Registration Dossier

Administrative data

Description of key information

Repeated dose toxicity: oral. Key study. Method similar to OECD 407, GLP study. The NOEL for the subacute oral repeated dose toxicity was determined to be 100 mg/kg bw/day in male and female rats.

Repeated dose toxicity: dermal. Weight of evidence: In a 13-week subchronic dermal toxicity study performed on the test item by the NTP, method similar to OECD 411 (GLP study), the NOAEL for the subchronic dermal toxicity of the test item in rats was 3 mg/kg bw in males and 6 mg/kg bw in females. In a 13 -week subchronic dermal toxicity study performed on the test item by the NTP, method similar to OECD 411 (GLP study), the NOAEL for the subchronic dermal toxicity of the test item in mice was 3 mg/kg bw in males and in females. In a 16-day repeated dose dermal study in F-344 rats by the NTP (GLP study), the NOAEL was found to be ca. 12 mg/kg bw in rats. The same study performed in B6C3F1 male and female mice gave a NOAEL of ca. 8 mg/kg bw.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2, 1991 - November, 1991.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Source: Tama Chemical Industry Co., Ltd.
- Lot: 10705
- Purity of 99.7 %
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan Co., Ltd.
- Age at study initiation: 5 weeks
- Weight at study initiation: 170-197 (males), 137-158 (females)
- Housing: 2 per cage (same sex), in polycarbonate cages (265 W x 426 D x 200 H mm: Tokiwa Scientific Instruments Co., Ltd.) .) laid with animal bedding (Beta chip: Nippon Charles River Co., Ltd.). The cage, feeder and water bottle were sterilised once per week.
- Diet: MF: Oriental Yeast Co., Ltd. ad libitum.
- Water: tap water, irradiated with ultraviolet light and filtered with a 5 µm filter, ad libitum.
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25ºC
- Humidity (%): 40-70%
- Air changes (per hr): 12 times/per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark


IN-LIFE DATES: Obtained on October 2, 1991.
Route of administration:
oral: gavage
Vehicle:
olive oil
Remarks:
(Japanese Pharmacopoeia)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Olive oil (Japanese Pharmacopoeia) was added to the test substance, and the mixture was warmed at around 40 ºC and dissolved to a predetermined concentration;
the solution was prepared every 9 days, and it was kept in a cool dark place until immediately before administration.

VEHICLE
- Amount of vehicle (if gavage): the dosing volume was 5 ml/kg, based on the weight at the nearest measurement day.
- Purity: compliant with Japanese Pharmacopoeia standards.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stabilty of the solution (test item in olive oil) was analyzed, and it was confirmed that it was stable for 11 days after preparation, when stored in a cool dark place. No further details provided on the method. Accordingly, the test item solution was prepared every 9 days, and it was kept in a cool dark place until immediately before administration.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily (once in the morning)
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6 males / 6 females per group in 15 mg/kg and 100 mg/kg groups.
12 males / 12 females per group in control and 500 mg/kg group.
Control animals:
yes
Details on study design:
- Dose selection rationale: Dose levels were selected based on a preliminary oral toxicity study. Repeated oral administration of the test substance to SD rats at doses of 125, 250 and 500 mg/kg for 7 days resulted in toxic signs such as salivation, loss of locomotor activity, weight gain suppression, liver weight gain at 500 mg/kg; at 125 and 250 mg/kg, toxic signs such as spontaneous motor activity decrease were also observed. Based on this study, the high dose of this study was 500 mg/kg, the middle dose was 100 mg/kg, and the low dose was 15 mg/kg. In addition to this, a control group (only vehicle) was provided.
- Administration period: 28-days in the three doses-group and control group
- Post-exposure recovery period: 14 days in 6 males / 6 females for control and high dose groups (satellite groups).
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: daily for morbidity, mortality, behaviour (4 weeks for all groups, up to 42 days for both recovery groups).

DETAILED CLINICAL OBSERVATIONS
- Time schedule: detalied observations including palpation once a week (4 weeks for all groups, up to 42 days for both recovery groups).

BODY WEIGHT:
- Time schedule for examinations: at the beginning of treatment and once a week thereafter (4 weeks for all groups, up to 42 days for both recovery groups).

FOOD CONSUMPTION: Food consumption was measured, although it is not a feeding study.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food was weighed at the beginning of the treatment and once a week thereafter, and the mean food intake per mouse per period was calculated (4 weeks for all groups, up to 40 days for both recovery groups).

FOOD EFFICIENCY: No.

WATER CONSUMPTION AND COMPOUND INTAKE: Water consumption was measured, although it is not a drinking water study.
- Time schedule for examinations: the tare was measured once a week and compared (4 weeks for all groups, up to 42 days for both recovery groups).

OPHTHALMOSCOPIC EXAMINATION: No.

HAEMATOLOGY:
- Time schedule for collection of blood: suvirval animals at the time of each planned killing (28 or 42 days) were bled from posterior vena cava after anesthesia by pentobarbital sodium (Nembutal injection solution: Dynabot Corporation) intraperitoneally and examined on the following items: red blood and white blood cell count, platelet count, hemoglobin concentration, hematocrit value, leukocyte percentage, reticulocyte count, prothrombin time, activated partial thromboxane plastin time, mean red blood cell volume, mean red blood cell hemoglobin content and mean red blood cell hemoglobin concentration.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: in the remaining blood used for hematalogical examination the following parameters were examined: total protein, albumin, A/G ratio, glucose, triglyceride, total cholesterol, urea nitrogen, creatinine, calcium, inorganic phosphorus, GOT (AST), GPT (ALT), γ-GTP, ALP, sodium, potassium, chloride.

URINALYSIS:
- Time schedule for collection of urine: fresh urine of the survival animals was collected before the end of the administration period (3 weeks) and on the end of th recovery period. The following parameters were determined: pH, protein, glucose, ketones, bilirubin, urobilinogen, ocult blood, specific gravity, sediment, urine volume, sodium, potassium and chlorine.

NEUROBEHAVIOURAL EXAMINATION: No.
IMMUNOLOGY: No.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes.
Surviving animals, at the time of each planned killing, were exsanguined, sacrificied and necropsied. Dead animals were necropsied as soon as they were found. Brain, liver, kidney, adrenal gland, testis and ovary were weighed.

HISTOPATHOLOGY: Yes.
The following organs were collected, fixed and stored: Harder's glands, brain, pituitary, eyeball (including accessory gland), lung, stomach, thyroid (including parathyroid), heart, liver, spleen, kidney, adrenal gland, bladder, testis or ovary, bone marrow (femur). The testes suspected of being altered, heart, liver, kidney, spleen, adrenal glands and those duodena that had shown changes in the autopsy, were microscopical examined. All the organs that were found abnormal at necropsy were examined too. Examinations were made in all dosing groups.
Statistics:
The equidistance test by the Bartlett method was performed, one-way ANOVA was performed when the variance was uniform and Kruskal-Wallis test was performed when the variance was not uniform. Where the were significant differences between the groups, the method of Dunnett was applied. For uirne qualtitative test and urine sediments, Armitage's test was used.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Reduced lococmotor activity and salivation were observed in the 500 mg/kg group.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female of the 500 mg/kg group died on day 13 of the administration period and another died on day 4 of the recovery period. Animals showed crouching, prone position, ataxic gait, tiptoe gait, clonic convulsions and gasping before death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain was reduced in the 500mg/kg males and females group. A recovery tendency was observed in both sexs during the recovery period.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption decreased in 500 mg/kg group (males and females) in the first week of administration, but no signifcant differences were observed after that.
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Water intake was increased in the 500 mg/kg (males and females).
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Reduction of mean red blood cell hemoglobin level and reduction of prothrombin time was detected in males of the highest dose group. Shortening of activated partial thromboplastin time and increase of platelet count were observed in females (highest dose group).
No change was observed at the end of the recovery period.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A decrease in alkaline phospatase was observed in the 500 mg/kg males group and an increase in potassium in the 500 mg/kg males and females group.
At the end of the recovery period, an increase in alkaline phosphatase was observed in males (500 mg/kg). An increase in inorganic phosphorus in males was observed too, but it was within the physiological variation range.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Urine volume tended to increase in both sexes although no significant differences was observed. Urinary pH was increased in the 500 mg/kg females group.
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males showed an increase in the liver/body weight ratio while females showed an increase in both actual weight and liver/body weight ratio in the 500 mg/kg group. Females in the 500 mg/kg recovery group showed an increase in liver weight but the difference with the control group was reduced. The weight of adrenal glands in females was decreased but there were no differences in the adrenal weight/ body weight ratios.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There was an elargement of the duodenum with thickening of the mucosa in both sexes in the 500 mg/kg group. This change was not observed in the recovery group.

Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the surviving animals, changes in the liver and duodenum of the 500 mg / kg group due to the administration of the test substance were observed in both sexes: enlarged hepatocytes around the Glissons capsules, eosinophilic hepatocyte cytoplasm and thickening of the duodenal mucosa.
In addition, fading foci in the kidney cortex, cyst formation, localized yellowing in the liver and brown / dark red spots in the lung were observed in a few cases, but it was considered to be a contingent lesion.
Details on results:
After the recovery period, histological changes of the liver and duodenum dissapeared and other charged parameters tended to show values almost within the normal range.
Key result
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
clinical biochemistry
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
duodenum
Treatment related:
yes
Dose response relationship:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
not specified

Table 3. Body weight (group mean values)

 

Days after commencement/cessation of treatment

Dose level (mg/kg)

 

0

7

14

21

28/0

35/7

42/14

MALE

 

0

Mean

SD

N

185

8.5

12

255

14.2

12

322

19.4

12

382

26.8

12

423

34.5

12

457

47.3

6

484

48.5

6

15

Mean

SD

N

187

6.7

6

255

11.3

6

319

19.1

6

377

27.0

6

415

37.8

6

-

-

100

Mean

SD

N

186

5.2

6

253

6.7

6

325

8.7

6

383

12.6

6

424

13.6

6

-

-

500

Mean

SD

N

187

8.4

12

239

15.9

12

300

22.5

12

351

23.9

12

368

24.3

12

427

23.5

6

468

28.1

6

FEMALES

 

0

Mean

SD

N

146

5.4

12

176

9.5

12

205

13.2

12

231

15.4

12

249

17.3

12

259

21.5

6

271

23.6

6

15

Mean

SD

N

145

6.6

6

181

8.7

6

207

10.8

6

233

16.0

6

245

13.5

6

-

-

100

Mean

SD

N

146

6.1

6

182

5.0

6

211

5.0

6

234

5.4

6

251

3.6

6

-

-

500

Mean

SD

N

147

4.9

12

170

8.5

12

196

10.3

11

216

13.2

11

225

26.6

11

250

21.5

4

268

15.1

4

Table 4. Organ weight and ratio organ weight/body weight (6 animals per group per dose, treatment groups)

Dose level (mg/kg)

 

Final body weight (g)

Brain

Brain/

body weight

Liver

Liver/

body weight

Kidneys

Kidneys/

body weight

Adrenals(x10-3)

Adrenals /

body weight

Testes /Ovaries

(x10-3)

Test-ovar/body weight

 

 

 

(g)

%

(g)

%

(g)

%

(g)

%

(g)/ (mg)

 

MALE

 

 

 

 

 

 

 

 

 

 

 

 

0

Mean

SD

430

31.3

2.00

31.3

0.47

0.029

18.06

2.234

4.19

0.249

3.06

0.297

0.71

0.054

58.3

0.276

13.7

1.58

3.03

0.276

0.71

0.055

15

Mean

SD

415

37.6

1.97

0.089

0.48

0.051

17.61

2.925

4.22

0.316

3.01

0.210

0.73

0.021

61.3

7.57

14.8

1.69

3.24

0.464

0.79

0.178

100

Mean

SD

424

13.8

2.04

0.037

0.48

0.024

18.20

1.611

4.29

0.272

3.08

0.225

0.73

0.055

57.4

7.75

13.5

1.49

3.10

0.092

0.73

0.044

500

Mean

SD

4386

28.3

1.99

0.029

0.52

0.043

19.58

1.886

5.07 *

0.166

3.01

0.229

0.79

0.068

57.2

9.92

14.8

1.83

3.06

0.178

0.80

0.064

FEMALE

 

 

 

 

 

 

 

 

 

 

 

 

0

Mean

SD

251

18.0

1.92

0.065

0.77

0.058

9.54

1.031

3.60

.0236

1.89

0.059

0.76

0.056

64.9

7.60

26.0

3.42

95.5

9.64

38.1

3.36

15

Mean

SD

246

13.5

1.88

0.053

0.77

0.051

9.13

0.811

3.70

0.194

1.85

0.126

0.75

0.046

68.5

6.76

28.0

3.88

95.7

13.69

38.9

4.75

100

Mean

SD

251

2.6

1.90

0.052

0.76

0.024

9.62

0.539

3.84

0.244

1.89

0.258

0.76

0.106

67.0

6.61

26.8

2.78

83.3

14.11

33.2

5.48

500

Mean

SD

231

17.3

1.84

0.054

0.80

0.062

12.61*

0.796

5.43 *

0.328

1.87

0.109

0.81

0.039

63.9

7.25

27.9

4.95

86.0

13.53

37.2

4.67

* Significantly different from control value p < 0.01

Table 5. Organ weight and ratio organ weight/body weight (6 animals per group per dose, recovery groups)

Dose level (mg/kg)

 

Final body weight

Brain

Brain/

body weight

Liver

Liver/

body weight

Kidneys

Kidneys/

body weight

Adrenals(x10-3)

Adrenals /

body weight

Testes /Ovaries

(x10-3)

Test-ovar /body weight

 

 

(g)

(g)

%

(g)

%

(g)

%

(g)

%

(g)/ (mg)

 

MALE

 

 

 

 

 

 

 

 

 

 

 

 

0

Mean

SD

485

47.8

2.10

0.049

0.44

0.039

20.80

4.627

4.27

0.524

3.57

0.314

0.74

0.023

63.4

9.21

13.2

2.29

3.36

0.272

0.70

0.081

500

Mean

SD

467

27.8

2.11

0.077

0.45

0.020

20.39

2.464

4.35

0.260

3.33

0.280

0.71

0.028

57.1

9.62

12.3

2.64

3.22

0.245

0.69

0.036

FEMALE

 

 

 

 

 

 

 

 

 

 

 

 

0

Mean

SD

271

23.7

1.9

0.061

0.73

0.059

10.20

1.640

3.75

0.284

19.99

0.166

0.74

0.056

76.7

5.68

28.6

4.34

110.2

15.88

40.7

5.62

500

Mean

SD

268

15.0

1.91

0.043

0.71

0.038

12.58

0.638*

4.69 *

0.080

1.97

0.137

0.74

0.045

66.4 *

6.44

24.9

3.65

113.1

11.99

42.2

3.15

* Significantly different from control value p < 0.01

Table 6. Total incidence macroscopic and microscopic findings

MACROSCOPIC FINDINGS

 

28 DAYS

RECOVERY

 

SEX

MALE

FEMALE

MALE

FEMALE

 

DOSE LEVEL (MG/KG)

0

15

100

500

0

15

100

500

0

500

0

500

ORGAN FINDINGS

NUMBER OF ANIMALS

6

6

6

6

6

6

6

6

6

6

6

4

Duodenum

enlargement

0

0

0

5

0

0

0

4

0

0

0

0

Kidneys

Focal discoloration

Cyst

 

