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Diss Factsheets

Administrative data

Endpoint:
repeated dose toxicity: dermal, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Sep. to Oct. 1983
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1983
Report date:
1984

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:
1981
Deviations:
yes
Remarks:
Females weighed less than protocol requirements. Temperature occasionally fell below 19 °C by 1 °C. Humidity occasionally exceeded upper limit.
GLP compliance:
not specified
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
400-320-4
EC Name:
-
Cas Number:
94933-05-8
Molecular formula:
not applicable
IUPAC Name:
chromium(3+) trisodium bis(2-(2-{4-hydroxy-2-oxido-3-[2-(5-sulfonatonaphthalen-1-yl)diazen-1-yl]phenyl}diazen-1-yl)benzoate)
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Crl:CD(SD)BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Ltd., Manston Road, Margate, Kent, England
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: 148 to 245 g
- Housing: individually in stainless steel wire mesh cages (All Type Tools Ltd., Woolwich, England) suspended over cardboard-lined trays, replaced when necessary to maintain hygiene
- Diet: ad libitum except in periods of fasting before blood sampling; pelleted diet, SQC Rat and Mouse Maintenance Diet No. 1, Expanded (Special Diets Services Ltd., Stepfield, Witham, Essex, England) (tested for concentration of heavy metals, aflatoxins and insecticides)
- Water: ad libitum; mains water from glass bottles changed weekly and attached to cage (periodic analyses for heavy metals and chlorinated hydrocarbons supplied by local water authority; tested for polycyclic aromatic hydrocarbons)
- Acclimation period: 14 days after examination for external signs of good health

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 24 °C
- Humidity: 55 to 80 %
- Air changes: at least 15 per hour
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Type of coverage:
occlusive
Vehicle:
water
Remarks:
distilled water
Details on exposure:
TEST SITE
- Area of exposure: dorso-lumbar region
- Coverage: 60 x 40 mm
- Applicaion: using a ball-ended metal cannula and a plastic disposable syringe.
- Type of wrap if used: porous gauze dressing then covered with a strip of impervious, non-irritating plastic wrapping (Sleek, Smith & Nephew, Welwyn Garden City, Herts, England); sealing ensured
- Time intervals for shavings or clipplings: as often as was necessary to maintain the area free of hair

REMOVAL OF TEST SUBSTANCE
- Washing: wiped, no washing
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount applied: 1000 mg/kg bw/day
- Concentration: 4.0 mg/kg bw/day
- Constant concentration used: yes
- Preparation: prepared daily before administration

USE OF RESTRAINERS FOR PREVENTING INGESTION: no
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
6 hours
Frequency of treatment:
daily for 28 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
1000 mg/kg bw/day: 5 males and 5 females
Control (distilled water without addition of test item): 5 males and 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: a limit test using a dose level of 1000 mg/kg bw/day was allocated because of a lack of observable toxic effects at dose levels up to 2000 mg/kg bw in an acute dermal toxicity study (report no. 3521-252/32).
- Rationale for animal assignment: the selected animals were classed according to body weights, and were then allocated to groups using a stratified randomisation based on body weight.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
MORBIDITY / MORTALITY
- Time schedule: daily. At the beginning and end of each working day all animals were examined to detect any which were dead or moribund

CLINICAL CONDITIONS
- Time schedule: each animal was given a detailed clinical examination once daily for signs of ill health or overt toxicity. An individual record of clinical changes observed was maintained for each animal

DERMAL IRRITATION
- Time schedule for examinations: daily
- The presence of any signs of cutaneous irritation to the application site was assessed daily before dosing. The cutaneous reaction to treatment was scored according to the Draize scale (1959), specified in Table 1: Draize sccores for skin irritation in "Any other information on materials and methods", below.

