameters were noted between the controls and the 20 mg/kg/day group. At this dose level, some variation was encountered with one assessment of skin irritation. As these differences were very small and being the result of subjective clinical observations, no experimental significance is attached to them. The dose of 20 mg/kg/day applied over a period of 90 days may be considered as "no observable effect" level.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
20 mg/kg bw/day
Study duration:
chronic
Species:
rabbit

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 1980 to Jun 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
GLP compliance:
no
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animals KA 46 rabbits, New Zealand White strain equal numbers of males and females bred and raised at Tierfarm Sisseln
Initial body weight 2.3 to 3.0kg
Initial age 2 to 16 weeks
No. of animals per group 10 males, 10 females (5 males, 5 females (5 males, 5 females for a 14 day recovery period)
Individual identification By numbered ear tags and under cage number
Method of randomisation Animals were assigned to the different groups by means of random numbers generated by the IBM computer of CIBA-GEIGY Ltd., Basle, Switzerland
Husbandry T he experiment was carried out under Standard laboratory conditions.
"Temperature: 22 ± 2°C
Relative humidity: 55 ± 10%
15 - 17 air changes/hr."

Housing individually in stainless steel cages
"Neither insecticides nor chemicals were applied in the animal room, with the exception of a desinfectant:
Fungitext SB (Prod. No. 30071, CIBA-GEIGY AG; Switzerland)"
Food Pelleted standard diet, NAFAG No. 814/85 (NAFAG SA, Gossau/Switzerland) ad libitum
Food from the follwing batches was fed throughout the test:

February 25 to March 31, 1980: Tox. 52
April 1 to April 21, 1980: Tox. 57
April 22 to May 19, 1980: Tox. 12/80
May 19 to June 2, 1980: Tox. 21/80
June 3 to June 16, 1980: Tox. 28/80

Water Tap water from automatic drinking valves ad libitum (drinking water quality according to the specifications of the "Schweizerishes lebensmittelbuch"
Route and method
of application Dermal on the shaved skin of the back. Gauze patch 8.5 x 6.5 cm, covered with aluminium foil (10 x 8 cm). Cover with an occlusive dressing and fastened around the trunk with an adhesive elastic bandage. The area involved was equivalent to about 10% of the total body surface. The hairs were clipped from the dorsal surface, once per week throughout the study"
Preparation TK12271 was appllied as a 2% solution in physiological saline (20 mg/kg/day) and as a concentrate (100, 500 mg/kg/day).
Frequency of application Once/day (6 hrs) 5 times/week for 13 consecutive weeks.
Duration of application
period 90 days
Recovery period 14 days
Dose levels (mg/kg/day) 0 (Control)
20
100
500
Type of coverage:
occlusive
Vehicle:
physiological saline
Details on exposure:
TEST SITE
- Area of exposure: Skin of the back
- % coverage: Approx. 10%
- Type of wrap if used: 8.5 x 6.5 cm Gauze patch, covered with aluminium foil (10 x 8 cm), covered with an occlusive dressing
- Time intervals for shavings or clipplings: Weekly

REMOVAL OF TEST SUBSTANCE
- Washing (if done):
- Time after start of exposure:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):
- Constant volume or concentration used: yes/no
- For solids, paste formed: yes/no

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes/no
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once/day (6 hours) 5 times/week for 13 weeks
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control
Dose / conc.:
20 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
No. of animals per sex per dose:
10 males, 10 females per dose
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
Mortality Daily (a.m. and p.m.)
Symptoms: Daily
Food consumption Weekly Althought measured accurately, it is presented as approximate consumption only because of the unavoidable wastages by the rabbits.
Body weight Weekly
Local skin irritiation Daily, for the first 3 weeks of treatment, weekly thereafter, immediately prior to next application of the test substance according to the procedure described in the "Appraisal of the Safety of Chemicals in Food, Drugs and cosmetics" (1959), the US Association of Food and Druf Officials (AFDO)
Sacrifice and pathology:
At the end of the 3 month test period and after a recovery period of two weeks all control and test animals were bied under ntravenous anaesthesia with T 61 (HOECHST) and subjected to detailed autopsy. Adrenals, brain, gonads, heart, kidneys, liver, thyroid (with parathyroid) and pituitary were weighed.