0

0

 

1

0

 

0

0

 

0

0

 

0

0

 

0

0

 

0

1

 

0

0

 

0

0

 

0

0

 

0

0

 

0

0

Liver

Focal yellowish change

0

0

0

0

0

0

0

0

1

0

0

0

Lungs

Brownish/dark patch

0

0

0

0

0

0

1

0

0

1

0

0

MICROSCOPIC FINDINGS

 

28 DAYS

RECOVERY

 

SEX

MALE

FEMALE

MALE

FEMALE

 

DOSE LEVEL (MG/KG)

0

15

100

500

0

15

100

500

0

500

0

500

ORGAN FINDINGS

NUMBER OF ANIMALS

6

6

6

6

6

6

6

6

6

6

6

4

Liver

Enlarged/eosinophilic hepatocytes

Neutrophilic infiltration

Focal fatty change

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

5

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

6

 

1

 

0

 

0

 

0

 

1

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

Duodenum

thickening of mucosa

0

0

0

5

0

0

0

4

0

0

1/5

0

Heart

Focal myocardial degeneration

 

0

 

-

 

-

 

1

 

0

 

-

 

-

 

0

 

-

 

-

 

-

 

-

Kidneys

Basophilic change of tubular epithelium

Focal linphocytic in interstitium

Hyaline droplets in tubular epithelium

Cyst

 

3

 

1

0

 

0

 

 

0/1

 

0/1

1/1

 

0/1

 

 

-

 

 

2

 

0

0

 

0

 

 

1

 

1

0

 

0

 

-

 

0/1

 

0/1

0/1

 

1/1

 

2

 

2

0

 

0

 

-

-

-

-

Lungs

Focal hemorrhage into alveoli

0

0

0

0

0

0

1

0

0

1

0

0

- not examined

Conclusions:
The NOEL was determined to be 100 mg/kg bw/day in male and female rats.
Executive summary:

A 28-day repeated dose toxicity test was performed according toaccording to the Guidelines on Chemical Substitution Law (1986) (Guideline 28-Day Repeat Dose Toxicity Test of Chemical, Japan), similar to OECD 407 (GLP study). SD (Crj: CD) rats were treated with 0, 15, 100 and 500 mg/kg of the test substance by oral gavage for 28 days. 6 males and 6 females were selected for the groups of 15 and 100 mg/kg and 12 males/12 females for the control and 500 mg/kg groups. After the 28-days treatment, 6 males and 6 females for the control and 500 mg/kg groups were left to study their recovery over a period of 14 days. Animals of both sexes in the 500 mg/kg group showed reduced locomotor activity, salivation, inhibition of body weight gain, decreased food consumption and increased water intake. Two females in the 500 mg/kg dose group died, one during treatment and one during the recovery period. A decrease in alkaline phosphatase was also observed in males in the 500 mg / kg group. Pathological examination revealed increased relative liver weight, enlargement of the duodenum due to mucosal thickening and hepatocellular swelling in both sexes of the higher dose group. Histological changes in the liver and duodenum disappeared due to discontinuation of administration, and other changes also disappeared or showed a recovery tendency after the 14 days post-exposure. No effects were observed in the 100 and 15 mg/kg group in both males and females. Based on the above results, the NOEL under the test conditions was considered to be 100 mg/kg/day in both sexes.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study is a GLP study and has Klimisch score 2.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
June 29, 1994 - July 15, 1994
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
yes
Remarks:
Treatment 5 days per week for 16 days. No haemathology or biochemistry analysis.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Aldrich Chemical Company (Milwaukee, WI), lot 00929TZ
- Purity: 99.5% (determined by GC)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, under a nitrogen head space, protected from light
- Stability under test conditions: Periodic analyses of the bulk chemical were performed by the study laboratories during the studies using GC: no degradation of the bulk chemical was detected.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Dose formulations were prepared by mixing dicyclohexylcarbodiimide and anhydrous ethanol to give the required concentration.
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Laboratory Animals and Services (Germantown, NY).
- Age at study initiation: 8 weeks
- Weight at study initiation: males 25.9 -28.1 g; females 19.9 - 21.3 g
- Fasting period before study: not specified
- Housing: individually housed
- Diet: ad libitum, NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA),
- Water: ad libitum, tap water (Washington Suburban Sanitary Commission Potomac Plant)
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.22 ± 0.16 ºC
- Humidity (%): 35-65%
- Air changes (per hr): at least 10 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark
Type of coverage:
not specified
Vehicle:
ethanol
Details on exposure:
TEST SITE
- Area of exposure: clipped dorsal skin, from the posterior of the scapulae to the base of the tail
- % coverage: not specified
- Type of wrap if used: not specified.

REMOVAL OF TEST SUBSTANCE: not specified

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0, 0.2, 0.6, 1.7, 5, or 15 mg/animal in 0.1 ml ethanol
- Concentration (if solution): 0, 2, 6, 17, 50, or 150 mg dicyclohexylcarbodiimide per ml of ethanol. 0.6 mg/animal approximately corresponded to 24 mg/kg body weight.
- For solids, paste formed: no, test substance was diluted in anhydrous ethanol to give the required concentration

VEHICLE
- Vehicle (if other than water): anhydrous ethanol
- Amount(s) applied (volume or weight with unit): 0.1 ml
- Lot/batch no. (if required): not specified
- Purity: greater than 99.9 %

PREPARATION OF DOSING SOLUTIONS
- A weighed amount of dicyclohexylcarbodiimide was dissolved in absolute ethanol to prepare the highest dose concentration formulation; lower dose formulations were prepared by diluting aliquots of this preparation with absolute ethanol. Dose formulations were prepared twice. The dose formulations were stored in sealed vials under a headspace of inert gas for up to 28 days at room temperature. Stability studies of 0.38, 2, and 7 mg/ml dose formulations of lot 00929TZ were conducted by the study laboratory using GC. Stability was confirmed for up to 35 days for dose formulations stored at room temperature in sealed containers under a nitrogen headspace and for up to 3 hours when exposed to light and air at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulations were prepared twice during the study and were analyzed once by GC . All five dose formulations were within 10% of the target concentrations.
Duration of treatment / exposure:
16 days
Frequency of treatment:
5 days per week
Dose / conc.:
0 other: mg / animal
Remarks:
control group
Dose / conc.:
0.2 other: mg / animal
Dose / conc.:
0.6 other: mg / animal
Remarks:
ca. 24 mg/kg bw
Dose / conc.:
1.7 other: mg / animal
Dose / conc.:
5 other: mg / animal
Dose / conc.:
15 other: mg / animal
No. of animals per sex per dose:
5 males and 5 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment: animals were distributed randomly into groups of approximately equal initial mean body weights.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical findings (including dermal irritation) were recorded initially, on day 8 and at the end of the studies.

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed initially, on day 8 and at the end of the studies

FOOD CONSUMPTION: No data

FOOD EFFICIENCY: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: No data

CLINICAL CHEMISTRY: No data

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsies were performed on all animals. Organs weighed were the adrenal gland, heart, right kidney, liver, lung, right testis, and thymus.

HISTOPATHOLOGY: Yes
Histopathology was performed on vehicle control, 0.2, 0.6 and 1.7 mg/animal mice. In addition to gross lesions and tissue masses, the following tissues were examined: brain, kidney, liver, lung, skin, and spinal cord.
Statistics:
Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs of toxicity at doses of 0.6 mg or greater.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Skin irritation at the site of application in all dosed groups.
Mortality:
mortality observed, treatment-related
Description (incidence):
One 0.6 mg female mouse and all mice in the 1.7, 5, and 15 mg groups died before the end of the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Final mean body weights of the 0.6 mg groups were significantly less than those of the vehicle controls, and animals in these groups generally lost weight during the study.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Heart weights of 0.6 mg groups of mice were significantly greater than those of the vehicle controls; thymus weights in these groups were significantly less than those of the vehicle controls.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathologic examination of mice dosed with 1.7 mg dicyclohexylcarbodiimide or less revealed lesions of the skin at the site of application including epidermal hyperplasia, epidermal necrosis, and acute or chronic active dermal inflammation (data not shown).
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
At a dose of 0.6 mg or greater, clinical signs of toxicity appeared. Final mean body weights were significantly lower than the control, heart weights were significantly greater and thymus weights were significantly lower.
Key result
Dose descriptor:
NOAEL
Effect level:
0.2 other: mg/animal
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
Remarks on result:
other: ca. 8 mg/kg bw
Key result
Critical effects observed:
no

Table 1. Survival and body weights of B6C3F1 mice.

Dose

mg/animal

Survival

Mean body weight

Final weight Relative to controls

(%)

Initial

Final

Change

MALE

0

5/5

27.0 ± 1.1

28.8 ± 1.1

1.8 ± 0.0

 

0.2

5/5

26.7 ± 0.7

28.3 ± 0.5

1.6 ± 0.3

98

0.6

5/5

27.0 ± 0.5

26.0 ± 0.5*

-1.0 ± 0.4 *

90

1.7

0/5

26.5 ± 0.6

-

-

 

5

0/5

26.6 ± 0.7

-

-

 

15

0/5

26.5 ± 0.7

-

-

 

FEMALE

0

5/5

20.6 ± 0.6

24.0 ± 0.5

3.4 ± 0.3

 

0.2

5/5

20.4 ± 0.5

23.9 ± 0.4

3.5 ± 0.4

100

0.6

4/5

20.8 ± 0.7

21.3 ± 0.8 *

-0.1 ± 0.5*

89

1.7

0/5

21.0 ± 0.3

-

-

 

5

0/5

20.8 ± 0.3

-

-

 

15

0/5

20.6 ± 0.6

-

-

 

* Significantly different from the vehicle control group by Dunnett’s or Williams’ test

Conclusions:
The NOAEL for the test substance was 0.2 mg/animal (ca. 8 mg/kg bw) in male and females mice after 16 days of dermal exposure.
Executive summary:

Groups of five male and five female mice were dermally treated with 0.1 ml ethanol containing 0, 0.2, 0.6, 1.7, 5 or 15 mg dicyclohexylcarbodiimide, 5 days per week (16 days of dosing). The control animals were treated with the vehcicle only (ethanol). One 0.6 mg female mouse and all mice in the 1.7, 5, and 15 mg groups died before the end of the study. Final mean body weights of the 0.6 mg groups were significantly less than those of the vehicle controls, and animals in these groups generally lost weight during the study. Clinical findings included skin irritation at the site of application in all dosed groups and clinical signs of toxicity at doses of 0.6 mg or greater. Heart weights of 0.6 mg groups of mice were significantly greater than those of the vehicle controls; thymus weights in these groups were significantly less than those of the vehicle controls. Histopathologic examination of mice dosed with 1.7 mg dicyclohexylcarbodiimide or less revealed lesions of the skin at the site of application including epidermal hyperplasia, epidermal necrosis, and acute or chronic active dermal inflammation. Based on these results, the NOAEL for the test substance was 0.2 mg/animal (ca. 8 mg/kg bw) in male and females mice after 16 days of dermal exposure.

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
June 28, 1994 - July 14, 1994.
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
yes
Remarks:
Treatment 5 days per week for 16 days. No haemathology or biochemistry analysis.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Aldrich Chemical Company (Milwaukee, WI), lot 00929TZ
- Purity: 99.5% (determined by GC)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, under a nitrogen head space, protected from light
- Stability under test conditions: Periodic analyses of the bulk chemical were performed by the study laboratories during the studies using GC: no degradation of the bulk chemical was detected.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Dose formulations were prepared by mixing dicyclohexylcarbodiimide and anhydrous ethanol to give the required concentration.
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Laboratory Animals and Services (Germantown, NY).
- Age at study initiation: 9 weeks old
- Weight at study initiation: males 201-220 g; females 145-148 g
- Fasting period before study: not specified
- Housing: individually housed, in polycarbonate cages (Lab Products, Inc., Seaford, DE) with bedding of heat-treated Sani-chips (R) (P.J. Murphy Forest Products Corp., Montville, NJ), changed weekly.
- Diet: ad libitum; NIH-07 open formula pelleted diet (Zeigler Brothers, Inc.,Gardners, PA),
- Water:ad libitum; Tap water (Washington Suburban Sanitary Commission Potomac Plant)
- Acclimation period: 26 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.22 ± 1.6 ºC
- Humidity (%): 35-65%
- Air changes (per hr): at least 10 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark
Type of coverage:
semiocclusive
Vehicle:
ethanol
Details on exposure:
TEST SITE
- Area of exposure: clipped dorsal skin, from the posterior of the scapulae to the base of the tail
- % coverage: not specified
- Type of wrap if used: not specified.

REMOVAL OF TEST SUBSTANCE: not specified

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0, 0.6, 1.8, 5.1, 15, or 45 mg/animal in 0.3 ml of ethanol.
- Concentration (if solution): 0, 2, 6, 17, 50, or 150 mg dicyclohexylcarbodiimide per ml of ethanol.
- For solids, paste formed: no, test substance was diluted in anhydrous ethanol to give the required concentration

VEHICLE
- Vehicle (if other than water): anhydrous ethanol
- Amount(s) applied (volume or weight with unit): 0.3 ml
- Lot/batch no. (if required): not specified
- Purity: greater than 99.9 %

PREPARATION OF DOSING SOLUTIONS
- A weighed amount of test item was dissolved in absolute ethanol to prepare the highest dose concentration formulation; lower dose formulations were prepared by diluting aliquots of this preparation with absolute ethanol. Dose formulations were prepared twice. The dose formulations were stored in sealed vials under a headspace of inert gas for up to 28 days at room temperature. Stability studies of 0.38, 2, and 7 mg/ml dose formulations of lot 00929TZ were conducted by the study laboratory using GC. Stability was confirmed for up to 35 days for dose formulations stored at room temperature in sealed containers under a nitrogen headspace and for up to 3 hours when exposed to light and air at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulations were prepared twice during the study and were analyzed once by GC . All five dose formulations were within 10% of the target concentrations.
Duration of treatment / exposure:
16 days
Frequency of treatment:
5 days per week
Dose / conc.:
0 other: mg / animal
Dose / conc.:
0.6 other: mg / animal
Dose / conc.:
1.8 other: mg / animal
Dose / conc.:
5.1 other: mg / animal
Dose / conc.:
15 other: mg / animal
Dose / conc.:
45 other: mg / animal
No. of animals per sex per dose:
5 males and 5 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment: animals were distributed randomly into groups of approximately equal initial mean body weights.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical findings (including dermal irritation) were recorded initially, on day 8 and at the end of the studies.

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed initially, on day 8 and at the end of the studies

FOOD CONSUMPTION: No data

FOOD EFFICIENCY: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: No data

CLINICAL CHEMISTRY: No data

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsies were performed on all animals. Organs weighed were the adrenal gland, heart, right kidney, liver, lung, right testis, and thymus.

