Registration Dossier

Administrative data

Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1987-11-19 - 1987-12-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report Date:
1987

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPP 81-2 (Acute Dermal Toxicity)
Version / remarks:
revised Nov. 1984
Deviations:
no
GLP compliance:
yes
Remarks:
US FDA (21 CFR 58) and US EPA (40 CFR 160 and 40 CFR 792)
Test type:
fixed dose procedure
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Appearance: Black powder
Bulk density: 0.21 g/mL
Solubility: Water: approximately 1 ppm; acetone: 0.81 g/100mL (20 °C); chloroform: 2 g/100mL (20 °C)
Storage: At ambient temperature in the dark in closed container
Stability: At least 14 days at 55 °C; poor in organic solvents

Test animals

Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
Adult rabbits of the NZW strain (9-10 weeks old at study start, SPF-quality) were supplied by the Broekman Institute, Someren, The Netherlands. Upon receipt each animal was identified with an ear tag. At least five days prior to dosing (acclimatization period) the animals were individually housed in metal cages (RUCO, Valkenswaard, The Netherlands). The animals had free access to tap-water (via automatic nozzles) and were held on a restricted diet of a standard laboratory animal feed (LK-01) obtained from Hope Farms, Woerden, The Netherlands. Certificates of analysis for both diet and drinking water are retained in the NOTOX archives.

ENVIRONMENTAL CONDITIONS
The animal room was air-conditioned, with the temperature maintained within the range of 20-21°C and the relative humidity within the range of 55-65% during the study. The artificial light cycle was 12 hours light, 12 hours dark.

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
methylcellulose
Details on dermal exposure:
Dosing
On the day of dosing the test substance was suspended in 1% aqueous methyl cellulose (Boom B.V., Meppel , The Netherlands) and was evenly spread on a surgical gauze (6x10 cm) that was fixed to successively micropore and flexible bandage (Coban, 3M, St. Paul, Minn., USA). This patch was applied to the clipped area of each animal and fixed with adhesive tape (Sleek, Smith and Nephew, Hull, U.K.). The dose volume was 7.5 ml/kg body weight. The dose level was 4000 mg/kg body weight. The day of dosing was designated as day 0.

Observations
Cage-side observations were performed on the day of dosing (approximately once every two hours) and once daily thereafter for 14 days. Any signs of toxicity were recorded along with the time of onset and duration. Individual body weights were measured weekly. At the end of the study (day 14) all animals were killed with euthesate and subjected to necropsy. In addition, observations regarding changes of the treated skin surface were performed following bandage removal and on day 4, 7, and 14.
Duration of exposure:
24 hours
Doses:
4000 mg/kg bw
No. of animals per sex per dose:
5 males and 5 females
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: daily (Observations regarding changes of the treated skin surface were performed following bandage removal and on day 4, 7, and 14.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight,organ weights, histopathology, other:
Statistics:
not reported

Results and discussion

Preliminary study:
Upon request of the sponsor the test could be performed as a limit test. One group of animals, comprising 5 males and 5 females, was treated with a single dermal dose of test substance at 4000 mg/kg body weight.
Effect levels
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 4 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortalities occurred during the 14-day observation period.
Clinical signs:
Six animals showed a closed 3rd eyelid which was red or swollen in some animals (Table 1). The treated skin surface of the animals revealed slight erythema and oedema, which disappeared by the end of the first week (Table 2). In addition the treated surface was black discoloured due to staining by the test substance.
Body weight:
All animals showed body weight gain during the study period (Table 3).
Gross pathology:
Macroscopic abnormalities at necropsy at the end of the observation period included local dark red areas in the lungs, enlarged spleen, accessory spleen tissue in the pancreas, dilated ileum and some petechiae in the dorso-lateral muscles (Table 1).
Other findings:
not reported

Any other information on results incl. tables

Table 1: Individual Animal Observations
Acute Dermal Toxicity study in the rabbit - Test item at 4000 mg/kg
Physical/Clinical Parameter Animal Number/Sex
3202
M
3204
M
3206
M
3208
M
3210
M
3250
F
3252
F
3254
F
3258
F
3260
F
No effects of treatment DPD 14 14 14
3rd eyelid
- closed DPD 2-4 2
- closed and swollen DPD 1
- closed and red DPD 1 1
Dead KIL X X X X X X X X X X
Necropsy findings:
- no gross lesions X X X X X X X
- Lungs local dark red areas X
- Enlarged spleen X
- Accessory spleen tissue in the pancreas X
- diated ileum X
- some small petechiae in dorso-lateral muscles X X
Glossary of terms for lsit of individuel findings:
DPD = Days post dosing
KIL = Killed at necropsy
KGL = no gross lesions
X = positive

Table 2: Group Incidences for irritation of the treated skin surface
Acute dermal toxicity study in the rabbit - Test iteml at 4000 mg/kg body weight
Observation Day of Evaluation
1* 4 7 14
No abnomalities 0 3 5 10
Sligth erythema 9 0 0 0
Oedema 9 2 0 0
Black discolouration 9 6 5 0
* Following bandage removal

Table 3: Indivudual body weights and dose volumes
Acute dermal toxicity study in the rabbit - Test item at 4000 mg/kg body weight
Dose Level (mg/kg) Animal number Body Weight (g) on: Body weight Gain (g) Day 0-14 Dose Volume (mL)
Day 0* Day 7 Day 14
4000
(Males)
3202 2196 2343 2536 340 16.5
3204 2395 2402 2513 118 18.0
3206 1804 1920 2174 370 13.5
3208 2065 2237 2358 293 15.5
3210 1991 2125 2290 299 14.9
Mean 2090 2205 2347 284
S.D. 221.6 191.4 152.4 98.0
N 5 5 5 5
4000
(Females)
3250 2145 2279 2347 202 16.1
3252 2152 2283 2402 250 16.1
3254 2277 2352 2470 193 17.1
3258 2172 2215 2356 184 16.3
3260 2349 2424 2502 153 17.6
Mean 2219 2313 2415 196
S.D. 90.1 83.4 68.7 35.2
N 5 5 5 5
* Day of dosing

Applicant's summary and conclusion

Interpretation of results:
other: EU GHS criteria not met
Conclusions:
Under the conditions used in this study, the LD50 value for both males and females was noted as exceeding 4000 mg/kg.
Executive summary:

The test material has been assessed in an acute dermal toxicity study in accordance with U.S. EPA FIFRA Guideline 81 -2 and in compliance with GLP. One group of NZW rabbits, comprising 5 males and 5 females, was treated with a single dermal dose of the test item at 4000 mg/kg body weight for 24 hours. No mortalities occurred during the 14-day observation period. A sign of toxicity was a closed and red or swollen 3rd eyelid. The treated skin surface of the animals revealed slight erythema and oedema, which disappeared by the end of the first week. In addition the treated surface was black discoloured due to staining by the test substance. All animals showed body weight gain during the study period. Macroscopic abnormalities noted in animals at necropsy included local dark red areas in the lungs, enlarged spleen, accessory spleen tissue in the pancreas, dilated ileum and petechiae in the dorso-lateral muscles. The LD50 value for both males and females was noted as exceeding 4000 mg/kg body weight.