The following organs and tissues were preserved in 10 % neutral formalin: brain (cerebrum, cerebellum, brainstem), spinal cord, sciatic nerve, eye, pituitary, heart, thymus, thyroid (with parathyroid if present in plane section), trachea, lungs (with mainstem bronchi), spleen, bone marrow, lymph nodes (cervical, mesenteric), salivary gland, oesophagus, stomach, small and large intestine, pancreas, liver, adrenal glands, kidneys, urinary bladder, testes, epididymis and prostate or ovaries and uterus, skeletal muscle, skin (sections from treated and untreated areas) and appropriate tissue of any animals with signs of gross lesions. The fixed tissue samples were embedded in paraplast, sectioned at 3-5 um, stained with haematoxylin and eosin and subjected to microscopical examination.
Statistics:
For each time point and parameter a uni-variate statistical analysis was conducted. Due to the routine manner of the analysis system parameter free methods were applied. Each treated group was compared to the control group in respect of dispersion and displacement*. In addition a trend test** was applied considering all groups.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The control and 20 mg/kg/day groups failed to show general symptoms of treatment.
For the 100 mg/kg/day group, starting with day 7 of the test period the rabbits showed slight sedation, ruffled fur, ataxia and partially dispnoe and tremor. Ruffled fur and sedation were still seen in the animals which remained for recovery.
For the 500 mg/kg/day group, Starting with day 4 of the test all rabits showed ataxia, dyspnea, sedation, curved position, ruffled fur and later tremor.

At the end of the recovery period curved position, ruffled fur and sedation were seen in the rabbits which remained for the recovery period. No death occured during the test and recovery period.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
The mean skin irritations at the application sites were

Application period Recovery period
control group none none
20 mg/kg/day minimal (Index 0.1) none
100 mg/kg/day extreme (index 6.3) marked (index 5.4)
500 mg/kg/day extreme (index 7.1) extreme (index 6.5)
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
With the exeption of a significant (Sign. L. = 0.01) negative trend in the bodyweights of the male rabbits during weeks 6 to 13 of the test period, all the other values were comparable to those of the controls. Additionally the male rabbits of group 4 showed a significant difference in bodyweights. ( Sign. L. = 0.01 ). during weeks 9 to 11 and 13. The decrease of all bodyweights during weeks 4 to 6 was due to the change in water quality as described above. During weeks 14 and 15 of the recovery period a negative trend (Sign. L = 0.01) from the control to the highest dose group in the male rabbits was still present.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
For each time point and parameter a uni-variate statistical analysis was conducted. Due to the routine manner of the analysis system parameter free methods were applied. Each treated group was compared to the control group in respect of dispersion and displacement*. In addition a trend test** was applied considering all groups.
Food efficiency:
no effects observed
Description (incidence and severity):
With the exeption of small intergroup differences the food utilization of the rabbits of the test groups was comparable to that of the controls during the test period.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Thrombocyte count:
The males of the 500 mg/kg/day groups at 6 and 12 weeks of treatment and the females of the 500 mg/kg/day group at 12 weeks of treatrment had increased thrombocyte counts and a positive trend from the control to the highest dosage group. Total Leucocyte count
In the males, a decrease was observed in the 20 mg/kg/day group at week 6. Increases were seen in both sexes of the 500 mg/kg/day group at week 6; positive trends from the control to the highest dosage group were observed at 6 and 12 weeks in the males and at week 12 in the females.
Differential Leucocyte count
Band Neutrophils; Decreased counts were seen in the males of the 100 and 500 mg/kg/day group at week 6.
Segmented Neutrophilst Increased counts were found in the males f the 100 mg/kg/day group at week 12 and in the 500 mg/kg/day group at 6 and 12 weeks. A positive trend from the control to the highest dosage group was also observed at 6 and 12 weeks. In the females increases were found in the 100 mg/kg/day group at weeks 6 and 12 and in the 500 mg/kg/day group at week 6. At week 6 a positive trend from the control to the highest dosage group was also seen.
Eosinophils: The females of the 100 mg/kg/day group had lówer values at week 12. 7.
Basophils: At week 12 a decrease was observed in the females ofthe 100 mg/kg/day group.
Lymphocytes: Decreased counts were seen in the male 100 mg/kg group at week 12 as well as in the 500 mg/kg group at both terms, on which also a negative trend from the control to the highest dosage group was apparent. In the females a decrease was found only in the 500 mg/kg group at week 6 with a simultaneous negative trend from the control to the highest dosage group. The haematological changes can be summarized as slight thrombocytosis, moderate leucocytosis with an absolute
neutrophilia, effects seen mainly in the 500 mg/kg/day groups and somewhat less pronounced in the 100 mg/kg/day groups. In view of the marked dermal irritations these findings can be considered as normal reactions to the local injuries. They are therefore rather consequences of the injury caused by TK 12271 than systemic effects attributable to the compound.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Glucose-concentration
Lower values were observed in the males of the 20 and 100 mg/kg/day group at week 6 and at 6 and 12 weeks in the 500 mg/kg/day group. A negative trend from the control to the highest dosage group was found at both test dates. At week 12 lower values were seen in the females of all treatment groups. These decreases are small and almost situated around the lower physiological limits.