HISTOPATHOLOGY: Yes
Histopathology was performed on the groups receiving vehicle control, 0.6, 1.8, and 5.1 mg/animal of test item. In addition to gross lesions and tissue masses, the following tissues were examined: brain, kidney, liver, lung, skin, and spinal cord.
Statistics:
Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs of toxicity in 5.1 mg or greater groups included lethargy, ruffled fur, abnormal breathing, coma, and thinness.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Skin irritation at the site of application were seen at doses of 1.8 mg or greater. At the highest doses, the skin lesions at the site of application included epidermal hyperplasia, necrosis, and chronic inflammation in the dermis.
Mortality:
mortality observed, treatment-related
Description (incidence):
All males and females in the 15 and 45 mg groups, four 5.1 mg males, and all 5.1 mg females died before the end of the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Of the surviving groups (0 trhough 1.8 mg/animal), final mean body weights were similar to those of the vehicle controls.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Organ weights of surviving groups of dosed rats were generally similar to those of the vehicle control groups.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histologic examination of rats dosed with 5.1 mg dicyclohexylcarbodiimide or less revealed test article-related lesions of the skin at the site of application including epidermal hyperplasia, epidermal necrosis, or chronic active inflammation in the dermis (data not shown in the report).
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
At doses of 5.1 mg/animal or greater, clinical signs of toxicity were observed, which included lethargy, ruffled fur, abnormal breathing, coma and thinness. At a dose of 5.1 mg/animal, four males and all females died; at higher doses, all animals died before the end of the study.
Key result
Dose descriptor:
NOAEL
Effect level:
1.8 other: mg/animal
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
Remarks on result:
other: ca. 12 mg/kg bw
Dose descriptor:
NOEL
Effect level:
0.6 other: mg / animal
Based on:
test mat.
Sex:
male/female
Basis for effect level:
dermal irritation
Remarks on result:
other: ca. 4 mg/kg bw
Key result
Critical effects observed:
no

Table 1. Survival and body weights

Dose

mg/animal

Survival a

Mean body weight b

(g)

Final weight Relative to controls

(%)

Initial

Final

Change

MALE

0

5/5

207 ± 6

258 ± 9

51 ± 2

 

0.6

5/5

209 ± 5

261 ± 8

52 ± 3

101

1.8

5/5

210 ± 7

254 ± 9

45 ± 3

99

5.1

1/5c

209 ± 6

191

-23

74

15

0/5d

208 ± 5

-

-

 

45

0/5e

211 ± 9

-

-

 

FEMALE

0

5/5

144 ± 4

165 ± 3

21 ± 2

 

0.6

5/5

142 ± 5

164 ± 8

22 ± 3

99

1.8

5/5

143 ± 3

155 ± 5

12 ± 3

94

5.1

0/5f

143 ± 6

-

-

 

15

0/5d

144 ± 4

-

-

 

45

0/5e

141 ± 4

-

-

 

*a: Number of animals surviving at 3 weeks / number initially in group; b: weights and weight changes are given as mean ± standard error. Subsequent calculations are based on animals surviving to the end of the study. Differences from the vehicle control group are not significant by Dunnet's test.; c: day of death: 10, 11, 11, 11; d: day of death: 3 or 4; e: day of death: 2; f: day of death: 9, 9, 9, 10, 11.

Conclusions:
The NOAEL for the test item was determined to be 1.8 mg/animal (ca. 12 mg/kg bw) in male and females rats after 16 days of dermal exposure.
Executive summary:

Groups of five male and five female rats were dermally administered 0.3 ml ethanol containing 0, 0.6, 1.8, 5.1, 15, or 45 mg dicyclohexylcarbodiimide, 5 days per week (16 days of dosing). All males and females in the 15 and 45 mg groups, four 5.1 mg males, and all 5.1 mg females died before the end of the study. Of the surviving groups, final mean body weights were similar to those of the vehicle controls, although the one surviving 5.1 mg male rat lost weight during the study. Clinical findings included skin irritation at hte site of application at doses of 1.8 mg or greater. Histopathologic examination of rats dosed with 5.1 mg dicyclohexylcarbodiimide or less revealed treatment-related lesions of the skin at the site of application including epidermal hyperplasia, epidermal necrosis, or chronic active inflammation in the dermis. Based on these results, the NOAEL for the test item was determined to be 1.8 mg/animal (ca. 12 mg/kg bw) in male and females rats after 16 days of dermal exposure under test conditions.

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
September 20, 1994 - December 19, 1994.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Version / remarks:
The study was comparable to this Guideline with certain differences.
Deviations:
yes
Remarks:
Animals were treated 5 days a week. No ophtalmological examination was performed.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Aldrich Chemical Company (Milwaukee, WI), lot 00929TZ
- Purity: 99.5% (determined by GC)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, under a nitrogen head space, protected from light.
- Stability under test conditions: Periodic analyses of the bulk chemical were performed by the study laboratories during the studies using GC: no degradation of the bulk chemical was detected.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Dose formulations were prepared by mixing dicyclohexylcarbodiimide and anhydrous ethanol to give the required concentration.
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Laboratory Animals and Services (Germantown, NY).
- Age at study initiation: 7 weeks old
- Weight at study initiation: males 115-124 g; females 100-106 g
- Fasting period before study: not specified
- Housing: individually housed
- Diet: ad libitum; NIH-07 open formula pelleted diet (Zeigler Brothers, Inc.,Gardners, PA),
- Water:ad libitum; Tap water (Washington Suburban Sanitary Commission Potomac Plant)
- Acclimation period: 12 days (males) or 13 days (females)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.22 ± 0.16 ºC
- Humidity (%): 35-65%
- Air changes (per hr): at least 10 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark
Type of coverage:
not specified
Vehicle:
ethanol
Details on exposure:
TEST SITE
- Area of exposure: clipped dorsal skin, from the posterior of the scapulae to the base of the tail
- % coverage: not specified
- Type of wrap if used: not specified

REMOVAL OF TEST SUBSTANCE: not specified

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0, 0.75, 1.5, 3, 6, or 12 mg/kg body weight in 0.5 ml/kg ethanol
- For solids, paste formed: no, test substance was diluted in anhydrous ethanol to give the required concentration

VEHICLE
- Vehicle (if other than water): anhydrous ethanol
- Amount(s) applied (volume or weight with unit): 0.5 ml/kg
- Lot/batch no. (if required): not specified
- Purity: greater than 99.9 %

PREPARATION OF DOSING SOLUTIONS
- A weighed amount of test item was dissolved in absolute ethanol to prepare the highest dose concentration formulation; lower dose formulations were prepared by diluting aliquots of this preparation with absolute ethanol. Dose formulations were prepared at least every 2 weeks . The dose formulations were stored in sealed vials under a headspace of inert gas for up to 28 days at room temperature. Stability studies of 0.38, 2, and 7 mg/ml dose formulations of lot 00929TZ were conducted by the study laboratory using GC. Stability was confirmed for up to 35 days for dose formulations stored at room temperature in sealed containers under a nitrogen headspace and for up to 3 hours when exposed to light and air at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Periodic analyses of the dose formulations of dicyclohexylcarbodiimide were conducted by the study laboratories using GC. The dose formulations were analyzed at the beginning, midpoint, and end of the studies; animal room samples of these dose formulations were also analyzed. Of the dose formulations analyzed, all 15 were within 10% of the target concentration; all 13 animal room samples were within 10% of the target concentrations.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
5 days per week
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control group
Dose / conc.:
0.75 mg/kg bw/day (nominal)
Dose / conc.:
1.5 mg/kg bw/day (nominal)
Dose / conc.:
3 mg/kg bw/day (nominal)
Dose / conc.:
6 mg/kg bw/day (nominal)
Dose / conc.:
12 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 males and 10 females per dose per group (core study and satellites)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a previous 16-days study was found that at the dose of 1.8 mg/animal could be seen clinical signs of toxicity. Doses greater than this dose resulted in mortality. So this dose was selected as the highest dose, which approximately corresponded to 12 mg/kg body weight.

Dose selection rationale: In a previous 16-day study, it was found that 1.8 mg/animal resulted in clinical signs of toxicity and greater doses resulted in mortality. Therefore, this dose was selected as the highest dose, which approximately corresponds to 12 mg/kg body weight.
- Rationale for selecting satellite groups: groups of 10 males and 10 females received the same doses as the core study animals during 22 days (clinical pathology rats), and blood was collected on days 3 and 22; no necropsy was performed on these animals. No justification provided.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical findings (including dermal irritation) recorded initially, weekly, and at the end of the studies.

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed initially, weekly, and at the end of the studies.

FOOD CONSUMPTION: No data

FOOD EFFICIENCY: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: from clinical pathology study rats, on days 3 and 22; from core study rats, at study termination.
- Anaesthetic used for blood collection: Yes, animals were anesthetized with a 70% CO2/30% O2 mixture.
- Animals fasted: No data
- How many animals: all animals of each group and study.
- Parameters examined: erythrocyte, reticulocyte, nucleated erythrocyte, platelet and leukocyte counts, automated and manual hematocrit values, hemoglobin concentration, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration. Differential leukocyte counts and erythrocyte and leukocyte morphologies were determined too.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: from clinical pathology study rats on days 3 and 22; from core study rats at study termination.
- Anaesthetic used for blood collection: Yes, animals were anesthetized with a 70% CO2/30% O2 mixture.
- Animals fasted: No data
- How many animals: all animals of each group and study.
- Parameters checked: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile acids.

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

OTHER: SPERM MOTILITY AND VAGINAL CITOLOGY
- Time schedule for collection: at the end of the core study in males, 12 consecutive days prior to the end in females
- How many animals: animals from vehicle control, 1.5, 3 and 6 mg/kg core study groups (males and females)
- Parameters checked: sperm motility evaluation: spermatid heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility and concentration. The left cauda, left epididymis, and left testis were weighed. Vaginal citology evaluations: relative numbers of leukocytes, nucleated epithelial cells, and large squamous epithelial cells were determined and used to ascertain estrous cycle stage (i.e., diestrus, proestrus, estrus, and metestrus) and to obtain the percentage of time spent in the various estrous cycle stages and the estrous cycle length.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes.
Necropsies were performed on all core study animals. Organs weighed were the heart, right kidney, liver, lung, right testis, and thymus.

HISTOPATHOLOGY: Yes.
Complete histopathology was performed on vehicle control, 6, and 12 mg/kg core study rats. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, heart with aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, uterus, and Zymbal’s gland. In addition, the bone marrow, spleen of male rats and skin were examined in all remaining dosed groups.
Statistics:
Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Hematology, clinical chemistry, spermatid, and epididymal spermatozoal data, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) (as modified by Williams, 1986) and Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test). Prior to statistical analysis, extreme values identified by the outlier test of Dixon and Massey (1957) were examined by NTP personnel, and implausible values were eliminated from the analysis. Average severity values were analyzed for significance with the Mann-Whitney U test (Hollander and Wolfe, 1973). Because vaginal cytology data are proportions (the proportion of the observation period that an animal was in a given estrous stage), an arcsine transformation was used to bring the data into closer conformance with a normality assumption. Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across exposure concentrations.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All 12 mg/kg males and females showed lethargy, tremors, ataxia, ruffled fur, a thin appearance, nasal/eye discharge, and head tilt.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Skin irrititation at the site of application was observed in all groups at doses of 3 mg/kg or higher.
Mortality:
mortality observed, treatment-related
Description (incidence):
All 12 mg/kg male and female rats died or were found moribund and sacrificed prior to day 45.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Final mean body weight and body weight gain of 6 mg/kg males were significantly less than those of the vehicle controls.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
On Day 3: increased neutrophil counts were seen in 6 mg/kg or greater (males and females).
On Day 22: increases in leukocyte, neutrophil, and band neutrophil counts occurred in 3 mg/kg males and 6 mg/kg or greater males and females; monocyte and eosinophil counts were also increased. Minimal decreases in hematocrit, hemoglobin and mean cell volume values and erythrocyte counts occurred in 6 mg/kg males and 12 mg/kg males and females.
On Week 13: increased neutrophil counts were observed only in 6 mg/kg females. Minimal decreases in hematocrit, hemoglobin and mean cell volume values and erythrocyte counts occurred in 6 mg/kg females too.
The leukocyte changes and the decreases in the erythron would be consistent with the progression of an inflammatory process from the acute to chronic phases and would be a response consistent with the development of skin necrosis and chronic active inflammation observed morphologically.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
On Day 22: small decreases in albumin concentration occurred in 3 mg/kg or greater males and 6 mg/kg or greater females.
On Week 13: small decreases in albumin concentration occurred in 3 mg/kg or greater males and 6 mg/kg or greater females too.
The albumin decrease could be a secondary response to chronic inflammation.
Small, transient increases in serum alanine aminotransferase (ALT) activity occurred on days 3 and 22 in treated males and females but not on week 13. As no other marker of hepatocellular injury was affected, it was considered a transient induction of ALT production by the liver.
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute weights of the heart, kidney, and liver of 3 and 6 mg/kg females were generally greater than those of the vehicle controls.
Relative weights of the heart and kidney of 3 and 6 mg/kg males were significantly greater than those of the vehicle controls.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Gross lesions of the skin at the site of application were detected in all animals dosed with 3 mg/kg or greater.
Epidermal hyperplasia occurred in all treated groups, and the incidences of this lesion were significantly increased in 3 mg/kg or greater males and 1.5 mg/kg or greater females; the incidences and severities of the lesion increased in a dose-related fashion.
Chronic active inflammation occurred in most of the dosed groups, and the incidences of this lesion were significantly increased in 6 and 12 mg/kg males and 1.5 mg/kg or greater females. The incidences of sebaceous gland hyperplasia were significantly increased in 3 mg/kg or greater males and 6 and 12 mg/kg females, and the incidence of epidermal necrosis was significantly increased in male rats administered 12 mg/kg. Incidences of sebaceous gland hyperplasia were considered to be a secondary effect of inflammation rather than a direct compound effect.
On the surface of the skin there was necrosis with marked acute inflammation consisting of polymorphonuclear cells with edema and coagulated serum admixed with cell debris (crusts). Deeper in the dermis, mononuclear cells mixed with proliferating fibroblasts were present, which suggested a more chronic feature of the inflammatory lesion.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The skin lesions were microscopically similar to the same lesions seen at the site of aplication (gross pathology) although most were milder in severity: epidermal necrosis, chronic active inflammation and epidermal hyperplasia.
Slightly increased incidences of minimal hematopoietic cell proliferation occurred in the spleen of all dosed groups of male rats; this finding was considered secondary to the skin lesions at the site of application.
The incidences of diffuse bone marrow hyperplasia in 12 mg/kg males and females were significantly greater than those in the vehicle controls. This finding was also considered secondary to the skin lesions at the site of application.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Reproductive tissue parameters in males or vaginal cytology parameters in females were not affected.
Key result
Dose descriptor:
NOAEL
Effect level:
3 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
mortality
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
6 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
Key result
Critical effects observed:
no

Table 1. Survival and body weights

Dose

mg/kg bw

Survival

Mean body weight

Final weight Relative to controls

(%)

Initial

Final

Change

MALE

0

10/10

121 ± 3

330 ± 5

209 ± 5

 

0.75

10/10

120 ± 2

337 ± 6

216 ± 6

102

1.5

10/10

120 ± 3

334 ± 6

213 ± 5

101

3

10/10

120 ± 3

325 ± 7

205 ± 6

99

6

10/10

118 ± 2

300 ± 4*

182 ± 3*

91

12

0/10

118 ± 3

-

-

 

FEMALE

0

10/10

102 ± 2

186 ± 3

84 ± 2

 

