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Toxicological information

Repeated dose toxicity: dermal

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
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.

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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).

Results and discussion

Results of examinations

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.

Effect levels

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

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.