Cholesterol-concentration
The Cholesterol-concentration was increased in the males of the 100 mg/kg/day group at week 12, and in the 500 mg/kg/day group at weeks 6 and 12; a positive trend from the control to the highest dosage group was observed at weeks 6 and 12. The changes in the 500 mg/kg/day group are obvious and must be considered as effects of the application of TK 12271.

Total Protein Concentration
At week 12 in the male 500 mg/kg/day group an increase as well as a positive trend from the control to the highest dosage group was seen.

Electrophoresis relative
Whereas albumin was decreased in the males of the 100 mg/kg/day group at week 12 and in all animals of the 500 mg/kg/day group at the end of the test with negative trends from the controls to the highest dosage groups, the A1 and B Globulines were increased. For the A1 Globulines, this was seen in both checks of the 100 and 500 mg/kg/day group with the exception of the female 100 mg/kg/day group at week 12. The B-Globulins were increased in the 100 mg/kg/day groups at week 12 in the males and at week 6 in the females and at both terms in all 500 mg/kg/day animals (500 mg/kg/day males week 12 not significant). Positives trends from the control to the highest dosage groups were also present in all cases. The changes in the Protein-Electrophoresis in the 100 and 500 mg/kg/day groups can be summarized as follows: Normoprotëinaemia, Hypalbuminaemia and Hyperglobulinaemia A and SB. A similar phenomenon is described in H.H. Marki in W. Siegenthaler, Klinische Pathophysiologie, G. Thieme Verlag, 1970, p 91 f for inflammatory processus. Therefore the changes observed in the 100 and 500 mg/kg/day groups are consequences of the irritation set by the test compound and not of systemic toxicity of TK 12271.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were slight differences in the individual weights of some organs among the treated and control animals as well. The analysis of the organ weights and organ to body weight ratios however revealed no consistent compound related trends oralterations. Organ weights and ratios for the compound treated rabbits were generally comparable to those of the control animals.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
All details regarding the gross anatomical observations are listed within the macroscopical findings in individual rabbits. In the skin at the site of dermal application of the tested compound severe local reaction with ulceration of the skin was seen in all rabbits from the 100 and 500 mg/kg/day dosage groups. After an additional recovery period of 14 days skin ulcers were still present in all (5/5) male and in 4/5 female rabbits from the 500 mg/kg/day and in 1/5 male and 1/5 female animals from the 100 mg/kg/day dosage groups. In the remaining rabbits of both these groups thickened skin at the site of dermal application of the tested compound was noted. No gross skin lesion were observed in rabbits treated with 20 mg/kg/day and in control animals. All other gross changes seen in a few control and treated rabbits were incidental in nature and not related to the treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the skin at the site of local administration of the tested compound in all male and female rabbits from the 500 mg/kg/day group there were severe local reactive changes showing large ulcers with necrosis, haemorrhages and suppuration at the base of the ulcers and proliferation of granulation tissue and initial fibrosis in the vicinity. Similar changes, however in some animals little less pronounced, were found also in all male and female rabbits from the 100 mg/kg/day group.