0.6

10/10

102 ± 2

191 ± 3

89 ± 2

103

1.8

10/10

104 ± 2

190 ±4

86 ± 3

102

5.1

10/10

104 ± 2

193 ± 3

89 ± 2

104

15

10/10

103 ± 2

192 ± 4

89 ± 3

103

45

0/10

103 ± 2

-

-

 

* significantly different

Table 2. Incidences of Nonneoplastic lesions of the skin (site of application)

 

Vehicle control

0.75 mg/kg

1.5 mg/kg

3 mg/kg

6 mg/kg

12 mg/kg

MALE

 

Number examined microscopically

10

10

10

10

10

10

Epidermis, hyperplasia

0

1

3

9*

10*

10*

Epidermis, necrosis

0

0

0

0

0

6

Sebaceous gland hyperplasia

0

1

1

4

7

9

Inflammation, chronic active

0

1

0

1

7

9

FEMALE

 

Number examined microscopically

10

10

10

10

10

10

Epidermis, hyperplasia

0

3

7*

7*

10*

10*

Epidermis, necrosis

0

0

0

0

1

3

Sebaceous gland hyperplasia

0

1

0

0

5*

7*

Inflammation, chronic active

0

0

4*

4*

9*

9*

* signficantly different

Table 3. Hematology and clinical chemistry parameters found to be altered in males

MALE

Vehicle control

0.75 mg/kg

1.5 mg/kg

3 mg/kg

6 mg/kg

12 mg/kg

Hematology

 

Automated hematocrit

Day 3

Day 22

Week 13

40.9±0.4

45.9±0.4

44.1±0.1

41.1±0.7

45.0±0.5

43.9±0.7

42.2±0.4

45.4±0.6

44.4±0.4

41.2±0.6

44.6±0.4

44.5±0.3

40.4±0.4

43.7±0.3*

44.5±.04

41.5±0.8

44.5±0.5*

-

Hemoglobin (g/dl)

Day 3

Day 22

Week 13

14.6±0.1

16.2±0.2

15.7±0.1

14.7±0.2

16.1±0.2

15.5±0.2

14.8±0.1

16.1±0.1

15.6±0.1

14.7±0.2

15.9±0.1

15.7±0.1

14.5±0.2

15.5±0.1*

15.6±0.1

14.6±0.2

15.6±0.2

-

Mean cell volume

Day 3

Day 22

Week 13

62.5±0.4

59.9±0.4

50.7±0.2

61.7±0.4

59.6±0.2

50.3±0.2

62.8±0.3

59.8±0.3

50.4±0.2

61.9±0.4

59.2±0.5

50.7±0.1

62.2±0.5

59.3±0.3

50.4±0.1

62.5±0.4

58.4±0.2*

-

Mean cell hemoglobin (pg)

Day 3

Day 22

Week 13

22.3±0.1

21.1±0.2

18.0±0.1

22.1±0.1

21.3±0.1

17.8±0.1

22.0±0.1

21.2±0.2

17.7±0.1

22.1±0.2

21.1±0.1

17.9±0.1

22.3±0.1

21.0±0.1

17.7±0.1

22.0±0.2

20.4±0.1*

-

Leukocytes (103/µl)

Day 3

Day 22

Week 13

10.85±0.2

9.39±0.2

8.26±0.6

10.06±0.34

8.76±0.6

7.88±0.4

9.87±0.3

9.88±0.7

7.20±0.6

10.24±0.5

10.80±0.8

9.19±0.9

10.82±0.3

19.94±1.5*

9.63±0.5

12.35±0.3

24.65±1.8*

-

Segmented neutrophils (103/µl)

Day 3

Day 22

Week 13

1.05±0.1

0.96±0.1

1.18±0.1

0.97±0.1

1.24±0.1

1.14±0.2

1.16±0.1

1.15±0.1

1.02±0.2

0.99±0.1

2.14±0.3*

1.87±0.6

1.74±0.2

9.71±1.0*

1.80±0.2

2.47±0.2*

14.24±1.2*

-

Bands (103/µl)

Day 3

Day 22

Week 13

0.00±0.0

0.00±0.0

0.00±0.0

0.00±0.0

0.02±0.01

0.01±0.01

0.01±0.01

0.03±0.01

0.02±0.02

0.00±0.0

0.13±0.05*

0.01±0.01

0.01±0.10

0.73±0.12*

0.02±0.02

0.05±0.02

0.75±0.06*

0.00±0.0

Monocytes (103/µl)

Day 3

Day 22

Week 13

0.22±0.02

0.22±0.03

0.19±0.44

0.12±0.03

0.24±0.04

0.22±0.07

0.25±0.04

0.24±0.04

0.15±0.06

0.16±0.06

0.22±0.05

0.16±0.04

0.23±0.05

0.58±0.21

0.19±0.05

0.29±0.06

0.62±0.11*

-

Eosinophils (103/µl)

Day 3

Day 22

Week 13

0.0.3±0.02

0.04±0.02

0.03±0.02

0.03±0.02

0.04±0.01

0.08±0.03

0.06±0.22

0.04±0.22

0.11±0.04

0.02±0.01

0.06±0.03

0.02±0.02

0.03±0.02

0.07±0.04

0.08±0.04

0.04±0.02

0.24±0.06*

-

Clinical chemistry

 

Total protein

Day 3

Day 22

Week 13

5.7±0.1

6.2±0.1

6.9±0.1

5.7±0.1

6.1±0.1

6.9±0.1

5.8±0.1

6.0±0.1

7.1±0.1

5.8±0.1

6.1±0.1

6.6±0.3

5.6±0.0

6.0±0.1

6.8±0.1

5.6±0.1

5.8±0.1*

-

Albumin

Day 3

Day 22

Week 13

4.6±0.1

4.8±0.0

5.0±0.1

4.7±0.1

4.8±0.0

4.9±0.1

4.7±0.1

4.7±0.1

5.0±0.0

4.7±0.0

4.6±0.1*

4.5±0.2

4.6±0.0

4.3±0.0*

4.7±0.1*

4.5±0.0

4.0±0.1

-

Alanine aminotransferase (IU/l)

Day 3

Day 22

Week 13

 

 

51±1

53±2

57±3

 

 

48±1

54±1

60±2

 

 

53±1

52±2

63±3

 

 

53±1

62±1*

63±5

 

 

61±2*

68±3*

68±4

 

 

76±3*

77±4*

-

Alkaline phosphatase (IU/l)

Day 3

Day 22

Week 13

762±26

520±9

262±5

752±20

532±10

276±12

761±22

501±13

275±3

767±11

527±11

259±20

736±16

462±12*

290±6*

728±12

442±15*

-

Table 4. Hematology and clinical chemistry parameters found to be altered in females

FEMALE

Vehicle control

0.75 mg/kg

1.5 mg/kg

3 mg/kg

6 mg/kg

12 mg/kg

Hematology

 

Automated hematocrit

Day 3

Day 22

Week 13

42.8±0.6

46.7±0.4

45.6±0.3

43.2±0.4

46.9±0.3

44.6±0.6

43.3±0.4

47.2±0.3

45.2±0.4

43.3±0.5

47.1±0.8

43.3±0.4*

43.7±0.3

45.5±0.3

43.0±.0.5*

43.3±0.5

46.1±0.5

-

Hemoglobin (g/dl)

Day 3

Day 22

Week 13

15.2±0.2

16.2±0.2

15.8±0.1

15.3±0.1

16.5±0.1

15.6±0.2

15.3±0.1

16.4±0.1

15.8±0.1

15.3±0.1

16.3±0.3

15.3±0.2*

15.5±0.1

15.9±0.1

15.1±0.2*

15.3±0.2

15.7±0.1*

-

Erythrocites (106/µl)

Day 3

Day 22

Week 13

6.94±0.09

7.60±0.06

8.08±0.06

7.02±0.06

7.64±0.05

7.92±0.10

7.03±0.07

7.66±0.06

8.03±0.06

7.03±0.08

7.69±0.13

7.68±0.07*

7.09±0.07

7.52±0.05

7.74±0.09*

7.01±0.09

7.67±0.12

-

Mean cell volume

Day 3

Day 22

Week 13

61.6±0.2

61.4±0.3

56.3±0.1

61.5±0.1

61.4±0.2

56.3±0.1

61.6±0.2

61.7±0.2

56.3±0.1

61.6±0.2

61.2±0.2

56.4±0.1

61.6±0.2

60.6±0.2*

55.6±0.2*

61.8±0.3

60.1±0.3*

-

Mean cell hemoglobin (pg)

Day 3

Day 22

Week 13

21.9±0.1

21.3±0.1

19.6±0.1

21.7±0.1

21.6±0.1

19.7±0.1

21.8±0.1

21.5±0.1

19.7±0.1

21.7±0.1

21.2±0.1

19.9±0.1

21.8±0.1

21.1±0.1

19.5±0.1

21.9±0.1

20.±0.2*

-

Platelets (103/µl)

Day 3

Day 22

Week 13

785.9±18.3

731.2±21.5

649.0±25.6

797.6±18.7

786.7±19.0

653.7±14.2

788.3±21.5

747.8±22.5

631.7±29.4

781.4±26.3

637.8±36.3

710.4±26.7*

833.7±32.6

701.1±16.4

718.9±25.1*

808.1±19.5

707.3±21.4

-

Leukocytes (103/µl)

Day 3

Day 22

Week 13

10.79±0.5

7.62±0.2

6.60±0.3

11.49±0.3

8.76±0.6

7.88±0.4

9.87±0.3

9.88±0.7

7.20±0.6

10.24±0.5

10.80±0.8

9.19±0.9

10.82±0.3

19.94±1.5*

9.63±0.5

12.35±0.3

24.65±1.8*

-

Segmented neutrophils (103/µl)

Day 3

Day 22

Week 13

1.05±0.1

0.96±0.1

1.18±0.1

0.97±0.1

1.24±0.1

1.14±0.2

1.16±0.1

1.15±0.1

1.02±0.2

0.99±0.1

2.14±0.3*

1.87±0.6

1.74±0.2

9.71±1.0*

1.80±0.2

2.47±0.2*

14.24±1.2*

-

Bands (103/µl)

Day 3

Day 22

Week 13

0.00±0.0

0.00±0.0

0.00±0.0

0.00±0.0

0.02±0.01

0.01±0.01

0.01±0.01

0.03±0.01

0.02±0.02

0.00±0.0

0.13±0.05*

0.01±0.01

0.01±0.10

0.73±0.12*

0.02±0.02

0.05±0.02

0.75±0.06*

0.00±0.0

Monocytes (103/µl)

Day 3

Day 22

Week 13

0.22±0.02

0.22±0.03

0.19±0.44

0.12±0.03

0.24±0.04

0.22±0.07

0.25±0.04

0.24±0.04

0.15±0.06

0.16±0.06

0.22±0.05

0.16±0.04

0.23±0.05

0.58±0.21

0.19±0.05

0.29±0.06

0.62±0.11*

-

Eosinophils (103/µl)

Day 3

Day 22

Week 13

0.0.3±0.02

0.04±0.02

0.03±0.02

0.03±0.02

0.04±0.01

0.08±0.03

0.06±0.22

0.04±0.22

0.11±0.04

0.02±0.01

0.06±0.03

0.02±0.02

0.03±0.02

0.07±0.04

0.08±0.04

0.04±0.02

0.24±0.06*

-

Clinical chemistry

 

Total protein

Day 3

Day 22

Week 13

5.7±0.1

6.2±0.1

6.9±0.1

5.7±0.1

6.1±0.1

6.9±0.1

5.8±0.1

6.0±0.1

7.1±0.1

5.8±0.1

6.1±0.1

6.6±0.3

5.6±0.0

6.0±0.1

6.8±0.1

5.6±0.1

5.8±0.1*

-

Albumin

Day 3

Day 22

Week 13

4.6±0.1

4.8±0.0

5.0±0.1

4.7±0.1

4.8±0.0

4.9±0.1

4.7±0.1

4.7±0.1

5.0±0.0

4.7±0.0

4.6±0.1*

4.5±0.2

4.6±0.0

4.3±0.0*

4.7±0.1*

4.5±0.0

4.0±0.1

-

Alanine aminotransferase (IU/l)

Day 3

Day 22

Week 13

 

 

51±1

53±2

57±3

 

 

48±1

54±1

60±2

 

 

53±1

52±2

63±3

 

 

53±1

62±1*

63±5

 

 

61±2*

68±3*

68±4

 

 

76±3*

77±4*

-

Alkaline phosphatase (IU/l)

Day 3

Day 22

Week 13

762±26

520±9

262±5

752±20

532±10

276±12

761±22

501±13

275±3

767±11

527±11

259±20

736±16

462±12*

290±6*

728±12

442±15*

-

Conclusions:
The NOAEL for the subchronic dermal toxicity of the test item in rats was 3 mg/kg bw in males and 6 mg/kg bw in females.
Executive summary:

In a 13 -week subchronic dermal toxicity study performed on the test item by the NTP (GLP study), groups of 10 male and 10 female F-344 rats were dermally administered 0, 0.75, 1.5, 3, 6 or 12 mg/kg bw of test item in ethanol (0.5 mL/kg), 5 days a week for 13 weeks. All 12 mg/kg male and female core study rats died or were found moribund and sacrificed prior to day 45. Final mean body weight and body weight gain of 6 mg/kg males were significantly less than those of the vehicle controls. The predominant clinical pathology changes suggest a secondary, treatmentrelated inflammatory leukogram and minimal decreased erythron of chronic inflammation that would be consistent with necrosis and chronic active inflammation of the skin. Significantly increased incidences of skin lesions at the site of application included epidermal hyperplasia in 3 mg/kg or greater males and 1.5 mg/kg or greater females, chronic active inflammation in 6 and 12 mg/kg males and 1.5 mg/kg or greater females, and epidermal necrosis in 12 mg/kg males. The incidences and severities of epidermal hyperplasia increased in a dose-related manner in both sexes of rats . Based on these results, the NOAEL for the subchronic dermal toxicity of the test item in rats was 3 mg/kg bw in males and 6 mg/kg bw in females.

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
September 22, 1994 - December 23, 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
Animals were treated 5 days a week. No ophtalmological or clinical biochemistry analysis was performed.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Aldrich Chemical Company (Milwaukee, WI), lot 00929TZ
- Purity: 99.5% (determined by GC)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, under a nitrogen head space, protected from light.
- Stability under test conditions: Periodic analyses of the bulk chemical were performed by the study laboratories during the studies using GC: no degradation of the bulk chemical was detected.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Dose formulations were prepared by mixing the test item and anhydrous ethanol to give the required concentration.
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Laboratory Animals and Services (Germantown, NY).
- Age at study initiation: 7 weeks old
- Weight at study initiation: males 24.2-24.9 g; females 19.1-20.3 g
- Fasting period before study: not specified
- Housing: individually housed
- Diet: ad libitum; NIH-07 open formula pelleted diet (Zeigler Brothers, Inc.,Gardners, PA).
- Water:ad libitum; Tap water (Washington Suburban Sanitary Commission Potomac Plant).
- Acclimation period: 14 days (males) or 15 days (females)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.22 ± 0.16 ºC
- Humidity (%): 35-65%
- Air changes (per hr): at least 10 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark
Type of coverage:
not specified
Vehicle:
ethanol
Details on exposure:
TEST SITE
- Area of exposure: clipped dorsal skin, from the posterior of the scapulae to the base of the tail
- % coverage: not specified
- Type of wrap if used: not specified

REMOVAL OF TEST SUBSTANCE: not specified

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0, 1.5, 3, 6, 12, or 24 mg/kg body weight in 2 ml/kg ethanol.
- For solids, paste formed: no, test substance was diluted in anhydrous ethanol to give the required concentration.