BODY WEIGHT
- Time schedule for examinations: individual body weights were recorded on the first day of test, at weekly intervals throughout the study and again on the day of necropsy

FOOD CONSUMPTION
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: yes
- Time schedule: the weight of food consumed individually by each animal was determined weekly

HAEMATOLOGY
- Time schedule for collection of blood: week 3
- Anaesthetic used for blood collection: yes, light anaesthesia (Diethyl ether, Analar grade, BDH, Poole, Dorset, England)
- Animals fasted: yes, 16 hours (overnight)
- How many animals: all (20)
- Procedure: parameters were measured on blood collected into tubes containing EDTA anticoagulant
- Parameters: see Table 2: Haematology parameters examined in "Any other information on materials and methods", below

CLINICAL CHEMISTRY
- Time schedule for collection of blood: week 3
- Animals fasted: yes, 16 hours (overnight)
- Anaesthetic used for blood collection: yes, light anaesthesia (Diethyl ether, Analar grade, BDH, Poole, Dorset, England)
- How many animals: all (20)
- Procedure: estimations were performed on plasma from blood collected into tubes containing lithium heparin anticoagulant.
- Parameters: see Table 3: Clinical chemistry parameters examined in "Any other information on materials and methods", below

FOOD EFFICIENCY: not monitored

WATER CONSUMPTION: not monitored

OPHTHALMOSCOPIC EXAMINATION: not monitored

URINALYSIS: not monitored

NEUROBEHAVIOURAL EXAMINATION: not monitored
Sacrifice and pathology:
NECROPSY
The animals were killed by an intraperitoneal injection of pentobarbitone sodium solution (Euthatal, 200 mg/mL) (May & Baker, Dagenham, Essex, England) following an overnight fast. A full external and internal examination was made and all lesions were recorded.

GROSS PATHOLOGY
Organ weights were measured before fixation, while still wet, of adrenals, kidneys, liver and testes.

HISTOPATHOLOGY
Samples of the following tissues from all animals were preserved in 10 % neutral buffered formalin: adrenals, kidneys, gross lesions, liver, treated skin (2 sites), untreated skin (2 sites). The tissues were then embedded in paraffin wax B. P., sectioned at a nominal thickness of 5 µm and stained with haematoxylin and eosin. Microscopic evaluation: sections of tissue were examined by the study pathologist.
Statistics:
Data were processed to give group mean values and standard deviations, where appropriate.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment-related clinical signs observed in either treatment or control animals.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
- Test animals: slight redness of the treated skin site was noted in some females exposed to the test item on days 2 to 4 and in 1 male on days 23 to 28. Slightly increased redness (grade 2) was noted on the treated skin site on female 16 on days 22 to 29 and in female 20 on days 11 to 26. Very slight oedema was also noted on female 20 on days 11 and 12. One female showed skin sores on its back probably due to the occlusive dressing.
- Control animals: no adverse skin redness was noted in control animals.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- Test animals: no mortality was recorded in the 28-day study period.
- Control animals: one conrol male was found dead on day 22.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Overall body weight gains during the treatment period were considered to be similar in all animals. No notable difference in body weight gain was observed between the test and control groups of animals.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No adverse effects on food intake observed among either test or control animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No changes in the haematologic parameters of treated animals that could be attributed to treatment with the test item.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No changes in the clinical chemistry parameters that could be attributed to treatment with the test item.
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
All absolute organ weights and organ weight to body weight ratios were considered similar for treated and control animals.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- Test animals: brown discolouration on the tail, attributed to contamination by the test item; sores and fur loss on the abdomen, attributed to the adhesive bandage.
- Control animals: sores and fur loss on the abdomen, attributed to the adhesive bandage; one male died of a urinary tract obstruction which was considered an isolated and not study-related finding.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Skin reactions in the treated skin site of control rats consisted primarily of low grade acanthosis. This was more noticeable in males than females. The skin sites exposed to the test item were generally similar to those of the controls. One male and 1 female had erosions, but these were minor focal lesions, probably of traumatic origin, and the rest of the treated skin area was similar to controls. Overall, there was no evidence of any significant skin irritation due to repeated percutaneous application of the test item.
Histopathology findings in the selected tissues examined were generally infrequent and of minor nature such as hyaline droplets in the kidney of males and leukocyte foci in the livers of both sexes. There were no findings of any unusual nature or incidence to suggest any systemic toxic effect due to test item administration.
Histopathological findings: neoplastic:
not specified

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
dermal irritation
gross pathology
haematology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Table 1: Mean body weight and standard deviation (S.D.)

Week of study Group and gender
1M 2M 1F 2F
start mean: 221.6 218.8 158.8 156.6
S.D.: 10.01 15.94 8.98 7.09
1 mean: 265.4 264.4 175.0 174.4
S.D.: 13.81 18.74 11.77 9.02
2 mean: 301.0 303.2 191.0 191.0
S.D.: 19.91 25.91 13.62 11.81
3 mean: 311.5 319.4 200.2 194.2
S.D.: 22.11 28.12 15.55 14.81
4 mean: 339.3 340.2 210.6 206.8
S.D.: 24.03 28.65 19.81 16.45

Table 2: Mean food consumption (g/week) and standard deviation (S.D.).