After an additional recovery period of 14 days histopathological signs of healing of the skin ulcers were obvious. There were reepithelialization under the crusts, fibrosis and chronic inflammatory cell infiltration in the dermis and subcutis and slight or moderate acanthosis and parakeratosis in the adjacent epidermis. Residual skin ulceration was found histologically in all (5/5) male and in 4/5 female rabbits from the 500 mg/kg/day and in 1/5 male and 1/5 female animals from the 100 mg/kg/day dosage groups.

No other microscopical findings which could be related to the dermal administration of the tested compound were noted in treated animals. With exception of the local dermal lesions all histopathological changes seen in some treated and control rabbits were incidental in nature and are attributed to spontaneously occurring diseases in rabbits.
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOEL
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
dermal irritation
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
20 mg/kg bw/day (actual dose received)
System:
other: Skin corrosion
Organ:
skin
Treatment related:
yes
Executive summary:

TK 12271 was administered dermally to rabbits 5 times per week over a period of 90 days at dosages of 0 (control), 20, 100 and 500 mg/kg/day. The test compound was applied under occlusive dressing for 6 hours per application. The animals were observed for an additional 14 days without treatment. The control and 20 mg/kg/day groups failed to show general symptoms of treatment. In the 100 and 500 mg/kg/day groups sedation, ruffled fur, ataxia and occasionally dispnoe and tremor was observed. The body weights of the males of the 100 and 500 mg/kg/day groups were slightly reduced in the second half of the application period. The mean skin irritations at the application sites were application period recovery period control group 20 mg/kg/day 100 mg/kg/day 500 mg/kg/day none minimal (index 0.1) extreme (index 6.3) extreme (index 7.1) non none marked (index 5.4) extreme (index 6.5) During the recovery period, a healing process was observable in the 100 mg/kg/day group, but not in the 500 mg/kg/day group.

The haematological examination revealed in the 500 mg/kg/day and partially in the 100 mg/kg/day groups a slight Thrombocytosis and a moderate Leucocytosis with an absolute neutrophilia. In the blood chemistry investigation, the same groups showed Normoprotein-aemia, Hyperalbuminaemia, Hyperglobulinamia A and SB. These observations are common to inflammatory processus and are therefore not to be considered as specific effect of the test compound. In addition to these observations the cholesterol concentration was increased in the males of the higher dosage groups.

Post mortem findings

At the site of dermal application of the test compound severe local changes such as ulceration of the skin with necrosis, haemorrhages and local suppuration were seen in all male and female rabbits treated with 500 mg/kg/day of TK 12271. Similar changes were observed also in all male and female animals of the 100 mg/kg/day group. After a recovery period of 14 days, a healing processus with fibrosis and reepitalisation was in progress. Residual skin ulcers were found in all male ( 5/5 ) and in 4/5 female rabbits of the 500 mg/kg/day group and in one male ( 1/5 ) and in one female of the 100 mg/kg/day group. No other macroscopical or histopathological changes which could be related to the dermal application of TK 12271 were observed.

No effect level

No differences in all observed parameters were noted between the controls and the 20 mg/kg/day group. At this dose level, some variation was encountered with one assessment of skin irritation. As these differences were very small and being the result of subjective clinical observations, no experimental significance is attached to them. The dose of 20 mg/kg/day applied over a period of 90 days may be considered as "no observable effect" level.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
Study duration:
chronic
Species:
rabbit

Additional information

Justification for classification or non-classification