VEHICLE
- Vehicle (if other than water): anhydrous ethanol
- Amount(s) applied (volume or weight with unit): 2 ml/kg
- Lot/batch no. (if required): not specified
- Purity: greater than 99.9 %

PREPARATION OF DOSING SOLUTIONS
- A weighed amount of test item was dissolved in absolute ethanol to prepare the highest dose concentration formulation; lower dose formulations were prepared by diluting aliquots of this preparation with absolute ethanol. Dose formulations were prepared at least every 2 weeks . The dose formulations were stored in sealed vials under a headspace of inert gas for up to 28 days at room temperature. Stability studies of 0.38, 2, and 7 mg/ml dose formulations of lot 00929TZ were conducted by the study laboratory using GC. Stability was confirmed for up to 35 days for dose formulations stored at room temperature in sealed containers under a nitrogen headspace and for up to 3 hours when exposed to light and air at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Periodic analyses of the dose formulations of dicyclohexylcarbodiimide were conducted by the study laboratories using GC. The dose formulations were analyzed at the beginning, midpoint, and end of the studies; animal room samples of these dose formulations were also analyzed. Of the dose formulations analyzed, all 15 were within 10% of the target concentration; all 13 animal room samples were within 10% of the target concentrations.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
5 doses per week
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control group
Dose / conc.:
1.5 mg/kg bw/day (nominal)
Dose / conc.:
3 mg/kg bw/day (nominal)
Dose / conc.:
6 mg/kg bw/day (nominal)
Dose / conc.:
12 mg/kg bw/day (nominal)
Dose / conc.:
24 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 males and 10 females per dose per group (core study)
Groups of 10 male and 10 female clinical pathology rats were given the same doses for 22 days.
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: In a previous 16-day study, it was found that doses greater than 1.8 mg/animal resulted in mortality. Therefore, this dose was selected as the highest dose, which approximately corresponds to 12 mg/kg body weight.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical findings (including dermal irritation) recorded initially, weekly, and at the end of the studies.

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed initially, weekly, and at the end of the studies.

FOOD CONSUMPTION: No data

FOOD EFFICIENCY: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: from clinical pathology study rats on days 3 and 22; from core study rats at study termination.
- Anaesthetic used for blood collection: Yes, animals were anesthetized with a 70% CO2/30% O2 mixture.
- Animals fasted: No data
- How many animals: all animals of each group and study.
- Parameters examined: erythrocyte, reticulocyte, nucleated erythrocyte, platelet and leukocyte counts, automated and manual hematocrit values, hemoglobin concentration, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration. Differential leukocyte counts and erythrocyte and leukocyte morphologies were determined too.

CLINICAL CHEMISTRY: No data

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

OTHER: SPERM MOTILITY AND VAGINAL CITOLOGY
- Time schedule for collection: at the end of the core study in males, 12 consecutive days prior to the end in females
- How many animals: animals from vehicle control, 1.5, 3 and 6 mg/kg core study groups (males and females)
- Parameters checked: sperm motility evaluation: spermatid heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility and concentration. The left cauda, left epididymis, and left testis were weighed. Vaginal citology evaluations: relative numbers of leukocytes, nucleated epithelial cells, and large squamous epithelial cells were determined and used to ascertain estrous cycle stage (i.e., diestrus, proestrus, estrus, and metestrus) and to obtain the percentage of time spent in the various estrous cycle stages and the estrous cycle length.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes.
Necropsies were performed on all core study animals. Organs weighed were the heart, right kidney, liver, lung, right testis, and thymus.

HISTOPATHOLOGY: Yes.
Complete histopathology was performed on vehicle control, 6, and 12 mg/kg core study rats. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, heart with aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, uterus, and Zymbal’s gland. In addition, the bone marrow, spleen of male rats and skin were examined in all remaining dosed groups.
Statistics:
Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Hematology, clinical chemistry, spermatid, and epididymal spermatozoal data, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) (as modified by Williams, 1986) and Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test). Prior to statistical analysis, extreme values identified by the outlier test of Dixon and Massey (1957) were examined by NTP personnel, and implausible values were eliminated from the analysis. Average severity values were analyzed for significance with the Mann-Whitney U test (Hollander and Wolfe, 1973). Because vaginal cytology data are proportions (the proportion of the observation period that an animal was in a given estrous stage), an arcsine transformation was used to bring the data into closer conformance with a normality assumption. Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across exposure concentrations.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Findings noted in 24 mg/kg animals included ataxia, lethargy, thinness, abnormal posture, ruffled fur, and head tilt.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
With the exception of two males and six females in the 24 mg/kg group, all animals administered 6 mg/kg or greater had skin irritation at the site of application.
Mortality:
mortality observed, treatment-related
Description (incidence):
All 24 mg/kg male and female mice died or were found (Table 6 and Figure 2). With the exception of two males moribund and sacrificed prior to day 16.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Final mean body weights of 6 and 12 mg/kg males and mean body weight gains of 6 and 12 mg/kg males and females were significantly less than those of the vehicle controls.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Evidence of an inflammatory process was indicated by increased leukocytes at week 13. The inflammatory leukogram, characterized by increased leukocyte and neutrophil counts, occurred in 12 mg/kg males and females. These leukocyte changes would be a response consistent with the development of the skin necrosis and chronic active inflammation observed morphologically. Mminimal decreases in the erythron (<10%), characterized by decreases in hematocrit and hemoglobin values and erythrocyte counts, occurred in 1.5 mg/kg or greater males and 12 mg/kg females. This minimal decrease in erythron would be consistent with the erythron decreases that can develop in response to chronic inflammation and would be considered a secondary response to skin inflammation.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute and relative lung weights of 3 mg/kg or greater male mice were generally less than those of the vehicle controls, and absolute and relative heart and liver weights of 6 and 12 mg/kg female mice were generally greater than those of the vehicle controls. The weight of the epididymis significantly decreased in 6 and 12 mg/kg males.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Chemical-related gross lesions of the skin at the site of application consisted of pink lesions, tan crusts, or areas of mottled irritation observed in all 12 mg/kg males and females, eight 24 mg/kg males, and five 24 mg/kg females. At the site of application, the incidences of epidermal hyperplasia were significantly increased in all dosed groups of males and females except those administered 24 mg/kg (not increased in this group because of early mortality). The incidences of chronic active inflammation were significantly increased in all dosed groups except 1.5 mg/kg females, and the incidences of epidermal necrosis were significantly increased in 24 mg/kg males and females. Epidermal necrosis also occurred in several animals administered 6 or 12 mg/kg. The severities of epidermal hyperplasia and chronic active inflammation generally increased in a doserelated fashion for both sexes.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Epidermal hyperplasia and inflammation and/or chronic active inflammation occurred in some of the dosed male and female mice. Incidences of hematopoietic cell proliferation in the spleen were increased in most dosed groups of males and females. Increased hematopoiesis can occur with inflammatory conditions due to the physiological need for more white blood cells. Therefore, this finding was considered secondary to the skin lesions at the site of application. Other nonneoplastic lesions occurred primarily in the 24 mg/kg groups and included bilateral atrophy of the preputial and clitoral glands, atrophy of the thymus, and hemorrhage of the brain. These lesions occurred in mice that died prior to 16 days on study and were considered secondary effects of inanition and moribundity.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The weight of the epididymis significantly decreased in 6 and 12 mg/kg males; significantly decreased epididymal spermatozoal motility in 6 mg/kg males was observed.
Vaginal cytology parameters in female mice were not affected by treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
3 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
Key result
Critical effects observed:
no

Table 1. Survival and body weights

Dose level (mg/kg)

Survival

Mean Body Weight

 

Initial

Final

Change

Final Weight

Relative to Controls

(%)

MALE

0

10/10

24.8 ± 0.6

33.7 ± 0.6

8.9 ± 0.4

 

1.5

10/10

24.8 ± 0.3

33.1 ± 0.7

8.3 ± 0.4

98

3

10/10

24.7 ± 0.4

33.0 ± 0.5

8.3 ± 0.2

98

6

10/10

24.9 ± 0.4

31.5 ± 0.5

6.6 ± 0.3

93

12

10/10

24.2 ± 0.5

30.4 ± 0.5

6.2 ± 0.4

90

24

0/10

24.5 ± 0.5

-

-

-

FEMALE

0

10/10

19.6 ± 0.3

28.8 ± 0.9

9.2 ± 0.7

 

1.5

10/10

19.1 ± 0.5

28.3 ± 1.1

9.2 ± 0.8

98

3

10/10

19.1 ± 0.4

27.8 ± 0.5

8.7 ± 0.5

97

6

10/10

20.3 ± 0.2

27.7 ± 0.3

7.3 ± 0.3

96

12

10/10

19.5 ± 0.3

26.5 ± 0.4

7.0 ± 0.3

92

24

0/10

19.5 ± 0.4

-

-

-

Table 2. Nonneoplastic Lestions of the Skin (Site of Application)

 

Vehicle

control

1.5

mg/kg

3

mg/kg

6

mg/kg

12

mg/kg

24

mg/kg

Male

No. Examined Microscopically

10

10

10

10

10

10

Epidermis, Hyperplasia

0

7

10

10

10

0

Epidermis, Necrosis

1

0

0

0

2

10

Inflammation, Chronic Active

0

4

10

10

10

10

Female

No. Examined Microscopically

10

10

10

10

10

10

Epidermis, Hyperplasia

0

5

9

10

10

3

Epidermis, Necrosis

0

0

0

2

3

10

Inflammation, Chronic Active

0

1

9

10

10

10

Table 3. Hematology Data for B6C3F1 Mice in the 13-Week Dermal Study.

 

Vehicle

Control

1.5

mg/kg

3

mg/kg

6

mg/kg

12

mg/kg

Male

 (n)

10

10

10

10

10

Automated

hematocrit (%)

52.9 ± 0.7

52.0 ± 0.7

51.1 ± 0.8

50.0 ± 0.6*

47.8 ± 0.6**

Manual

hematocrit (%)

53.0 ± 0.6

51.4 ± 0.5*

50.4 ± 0.6**

50.2 ± 0.6**

48.3 ± 0.7**

Hemoglobin

(g/dL)

16.9 ± 0.1

16.6 ± 0.2

16.4 ± 0.2*

16.1 ± 0.1**

15.3 ± 0.2**

Erythrocytes

(106/µL)

10.39 ± 0.13

10.23 ± 0.15

10.03 ± 0.15

9.89 ± 0.15

9.44 ± 0.11**

Reticulocytes

(106/µL)

0.20 ± 0.01

0.18 ± 0.01

0.19 ± 0.01

0.18 ± 0.01

0.18 ± 0.02

Nucleated erythrocytes

(103/µL)

0.10 ± 0.10

0.10 ± 0.10

0.00 ± 0.00

0.00 ± 0.00

0.00 ± 0.00

Mean cell volumen

(fL)

51.0 ± 0.2

50.9 ± 0.2

50.9 ± 0.2

50.6 ± 0.3

50.6 ± 0.2

Mean cell hemoglobin

(pg)

16.3 ± 0.2

16.3 ± 0.1

16.3 ± 0.1

16.3 ± 0.1

16.2 ± 0.1

Mean cell hemoglobin

concentration

(g/dL)

31.9 ± 0.3

32.0 ± 0.2

32.1 ± 0.2

32.2 ± 0.2

32.0 ± 0.2

Platelets

(103/µL)

637.2 ± 25.7

708.9 ± 36.1

664.6 ± 26.3

737.2 ± 15.0*

830.8 ± 25.3**

Leukocytes

(103/µL)

4.73 ± 0.45

5.98 ± 0.64

4.99 ± 0.39

6.11 ± 0.69

7.80 ± 0.63**

Segmented neutrophils

(103/µL)

0.41 ± 0.05

0.88 ± 0.25

0.52 ± 0.10

0.74 ± 0.17

1.27 ± 0.22**

Bands

(103/µL)

0.00 ± 0.00

0.00 ± 0.00

0.00 ± 0.00

0.00 ± 0.00

0.02 ± 0.01*

Lymphocytes

(103/µL)

4.24 ± 0.41

4.99 ± 0.46

4.38 ± 0.32

5.22 ± 0.55

6.34 ± 0.51**

Monocytes

(103/µL)

0.05 ± 0.01

0.08 ± 0.02

0.06 ± 0.02

0.11 ± 0.03

0.16 ± 0.05

Eosinophils

(103/µL)

0.03 ± 0.01

0.03 ± 0.01

0.03 ± 0.01

0.04 ± 0.01

0.02 ± 0.01

Female

Automated

hematocrit (%)

50.9 ± 0.7

50.8 ± 0.6

50.7 ± 0.6

48.7 ± 0.8

48.1 ± 0.6*

Manual

hematocrit (%)

51.1 ± 0.7

51.2 ± 0.4

51.4 ± 0.6

49.0 ± 0.7

49.0 ± 0.4

Hemoglobin

(g/dL)

16.3 ± 0.2

16.3 ± 0.1

16.3 ± 0.1

15.8 ± 0.2

15.7 ± 0.1*

Erythrocytes

(106/µL)

9.96 ± 0.14

9.92 ± 0.12

9.93 ± 0.12

9.55 ± 0.17

9.40 ± 0.11*

Reticulocytes

(106/µL)

0.19 ± 0.01

0.25 ± 0.02

0.20 ± 0.02

0.24 ± 0.02

0.20 ± 0.02

Nucleated erythrocytes

(103/µL)

0.10 ± 0.10

0.00 ± 0.00

0.10 ± 0.10

0.00 ± 0.00

0.00 ± 0.00

Mean cell volumen

(fL)

51.1 ± 0.1

51.3 ± 0.1

51.1 ± 0.2

51.0 ± 0.1

51.2 ± 0.2

Mean cell hemoglobin

(pg)

16.4 ± 0.1

16.4 ± 0.1

16.4 ± 0.1

16.5 ± 0.1

16.7 ± 0.1

Mean cell hemoglobin

concentration

(g/dL)

32.0 ± 0.2

32.1 ± 0.2

32.1 ± 0.1

32.4 ± 0.2

32.6 ± 0.2

Platelets

(103/µL)

607.9 ± 34.2

637.2 ± 25.6

616.5 ± 31.1

686.9 ± 36.9

751.2 ± 39.4*

Leukocytes

(103/µL)

4.05 ± 0.39

4.16 ± 0.26

4.38 ± 0.28

4.42 ± 0.25

5.76 ± 0.45**

Segmented neutrophils

(103/µL)

0.36 ± 0.03

0.43 ± 0.05

0.35 ± 0.04

0.50 ± 0.08

1.05 ± 0.23**

Bands

(103/µL)

0.00 ± 0.00

0.01 ± 0.00

0.00 ± 0.00

0.00 ± 0.00

0.02 ± 0.01

Lymphocytes

(103/µL)

3.59 ± 0.36

3.64 ± 0.26

3.92 ± 0.27

3.80 ± 0.25

4.52 ± 0.26

Monocytes

(103/µL)

0.00 ± 0.00

0.00 ± 0.00

0.00 ± 0.00

0.01 ± 0.01

0.02 ± 0.01

Eosinophils

(103/µL)

0.06 ± 0.01

0.06 ± 0.01

0.08 ± 0.01

0.06 ± 0.01

0.11 ± 0.02

Automated

hematocrit (%)

0.04 ± 0.01

0.03 ± 0.01

0.02 ± 0.01

0.05 ± 0.01

0.05 ± 0.01

Conclusions:
The NOAEL for the subchronic dermal toxicity of the test item in mice was 3 mg/kg bw in males and females.
Executive summary:

In a 13 -week subchronic dermal toxicity study performed on the test item by the NTP (GLP study), groups of 10 male and 10 female B6C3F1 mice were dermally administered 0, 1.5, 3, 6, 12 or 24 mg/kg bw of test item in ethanol (2 mL/kg), 5 days a week for 13 weeks. All 24 mg/kg male and female mice died
or were found moribund and sacrificed prior to day 16. Final mean body weights of 6 and 12 mg/kg males and mean body weight gains of 6 and 12 mg/kg males and females were significantly less than those of the vehicle controls. The predominant clinical pathology changes suggest a secondary, treatment-related inflammatory leukogram and minimal decreased erythron of chronic inflammation that would be consistent with necrosis and chronic active inflammation of the skin. Dermal administration of the test item significantly decreased the weight of the epididymis in 6 and 12 mg/kg males and significantly decreased epididymal spermatozoal motility in 6 mg/kg males. Significantly increased incidences of skin lesions at the site of application included epidermal hyperplasia in all dosed groups except those administered 24 mg/kg, chronic active inflammation in all dosed groups except 1.5 mg/kg females, and epidermal necrosis in 24 mg/kg males and females.
Based on these results, the NOAEL for the subchronic dermal toxicity of the test item in mice was 3 mg/kg bw in males and in females.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
3 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Four studies are available, all with a Klimisch score of 2.

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
June 29, 1994 - July 15, 1994
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
yes
Remarks:
Treatment 5 days per week for 16 days. No haemathology or biochemistry analysis.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Aldrich Chemical Company (Milwaukee, WI), lot 00929TZ
- Purity: 99.5% (determined by GC)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, under a nitrogen head space, protected from light
- Stability under test conditions: Periodic analyses of the bulk chemical were performed by the study laboratories during the studies using GC: no degradation of the bulk chemical was detected.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Dose formulations were prepared by mixing dicyclohexylcarbodiimide and anhydrous ethanol to give the required concentration.
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Laboratory Animals and Services (Germantown, NY).
- Age at study initiation: 8 weeks
- Weight at study initiation: males 25.9 -28.1 g; females 19.9 - 21.3 g
- Fasting period before study: not specified
- Housing: individually housed
- Diet: ad libitum, NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA),
- Water: ad libitum, tap water (Washington Suburban Sanitary Commission Potomac Plant)
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.22 ± 0.16 ºC
- Humidity (%): 35-65%
- Air changes (per hr): at least 10 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark
Type of coverage:
not specified
Vehicle:
ethanol
Details on exposure:
TEST SITE
- Area of exposure: clipped dorsal skin, from the posterior of the scapulae to the base of the tail
- % coverage: not specified
- Type of wrap if used: not specified.

REMOVAL OF TEST SUBSTANCE: not specified

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0, 0.2, 0.6, 1.7, 5, or 15 mg/animal in 0.1 ml ethanol
- Concentration (if solution): 0, 2, 6, 17, 50, or 150 mg dicyclohexylcarbodiimide per ml of ethanol. 0.6 mg/animal approximately corresponded to 24 mg/kg body weight.
- For solids, paste formed: no, test substance was diluted in anhydrous ethanol to give the required concentration

VEHICLE
- Vehicle (if other than water): anhydrous ethanol
- Amount(s) applied (volume or weight with unit): 0.1 ml
- Lot/batch no. (if required): not specified
- Purity: greater than 99.9 %

PREPARATION OF DOSING SOLUTIONS
- A weighed amount of dicyclohexylcarbodiimide was dissolved in absolute ethanol to prepare the highest dose concentration formulation; lower dose formulations were prepared by diluting aliquots of this preparation with absolute ethanol. Dose formulations were prepared twice. The dose formulations were stored in sealed vials under a headspace of inert gas for up to 28 days at room temperature. Stability studies of 0.38, 2, and 7 mg/ml dose formulations of lot 00929TZ were conducted by the study laboratory using GC. Stability was confirmed for up to 35 days for dose formulations stored at room temperature in sealed containers under a nitrogen headspace and for up to 3 hours when exposed to light and air at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulations were prepared twice during the study and were analyzed once by GC . All five dose formulations were within 10% of the target concentrations.
Duration of treatment / exposure:
16 days
Frequency of treatment:
5 days per week
Dose / conc.:
0 other: mg / animal
Remarks:
control group
Dose / conc.:
0.2 other: mg / animal
Dose / conc.:
0.6 other: mg / animal
Remarks:
ca. 24 mg/kg bw
Dose / conc.:
1.7 other: mg / animal
Dose / conc.:
5 other: mg / animal
Dose / conc.:
15 other: mg / animal
No. of animals per sex per dose:
5 males and 5 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment: animals were distributed randomly into groups of approximately equal initial mean body weights.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical findings (including dermal irritation) were recorded initially, on day 8 and at the end of the studies.

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed initially, on day 8 and at the end of the studies

FOOD CONSUMPTION: No data

FOOD EFFICIENCY: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: No data

CLINICAL CHEMISTRY: No data

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsies were performed on all animals. Organs weighed were the adrenal gland, heart, right kidney, liver, lung, right testis, and thymus.

HISTOPATHOLOGY: Yes
Histopathology was performed on vehicle control, 0.2, 0.6 and 1.7 mg/animal mice. In addition to gross lesions and tissue masses, the following tissues were examined: brain, kidney, liver, lung, skin, and spinal cord.
Statistics:
Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs of toxicity at doses of 0.6 mg or greater.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Skin irritation at the site of application in all dosed groups.
Mortality:
mortality observed, treatment-related
Description (incidence):
One 0.6 mg female mouse and all mice in the 1.7, 5, and 15 mg groups died before the end of the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Final mean body weights of the 0.6 mg groups were significantly less than those of the vehicle controls, and animals in these groups generally lost weight during the study.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Heart weights of 0.6 mg groups of mice were significantly greater than those of the vehicle controls; thymus weights in these groups were significantly less than those of the vehicle controls.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathologic examination of mice dosed with 1.7 mg dicyclohexylcarbodiimide or less revealed lesions of the skin at the site of application including epidermal hyperplasia, epidermal necrosis, and acute or chronic active dermal inflammation (data not shown).
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
At a dose of 0.6 mg or greater, clinical signs of toxicity appeared. Final mean body weights were significantly lower than the control, heart weights were significantly greater and thymus weights were significantly lower.
Key result
Dose descriptor:
NOAEL
Effect level:
0.2 other: mg/animal
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
Remarks on result:
other: ca. 8 mg/kg bw
Key result
Critical effects observed:
no

Table 1. Survival and body weights of B6C3F1 mice.

Dose

mg/animal

Survival

Mean body weight

Final weight Relative to controls

(%)

Initial

Final

Change

MALE

0

5/5

27.0 ± 1.1

28.8 ± 1.1

1.8 ± 0.0

 

0.2

5/5

26.7 ± 0.7

28.3 ± 0.5

1.6 ± 0.3

98

0.6

5/5

27.0 ± 0.5

26.0 ± 0.5*

-1.0 ± 0.4 *

90

1.7

0/5

26.5 ± 0.6

-

-

 

5

0/5

26.6 ± 0.7

-

-

 

15

0/5

26.5 ± 0.7

-

-

 

FEMALE

0

5/5

20.6 ± 0.6

24.0 ± 0.5

3.4 ± 0.3

 

0.2

5/5

20.4 ± 0.5

23.9 ± 0.4

3.5 ± 0.4

100

0.6

4/5

20.8 ± 0.7

21.3 ± 0.8 *

-0.1 ± 0.5*

89

1.7

0/5

21.0 ± 0.3

-

-

 

5

0/5

20.8 ± 0.3

-

-

 

15

0/5

20.6 ± 0.6

-

-

 

* Significantly different from the vehicle control group by Dunnett’s or Williams’ test

Conclusions:
The NOAEL for the test substance was 0.2 mg/animal (ca. 8 mg/kg bw) in male and females mice after 16 days of dermal exposure.
Executive summary:

Groups of five male and five female mice were dermally treated with 0.1 ml ethanol containing 0, 0.2, 0.6, 1.7, 5 or 15 mg dicyclohexylcarbodiimide, 5 days per week (16 days of dosing). The control animals were treated with the vehcicle only (ethanol). One 0.6 mg female mouse and all mice in the 1.7, 5, and 15 mg groups died before the end of the study. Final mean body weights of the 0.6 mg groups were significantly less than those of the vehicle controls, and animals in these groups generally lost weight during the study. Clinical findings included skin irritation at the site of application in all dosed groups and clinical signs of toxicity at doses of 0.6 mg or greater. Heart weights of 0.6 mg groups of mice were significantly greater than those of the vehicle controls; thymus weights in these groups were significantly less than those of the vehicle controls. Histopathologic examination of mice dosed with 1.7 mg dicyclohexylcarbodiimide or less revealed lesions of the skin at the site of application including epidermal hyperplasia, epidermal necrosis, and acute or chronic active dermal inflammation. Based on these results, the NOAEL for the test substance was 0.2 mg/animal (ca. 8 mg/kg bw) in male and females mice after 16 days of dermal exposure.

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
June 28, 1994 - July 14, 1994.
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
yes
Remarks:
Treatment 5 days per week for 16 days. No haemathology or biochemistry analysis.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Aldrich Chemical Company (Milwaukee, WI), lot 00929TZ
- Purity: 99.5% (determined by GC)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, under a nitrogen head space, protected from light
- Stability under test conditions: Periodic analyses of the bulk chemical were performed by the study laboratories during the studies using GC: no degradation of the bulk chemical was detected.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Dose formulations were prepared by mixing dicyclohexylcarbodiimide and anhydrous ethanol to give the required concentration.
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Laboratory Animals and Services (Germantown, NY).
- Age at study initiation: 9 weeks old
- Weight at study initiation: males 201-220 g; females 145-148 g
- Fasting period before study: not specified
- Housing: individually housed, in polycarbonate cages (Lab Products, Inc., Seaford, DE) with bedding of heat-treated Sani-chips (R) (P.J. Murphy Forest Products Corp., Montville, NJ), changed weekly.
- Diet: ad libitum; NIH-07 open formula pelleted diet (Zeigler Brothers, Inc.,Gardners, PA),
- Water:ad libitum; Tap water (Washington Suburban Sanitary Commission Potomac Plant)
- Acclimation period: 26 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.22 ± 1.6 ºC
- Humidity (%): 35-65%
- Air changes (per hr): at least 10 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark
Type of coverage:
semiocclusive
Vehicle:
ethanol
Details on exposure:
TEST SITE
- Area of exposure: clipped dorsal skin, from the posterior of the scapulae to the base of the tail
- % coverage: not specified
- Type of wrap if used: not specified.

REMOVAL OF TEST SUBSTANCE: not specified

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0, 0.6, 1.8, 5.1, 15, or 45 mg/animal in 0.3 ml of ethanol.
- Concentration (if solution): 0, 2, 6, 17, 50, or 150 mg dicyclohexylcarbodiimide per ml of ethanol.
- For solids, paste formed: no, test substance was diluted in anhydrous ethanol to give the required concentration

VEHICLE
- Vehicle (if other than water): anhydrous ethanol
- Amount(s) applied (volume or weight with unit): 0.3 ml
- Lot/batch no. (if required): not specified
- Purity: greater than 99.9 %

PREPARATION OF DOSING SOLUTIONS
- A weighed amount of test item was dissolved in absolute ethanol to prepare the highest dose concentration formulation; lower dose formulations were prepared by diluting aliquots of this preparation with absolute ethanol. Dose formulations were prepared twice. The dose formulations were stored in sealed vials under a headspace of inert gas for up to 28 days at room temperature. Stability studies of 0.38, 2, and 7 mg/ml dose formulations of lot 00929TZ were conducted by the study laboratory using GC. Stability was confirmed for up to 35 days for dose formulations stored at room temperature in sealed containers under a nitrogen headspace and for up to 3 hours when exposed to light and air at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulations were prepared twice during the study and were analyzed once by GC . All five dose formulations were within 10% of the target concentrations.
Duration of treatment / exposure:
16 days
Frequency of treatment:
5 days per week
Dose / conc.:
0 other: mg / animal
Dose / conc.:
0.6 other: mg / animal
Dose / conc.:
1.8 other: mg / animal
Dose / conc.:
5.1 other: mg / animal
Dose / conc.:
15 other: mg / animal
Dose / conc.:
45 other: mg / animal
No. of animals per sex per dose:
5 males and 5 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment: animals were distributed randomly into groups of approximately equal initial mean body weights.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical findings (including dermal irritation) were recorded initially, on day 8 and at the end of the studies.

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed initially, on day 8 and at the end of the studies

FOOD CONSUMPTION: No data

FOOD EFFICIENCY: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: No data

CLINICAL CHEMISTRY: No data

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsies were performed on all animals. Organs weighed were the adrenal gland, heart, right kidney, liver, lung, right testis, and thymus.

HISTOPATHOLOGY: Yes
Histopathology was performed on the groups receiving vehicle control, 0.6, 1.8, and 5.1 mg/animal of test item. In addition to gross lesions and tissue masses, the following tissues were examined: brain, kidney, liver, lung, skin, and spinal cord.
Statistics:
Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs of toxicity in 5.1 mg or greater groups included lethargy, ruffled fur, abnormal breathing, coma, and thinness.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Skin irritation at the site of application were seen at doses of 1.8 mg or greater. At the highest doses, the skin lesions at the site of application included epidermal hyperplasia, necrosis, and chronic inflammation in the dermis.
Mortality:
mortality observed, treatment-related
Description (incidence):
All males and females in the 15 and 45 mg groups, four 5.1 mg males, and all 5.1 mg females died before the end of the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Of the surviving groups (0 trhough 1.8 mg/animal), final mean body weights were similar to those of the vehicle controls.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Organ weights of surviving groups of dosed rats were generally similar to those of the vehicle control groups.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histologic examination of rats dosed with 5.1 mg dicyclohexylcarbodiimide or less revealed test article-related lesions of the skin at the site of application including epidermal hyperplasia, epidermal necrosis, or chronic active inflammation in the dermis (data not shown in the report).
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
At doses of 5.1 mg/animal or greater, clinical signs of toxicity were observed, which included lethargy, ruffled fur, abnormal breathing, coma and thinness. At a dose of 5.1 mg/animal, four males and all females died; at higher doses, all animals died before the end of the study.
Key result
Dose descriptor:
NOAEL
Effect level:
1.8 other: mg/animal
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
Remarks on result:
other: ca. 12 mg/kg bw
Dose descriptor:
NOEL
Effect level:
0.6 other: mg / animal
Based on:
test mat.
Sex:
male/female
Basis for effect level:
dermal irritation
Remarks on result:
other: ca. 4 mg/kg bw
Key result
Critical effects observed:
no

Table 1. Survival and body weights

Dose

mg/animal

Survival a

Mean body weight b

(g)

Final weight Relative to controls

(%)

Initial

Final

Change

MALE

0

5/5

207 ± 6

258 ± 9

51 ± 2

 

0.6

5/5

209 ± 5

261 ± 8

52 ± 3

101

1.8

5/5

210 ± 7

254 ± 9

45 ± 3

99

5.1

1/5c

209 ± 6

191

-23

74

15

0/5d

208 ± 5

-

-

 

45

0/5e

211 ± 9

-

-

 

FEMALE

0

5/5

144 ± 4

165 ± 3

21 ± 2

 

0.6

5/5

142 ± 5

164 ± 8

22 ± 3

99

1.8

5/5

143 ± 3

155 ± 5

12 ± 3

94

5.1

0/5f

143 ± 6

-

-

 

15

0/5d

144 ± 4

-

-

 

45

0/5e

141 ± 4

-

-

 

*a: Number of animals surviving at 3 weeks / number initially in group; b: weights and weight changes are given as mean ± standard error. Subsequent calculations are based on animals surviving to the end of the study. Differences from the vehicle control group are not significant by Dunnet's test.; c: day of death: 10, 11, 11, 11; d: day of death: 3 or 4; e: day of death: 2; f: day of death: 9, 9, 9, 10, 11.

Conclusions:
The NOAEL for the test item was determined to be 1.8 mg/animal (ca. 12 mg/kg bw) in male and females rats after 16 days of dermal exposure.
Executive summary:

Groups of five male and five female rats were dermally administered 0.3 ml ethanol containing 0, 0.6, 1.8, 5.1, 15, or 45 mg dicyclohexylcarbodiimide, 5 days per week (16 days of dosing). All males and females in the 15 and 45 mg groups, four 5.1 mg males, and all 5.1 mg females died before the end of the study. Of the surviving groups, final mean body weights were similar to those of the vehicle controls, although the one surviving 5.1 mg male rat lost weight during the study. Clinical findings included skin irritation at hte site of application at doses of 1.8 mg or greater. Histopathologic examination of rats dosed with 5.1 mg dicyclohexylcarbodiimide or less revealed treatment-related lesions of the skin at the site of application including epidermal hyperplasia, epidermal necrosis, or chronic active inflammation in the dermis. Based on these results, the NOAEL for the test item was determined to be 1.8 mg/animal (ca. 12 mg/kg bw) in male and females rats after 16 days of dermal exposure under test conditions.

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
September 20, 1994 - December 19, 1994.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Version / remarks:
The study was comparable to this Guideline with certain differences.
Deviations:
yes
Remarks:
Animals were treated 5 days a week. No ophtalmological examination was performed.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Aldrich Chemical Company (Milwaukee, WI), lot 00929TZ
- Purity: 99.5% (determined by GC)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, under a nitrogen head space, protected from light.
- Stability under test conditions: Periodic analyses of the bulk chemical were performed by the study laboratories during the studies using GC: no degradation of the bulk chemical was detected.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Dose formulations were prepared by mixing dicyclohexylcarbodiimide and anhydrous ethanol to give the required concentration.
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Laboratory Animals and Services (Germantown, NY).
- Age at study initiation: 7 weeks old
- Weight at study initiation: males 115-124 g; females 100-106 g
- Fasting period before study: not specified
- Housing: individually housed
- Diet: ad libitum; NIH-07 open formula pelleted diet (Zeigler Brothers, Inc.,Gardners, PA),
- Water:ad libitum; Tap water (Washington Suburban Sanitary Commission Potomac Plant)
- Acclimation period: 12 days (males) or 13 days (females)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.22 ± 0.16 ºC
- Humidity (%): 35-65%
- Air changes (per hr): at least 10 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark
Type of coverage:
not specified
Vehicle:
ethanol
Details on exposure:
TEST SITE
- Area of exposure: clipped dorsal skin, from the posterior of the scapulae to the base of the tail
- % coverage: not specified
- Type of wrap if used: not specified

REMOVAL OF TEST SUBSTANCE: not specified

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0, 0.75, 1.5, 3, 6, or 12 mg/kg body weight in 0.5 ml/kg ethanol
- For solids, paste formed: no, test substance was diluted in anhydrous ethanol to give the required concentration

VEHICLE
- Vehicle (if other than water): anhydrous ethanol
- Amount(s) applied (volume or weight with unit): 0.5 ml/kg
- Lot/batch no. (if required): not specified
- Purity: greater than 99.9 %

PREPARATION OF DOSING SOLUTIONS
- A weighed amount of test item was dissolved in absolute ethanol to prepare the highest dose concentration formulation; lower dose formulations were prepared by diluting aliquots of this preparation with absolute ethanol. Dose formulations were prepared at least every 2 weeks . The dose formulations were stored in sealed vials under a headspace of inert gas for up to 28 days at room temperature. Stability studies of 0.38, 2, and 7 mg/ml dose formulations of lot 00929TZ were conducted by the study laboratory using GC. Stability was confirmed for up to 35 days for dose formulations stored at room temperature in sealed containers under a nitrogen headspace and for up to 3 hours when exposed to light and air at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Periodic analyses of the dose formulations of dicyclohexylcarbodiimide were conducted by the study laboratories using GC. The dose formulations were analyzed at the beginning, midpoint, and end of the studies; animal room samples of these dose formulations were also analyzed. Of the dose formulations analyzed, all 15 were within 10% of the target concentration; all 13 animal room samples were within 10% of the target concentrations.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
5 days per week
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control group
Dose / conc.:
0.75 mg/kg bw/day (nominal)
Dose / conc.:
1.5 mg/kg bw/day (nominal)
Dose / conc.:
3 mg/kg bw/day (nominal)
Dose / conc.:
6 mg/kg bw/day (nominal)
Dose / conc.:
12 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 males and 10 females per dose per group (core study and satellites)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a previous 16-days study was found that at the dose of 1.8 mg/animal could be seen clinical signs of toxicity. Doses greater than this dose resulted in mortality. So this dose was selected as the highest dose, which approximately corresponded to 12 mg/kg body weight.

Dose selection rationale: In a previous 16-day study, it was found that 1.8 mg/animal resulted in clinical signs of toxicity and greater doses resulted in mortality. Therefore, this dose was selected as the highest dose, which approximately corresponds to 12 mg/kg body weight.
- Rationale for selecting satellite groups: groups of 10 males and 10 females received the same doses as the core study animals during 22 days (clinical pathology rats), and blood was collected on days 3 and 22; no necropsy was performed on these animals. No justification provided.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical findings (including dermal irritation) recorded initially, weekly, and at the end of the studies.

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed initially, weekly, and at the end of the studies.

FOOD CONSUMPTION: No data

FOOD EFFICIENCY: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: from clinical pathology study rats, on days 3 and 22; from core study rats, at study termination.
- Anaesthetic used for blood collection: Yes, animals were anesthetized with a 70% CO2/30% O2 mixture.
- Animals fasted: No data
- How many animals: all animals of each group and study.
- Parameters examined: erythrocyte, reticulocyte, nucleated erythrocyte, platelet and leukocyte counts, automated and manual hematocrit values, hemoglobin concentration, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration. Differential leukocyte counts and erythrocyte and leukocyte morphologies were determined too.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: from clinical pathology study rats on days 3 and 22; from core study rats at study termination.
- Anaesthetic used for blood collection: Yes, animals were anesthetized with a 70% CO2/30% O2 mixture.
- Animals fasted: No data
- How many animals: all animals of each group and study.
- Parameters checked: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile acids.

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

OTHER: SPERM MOTILITY AND VAGINAL CITOLOGY
- Time schedule for collection: at the end of the core study in males, 12 consecutive days prior to the end in females
- How many animals: animals from vehicle control, 1.5, 3 and 6 mg/kg core study groups (males and females)
- Parameters checked: sperm motility evaluation: spermatid heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility and concentration. The left cauda, left epididymis, and left testis were weighed. Vaginal citology evaluations: relative numbers of leukocytes, nucleated epithelial cells, and large squamous epithelial cells were determined and used to ascertain estrous cycle stage (i.e., diestrus, proestrus, estrus, and metestrus) and to obtain the percentage of time spent in the various estrous cycle stages and the estrous cycle length.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes.
Necropsies were performed on all core study animals. Organs weighed were the heart, right kidney, liver, lung, right testis, and thymus.

HISTOPATHOLOGY: Yes.
Complete histopathology was performed on vehicle control, 6, and 12 mg/kg core study rats. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, heart with aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, uterus, and Zymbal’s gland. In addition, the bone marrow, spleen of male rats and skin were examined in all remaining dosed groups.
Statistics:
Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Hematology, clinical chemistry, spermatid, and epididymal spermatozoal data, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) (as modified by Williams, 1986) and Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test). Prior to statistical analysis, extreme values identified by the outlier test of Dixon and Massey (1957) were examined by NTP personnel, and implausible values were eliminated from the analysis. Average severity values were analyzed for significance with the Mann-Whitney U test (Hollander and Wolfe, 1973). Because vaginal cytology data are proportions (the proportion of the observation period that an animal was in a given estrous stage), an arcsine transformation was used to bring the data into closer conformance with a normality assumption. Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across exposure concentrations.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All 12 mg/kg males and females showed lethargy, tremors, ataxia, ruffled fur, a thin appearance, nasal/eye discharge, and head tilt.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Skin irrititation at the site of application was observed in all groups at doses of 3 mg/kg or higher.
Mortality:
mortality observed, treatment-related
Description (incidence):
All 12 mg/kg male and female rats died or were found moribund and sacrificed prior to day 45.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Final mean body weight and body weight gain of 6 mg/kg males were significantly less than those of the vehicle controls.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
On Day 3: increased neutrophil counts were seen in 6 mg/kg or greater (males and females).
On Day 22: increases in leukocyte, neutrophil, and band neutrophil counts occurred in 3 mg/kg males and 6 mg/kg or greater males and females; monocyte and eosinophil counts were also increased. Minimal decreases in hematocrit, hemoglobin and mean cell volume values and erythrocyte counts occurred in 6 mg/kg males and 12 mg/kg males and females.
On Week 13: increased neutrophil counts were observed only in 6 mg/kg females. Minimal decreases in hematocrit, hemoglobin and mean cell volume values and erythrocyte counts occurred in 6 mg/kg females too.
The leukocyte changes and the decreases in the erythron would be consistent with the progression of an inflammatory process from the acute to chronic phases and would be a response consistent with the development of skin necrosis and chronic active inflammation observed morphologically.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
On Day 22: small decreases in albumin concentration occurred in 3 mg/kg or greater males and 6 mg/kg or greater females.
On Week 13: small decreases in albumin concentration occurred in 3 mg/kg or greater males and 6 mg/kg or greater females too.
The albumin decrease could be a secondary response to chronic inflammation.
Small, transient increases in serum alanine aminotransferase (ALT) activity occurred on days 3 and 22 in treated males and females but not on week 13. As no other marker of hepatocellular injury was affected, it was considered a transient induction of ALT production by the liver.
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute weights of the heart, kidney, and liver of 3 and 6 mg/kg females were generally greater than those of the vehicle controls.
Relative weights of the heart and kidney of 3 and 6 mg/kg males were significantly greater than those of the vehicle controls.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Gross lesions of the skin at the site of application were detected in all animals dosed with 3 mg/kg or greater.
Epidermal hyperplasia occurred in all treated groups, and the incidences of this lesion were significantly increased in 3 mg/kg or greater males and 1.5 mg/kg or greater females; the incidences and severities of the lesion increased in a dose-related fashion.
Chronic active inflammation occurred in most of the dosed groups, and the incidences of this lesion were significantly increased in 6 and 12 mg/kg males and 1.5 mg/kg or greater females. The incidences of sebaceous gland hyperplasia were significantly increased in 3 mg/kg or greater males and 6 and 12 mg/kg females, and the incidence of epidermal necrosis was significantly increased in male rats administered 12 mg/kg. Incidences of sebaceous gland hyperplasia were considered to be a secondary effect of inflammation rather than a direct compound effect.
On the surface of the skin there was necrosis with marked acute inflammation consisting of polymorphonuclear cells with edema and coagulated serum admixed with cell debris (crusts). Deeper in the dermis, mononuclear cells mixed with proliferating fibroblasts were present, which suggested a more chronic feature of the inflammatory lesion.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The skin lesions were microscopically similar to the same lesions seen at the site of aplication (gross pathology) although most were milder in severity: epidermal necrosis, chronic active inflammation and epidermal hyperplasia.
Slightly increased incidences of minimal hematopoietic cell proliferation occurred in the spleen of all dosed groups of male rats; this finding was considered secondary to the skin lesions at the site of application.
The incidences of diffuse bone marrow hyperplasia in 12 mg/kg males and females were significantly greater than those in the vehicle controls. This finding was also considered secondary to the skin lesions at the site of application.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Reproductive tissue parameters in males or vaginal cytology parameters in females were not affected.
Key result
Dose descriptor:
NOAEL
Effect level:
3 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
mortality
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
6 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
Key result
Critical effects observed:
no

Table 1. Survival and body weights

Dose

mg/kg bw

Survival

Mean body weight

Final weight Relative to controls

(%)

Initial

Final

Change

MALE

0

10/10

121 ± 3

330 ± 5

209 ± 5

 

0.75

10/10

120 ± 2

337 ± 6

216 ± 6

102

1.5

10/10

120 ± 3

334 ± 6

213 ± 5

101

3

10/10

120 ± 3

325 ± 7

205 ± 6

99

6

10/10

118 ± 2

300 ± 4*

182 ± 3*

91

12

0/10

118 ± 3

-

-

 

FEMALE

0

10/10

102 ± 2

186 ± 3

84 ± 2

 

0.6

10/10

102 ± 2

191 ± 3

89 ± 2

103

1.8

10/10

104 ± 2

190 ±4

86 ± 3

102

5.1

10/10

104 ± 2

193 ± 3

89 ± 2

104

15

10/10

103 ± 2

192 ± 4

89 ± 3

103

45

0/10

103 ± 2

-

-

 

* significantly different

Table 2. Incidences of Nonneoplastic lesions of the skin (site of application)

 

Vehicle control

0.75 mg/kg

1.5 mg/kg

3 mg/kg

6 mg/kg

12 mg/kg

MALE

 

Number examined microscopically

10

10

10

10

10

10

Epidermis, hyperplasia

0

1

3

9*

10*

10*

Epidermis, necrosis

0

0

0

0

0

6

Sebaceous gland hyperplasia

0

1

1

4

7

9

Inflammation, chronic active

0

1

0

1

7

9

FEMALE

 

Number examined microscopically

10

10

10

10

10

10

Epidermis, hyperplasia

0

3

7*

7*

10*

10*

Epidermis, necrosis

0

0

0

0

1

3

Sebaceous gland hyperplasia

0

1

0

0

5*

7*

Inflammation, chronic active

0

0

4*

4*

9*

9*

* signficantly different

Table 3. Hematology and clinical chemistry parameters found to be altered in males

MALE

Vehicle control

0.75 mg/kg

1.5 mg/kg

3 mg/kg

6 mg/kg

12 mg/kg

Hematology

 

Automated hematocrit

Day 3

Day 22

Week 13

40.9±0.4

45.9±0.4

44.1±0.1

41.1±0.7

45.0±0.5

43.9±0.7

42.2±0.4

45.4±0.6

44.4±0.4

41.2±0.6

44.6±0.4

44.5±0.3

40.4±0.4

43.7±0.3*

44.5±.04

41.5±0.8

44.5±0.5*

-

Hemoglobin (g/dl)

Day 3

Day 22

Week 13

14.6±0.1

16.2±0.2

15.7±0.1

14.7±0.2

16.1±0.2

15.5±0.2

14.8±0.1

16.1±0.1

15.6±0.1

14.7±0.2

15.9±0.1

15.7±0.1

14.5±0.2

15.5±0.1*

15.6±0.1

14.6±0.2

15.6±0.2

-

Mean cell volume

Day 3

Day 22

Week 13

62.5±0.4

59.9±0.4

50.7±0.2

61.7±0.4

59.6±0.2

50.3±0.2

62.8±0.3

59.8±0.3

50.4±0.2

61.9±0.4

59.2±0.5

50.7±0.1

62.2±0.5

59.3±0.3

50.4±0.1

62.5±0.4

58.4±0.2*

-

Mean cell hemoglobin (pg)

Day 3

Day 22

Week 13

22.3±0.1

21.1±0.2

18.0±0.1

22.1±0.1

21.3±0.1

17.8±0.1

22.0±0.1

21.2±0.2

17.7±0.1

22.1±0.2

21.1±0.1

17.9±0.1

22.3±0.1

21.0±0.1

17.7±0.1

22.0±0.2

20.4±0.1*

-

Leukocytes (103/µl)

Day 3

Day 22

Week 13

10.85±0.2

9.39±0.2

8.26±0.6

10.06±0.34

8.76±0.6

7.88±0.4

9.87±0.3

9.88±0.7

7.20±0.6

10.24±0.5

10.80±0.8

9.19±0.9

10.82±0.3

19.94±1.5*

9.63±0.5

12.35±0.3

24.65±1.8*

-

Segmented neutrophils (103/µl)

Day 3

Day 22

Week 13

1.05±0.1

0.96±0.1

1.18±0.1

0.97±0.1

1.24±0.1

1.14±0.2

1.16±0.1

1.15±0.1

1.02±0.2

0.99±0.1

2.14±0.3*

1.87±0.6

1.74±0.2

9.71±1.0*

1.80±0.2

2.47±0.2*

14.24±1.2*

-

Bands (103/µl)

Day 3

Day 22

Week 13

0.00±0.0

0.00±0.0

0.00±0.0

0.00±0.0

0.02±0.01

0.01±0.01

0.01±0.01

0.03±0.01

0.02±0.02

0.00±0.0

0.13±0.05*

0.01±0.01

0.01±0.10

0.73±0.12*

0.02±0.02

0.05±0.02

0.75±0.06*

0.00±0.0

Monocytes (103/µl)

Day 3

Day 22

Week 13

0.22±0.02

0.22±0.03

0.19±0.44

0.12±0.03

0.24±0.04

0.22±0.07

0.25±0.04

0.24±0.04

0.15±0.06

0.16±0.06

0.22±0.05

0.16±0.04

0.23±0.05

0.58±0.21

0.19±0.05

0.29±0.06

0.62±0.11*

-

Eosinophils (103/µl)

Day 3

Day 22

Week 13

0.0.3±0.02

0.04±0.02

0.03±0.02

0.03±0.02

0.04±0.01

0.08±0.03

0.06±0.22

0.04±0.22

0.11±0.04

0.02±0.01

0.06±0.03

0.02±0.02

0.03±0.02

0.07±0.04

0.08±0.04

0.04±0.02

0.24±0.06*

-

Clinical chemistry

 

Total protein

Day 3

Day 22

Week 13

5.7±0.1

6.2±0.1

6.9±0.1

5.7±0.1

6.1±0.1

6.9±0.1

5.8±0.1

6.0±0.1

7.1±0.1

5.8±0.1

6.1±0.1

6.6±0.3

5.6±0.0

6.0±0.1

6.8±0.1

5.6±0.1

5.8±0.1*

-

Albumin

Day 3

Day 22

Week 13

4.6±0.1

4.8±0.0

5.0±0.1

4.7±0.1

4.8±0.0

4.9±0.1

4.7±0.1

4.7±0.1

5.0±0.0

4.7±0.0

4.6±0.1*

4.5±0.2

4.6±0.0

4.3±0.0*

4.7±0.1*

4.5±0.0

4.0±0.1

-

Alanine aminotransferase (IU/l)

Day 3

Day 22

Week 13

 

 

51±1

53±2

57±3

 

 

48±1

54±1

60±2

 

 

53±1

52±2

63±3

 

 

53±1

62±1*

63±5

 

 

61±2*

68±3*

68±4

 

 

76±3*

77±4*

-

Alkaline phosphatase (IU/l)

Day 3

Day 22

Week 13

762±26

520±9

262±5

752±20

532±10

276±12

761±22

501±13

275±3

767±11

527±11

259±20

736±16

462±12*

290±6*

728±12

442±15*

-

Table 4. Hematology and clinical chemistry parameters found to be altered in females

FEMALE

Vehicle control

0.75 mg/kg

1.5 mg/kg

3 mg/kg

6 mg/kg

12 mg/kg

Hematology

 

Automated hematocrit

Day 3

Day 22

Week 13

42.8±0.6

46.7±0.4

45.6±0.3

43.2±0.4

46.9±0.3

44.6±0.6

43.3±0.4

47.2±0.3

45.2±0.4

43.3±0.5

47.1±0.8

43.3±0.4*

43.7±0.3

45.5±0.3

43.0±.0.5*

43.3±0.5

46.1±0.5

-

Hemoglobin (g/dl)

Day 3

Day 22

Week 13

15.2±0.2

16.2±0.2

15.8±0.1

15.3±0.1

16.5±0.1

15.6±0.2

15.3±0.1

16.4±0.1

15.8±0.1

15.3±0.1

16.3±0.3

15.3±0.2*

15.5±0.1

15.9±0.1

15.1±0.2*

15.3±0.2

15.7±0.1*

-

Erythrocites (106/µl)

Day 3

Day 22

Week 13

6.94±0.09

7.60±0.06

8.08±0.06

7.02±0.06

7.64±0.05

7.92±0.10

7.03±0.07

7.66±0.06

8.03±0.06

7.03±0.08

7.69±0.13

7.68±0.07*

7.09±0.07

7.52±0.05

7.74±0.09*

7.01±0.09

7.67±0.12

-

Mean cell volume

Day 3

Day 22

Week 13

61.6±0.2

61.4±0.3

56.3±0.1

61.5±0.1

61.4±0.2

56.3±0.1

61.6±0.2

61.7±0.2

56.3±0.1

61.6±0.2

61.2±0.2

56.4±0.1

61.6±0.2

60.6±0.2*

55.6±0.2*

61.8±0.3

60.1±0.3*

-

Mean cell hemoglobin (pg)

Day 3

Day 22

Week 13

21.9±0.1

21.3±0.1

19.6±0.1

21.7±0.1

21.6±0.1

19.7±0.1

21.8±0.1

21.5±0.1

19.7±0.1

21.7±0.1

21.2±0.1

19.9±0.1

21.8±0.1

21.1±0.1

19.5±0.1

21.9±0.1

20.±0.2*

-

Platelets (103/µl)

Day 3

Day 22

Week 13

785.9±18.3

731.2±21.5

649.0±25.6

797.6±18.7

786.7±19.0

653.7±14.2

788.3±21.5

747.8±22.5

631.7±29.4

781.4±26.3

637.8±36.3

710.4±26.7*

833.7±32.6

701.1±16.4

718.9±25.1*

808.1±19.5

707.3±21.4

-

Leukocytes (103/µl)

Day 3

Day 22

Week 13

10.79±0.5

7.62±0.2

6.60±0.3

11.49±0.3

8.76±0.6

7.88±0.4

9.87±0.3

9.88±0.7

7.20±0.6

10.24±0.5

10.80±0.8

9.19±0.9

10.82±0.3

19.94±1.5*

9.63±0.5

12.35±0.3

24.65±1.8*

-

Segmented neutrophils (103/µl)

Day 3

Day 22

Week 13

1.05±0.1

0.96±0.1

1.18±0.1

0.97±0.1

1.24±0.1

1.14±0.2

1.16±0.1

1.15±0.1

1.02±0.2

0.99±0.1

2.14±0.3*

1.87±0.6

1.74±0.2

9.71±1.0*

1.80±0.2

2.47±0.2*

14.24±1.2*

-

Bands (103/µl)

Day 3

Day 22

Week 13

0.00±0.0

0.00±0.0

0.00±0.0

0.00±0.0

0.02±0.01

0.01±0.01

0.01±0.01

0.03±0.01

0.02±0.02

0.00±0.0

0.13±0.05*

0.01±0.01

0.01±0.10

0.73±0.12*

0.02±0.02

0.05±0.02

0.75±0.06*

0.00±0.0

Monocytes (103/µl)

Day 3

Day 22

Week 13

0.22±0.02

0.22±0.03

0.19±0.44

0.12±0.03

0.24±0.04

0.22±0.07

0.25±0.04

0.24±0.04

0.15±0.06

0.16±0.06

0.22±0.05

0.16±0.04

0.23±0.05

0.58±0.21

0.19±0.05

0.29±0.06

0.62±0.11*

-

Eosinophils (103/µl)

Day 3

Day 22

Week 13

0.0.3±0.02

0.04±0.02

0.03±0.02

0.03±0.02

0.04±0.01

0.08±0.03

0.06±0.22

0.04±0.22

0.11±0.04

0.02±0.01

0.06±0.03

0.02±0.02

0.03±0.02

0.07±0.04

0.08±0.04

0.04±0.02

0.24±0.06*

-

Clinical chemistry

 

Total protein

Day 3

Day 22

Week 13

5.7±0.1

6.2±0.1

6.9±0.1

5.7±0.1

6.1±0.1

6.9±0.1

5.8±0.1

6.0±0.1

7.1±0.1

5.8±0.1

6.1±0.1

6.6±0.3

5.6±0.0

6.0±0.1

6.8±0.1

5.6±0.1

5.8±0.1*

-

Albumin

Day 3

Day 22

Week 13

4.6±0.1

4.8±0.0

5.0±0.1

4.7±0.1

4.8±0.0

4.9±0.1

4.7±0.1

4.7±0.1

5.0±0.0

4.7±0.0

4.6±0.1*

4.5±0.2

4.6±0.0

4.3±0.0*

4.7±0.1*

4.5±0.0

4.0±0.1

-

Alanine aminotransferase (IU/l)

Day 3

Day 22

Week 13

 

 

51±1

53±2

57±3

 

 

48±1

54±1

60±2

 

 

53±1

52±2

63±3

 

 

53±1

62±1*

63±5

 

 

61±2*

68±3*

68±4

 

 

76±3*

77±4*

-

Alkaline phosphatase (IU/l)

Day 3

Day 22

Week 13

762±26

520±9

262±5

752±20

532±10

276±12

761±22

501±13

275±3

767±11

527±11

259±20

736±16

462±12*

290±6*

728±12

442±15*

-

Conclusions:
The NOAEL for the subchronic dermal toxicity of the test item in rats was 3 mg/kg bw in males and 6 mg/kg bw in females.
Executive summary:

In a 13 -week subchronic dermal toxicity study performed on the test item by the NTP (GLP study), groups of 10 male and 10 female F-344 rats were dermally administered 0, 0.75, 1.5, 3, 6 or 12 mg/kg bw of test item in ethanol (0.5 mL/kg), 5 days a week for 13 weeks. All 12 mg/kg male and female core study rats died or were found moribund and sacrificed prior to day 45. Final mean body weight and body weight gain of 6 mg/kg males were significantly less than those of the vehicle controls. The predominant clinical pathology changes suggest a secondary, treatmentrelated inflammatory leukogram and minimal decreased erythron of chronic inflammation that would be consistent with necrosis and chronic active inflammation of the skin. Significantly increased incidences of skin lesions at the site of application included epidermal hyperplasia in 3 mg/kg or greater males and 1.5 mg/kg or greater females, chronic active inflammation in 6 and 12 mg/kg males and 1.5 mg/kg or greater females, and epidermal necrosis in 12 mg/kg males. The incidences and severities of epidermal hyperplasia increased in a dose-related manner in both sexes of rats . Based on these results, the NOAEL for the subchronic dermal toxicity of the test item in rats was 3 mg/kg bw in males and 6 mg/kg bw in females.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed

Additional information

Repeated dose toxicity: oral. Key study. Method similar to OECD 407, GLP study. SD (Crj: CD) rats were treated daily with 0, 15, 100 or 500 mg/kg of the test substance by oral gavage for 28 days. Animals of both sexes in the 500 mg/kg group showed reduced locomotor activity, salivation, inhibition of body weight gain, decreased food consumption and increased water intake. Two females in the 500 mg/kg dose group died, one during treatment and one during the recovery period. A decrease in alkaline phosphatase was also observed in males in the 500 mg / kg group. Pathological examination revealed increased relative liver weight, enlargement of the duodenum due to mucosal thickening and hepatocellular swelling in both sexes of the higher dose group. Histological changes in the liver and duodenum disappeared due to discontinuation of administration, and other changes also disappeared or showed a recovery tendency after the 14 days post-exposure. No effects were observed in the 100 and 15 mg/kg group in both males and females. Based on the above results, the NOEL under the test conditions was considered to be 100 mg/kg/day in both sexes.

Repeated dose toxicity: dermal. Weight of evidence: In a 13 -week subchronic dermal toxicity study performed on the test item by the NTP, method similar to OECD 411 (GLP study), groups of 10 male and 10 female F-344 rats were dermally administered 0, 0.75, 1.5, 3, 6 or 12 mg/kg bw of test item in ethanol (0.5 mL/kg), 5 days a week for 13 weeks. The NOAEL for the subchronic dermal toxicity of the test item in rats was 3 mg/kg bw in males and 6 mg/kg bw in females. In a 13 -week subchronic dermal toxicity study performed on the test item by the NTP, method similar to OECD 411 (GLP study), groups of 10 male and 10 female B6C3F1 mice were dermally administered 0, 1.5, 3, 6, 12 or 24 mg/kg bw of test item in ethanol (2 mL/kg), 5 days a week for 13 weeks. The NOAEL for the subchronic dermal toxicity of the test item in mice was 3 mg/kg bw in males and in females. In a 16-day repeated dose dermal study in F-344 rats by the NTP (GLP study), where male and female rats were dosed 5 days a week for 16 days, the NOAEL was found to be ca. 12 mg/kg bw in rats. The same study was also performed in B6C3F1 male and female mice, and the NOAEL was found to be ca. 8 mg/kg bw in mice.

Justification for classification or non-classification

Based on available information, the substance is not classified for specific target organ toxicity-repeated exposure (STOT-RE) according
to the CLP criteria; Regulation (EC) No 1272/2008.