Week of study Group and gender
1M 2M 1F 2F
1 mean: 230.4 201.2 159.8 193.2
S.D.: 19.82 12.32 15.74 89.50
2 mean: 218.4 213.2 158.0 155.8
S.D.: 18.69 22.71 15.60 13.52
3 mean: 200.2 212.4 170.2 149.8
S.D.: 45.07 19.09 18.62 11.82
4 mean: 236.5 219.2 176.8 163.2
S.D.: 8.19 8.29 18.89 14.13

Table 3: Individual body weights (g).

Group number Animal number Body weights (g) at week:
0 1 2 3 4
1M 1 216 266 313 329 350
2 234 268 297 305 333
3 220 277 310 - -
4 229 274 317 329 365
5 209 242 268 283 309
2M 6 213 268 313 336 354
7 205 245 278 290 303
8 245 290 325 336 351
9 222 272 327 347 374
10 209 247 273 388 319
1F 11 168 187 209 211 222
12 148 164 183 190 199
13 157 174 183 196 209
14 168 187 202 221 237
15 153 163 178 183 186
2F 16 153 180 199 205 222
17 152 175 191 190 209
18 159 165 181 182 197
19 151 166 178 180 184
20 168 186 206 214 222

Table 4: Individual food consumption (g/week).

Group number Animal number Consumption (g) at week:
1 2 3 4
1M 1 243 247 235 247
2 233 206 212 230
3 253 226 122 -
4 221 213 225 239
5 202 200 207 230
2M 6 208 233 231 225
7 187 298 194 218
8 216 219 220 213
9 205 234 227 230
10 190 182 190 210
1F 11 173 170 187 181
12 151 146 152 158
13 164 159 183 188
14 176 176 181 200
15 137 139 148 157
2F 16 352 169 167 180
17 165 155 144 159
18 136 143 142 148
19 151 142 139 153
20 162 170 157 176

Table 5: Individual skin irritation scores.

Group number Animal number Reaction Day of study
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
1M 1 erythema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
oedema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
2 erythema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
oedema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
3 erythema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 found dead in cage 6.10.83
oedema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
4 erythema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
oedema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
5 erythema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
oedema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
2M 6 erythema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
oedema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
7 erythema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
oedema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
8 erythema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1
oedema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
9 erythema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
oedema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
10 erythema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
oedema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
1F 11 erythema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
oedema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
12 erythema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
oedema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
13 erythema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
oedema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
14 erythema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
oedema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
15 erythema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
oedema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
2F 16 erythema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 2 2 2 2 2 2 2
oedema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
17 erythema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
oedema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
18 erythema 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
oedema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
19 erythema 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
oedema 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
20 erythema 0 0 1 0 0 0 0 0 0 0 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 0 0 0
oedema 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Table 6: Individual haematology data; occasion: week 3.

Group and gender Animal number Hb (g/dl) RBC (mil./cmm) PCV (%) MCH (pg) MCHC (g/dl) MCV (cum) PT WBC (1000/cmm) (%)
Sec Total N L M E B
1M 1 16.1 7.15 44 22.5 37 61 11.9 10.5 1.68(16) 8.61(82) .10(1) .10(1) .00(0)
2 15.9 7.09 43 22.4 37 60 12.7 17.6 .35(2) 17.07(97) .18(1) .00(0) .00(0)
3 16.4 7.11 45 23.1 36 64 12.9 13.9 1.67(12) 12.09(87) .14(1) .00(0) .00(0)
4 15.6 6.92 43 22.5 36 62 11.4 16.2 1.46(9) 14.58(90) .16(1) .00(0) .00(0)
5 16.6 7.77 43 21.4 38 56 11.7 10.7 1.60(15) 8.88(83) .21(2) .00(0) .00(0)
mean:  16.1 7.21 44 22.4 37 61 12.1 13.78 1.35(10) 12.25(89) .16(1) .02(0) .00(0)
S.D.:  0.4 0.33 1 0.6 1 3 0.6 3.19 .57(-) 3.65(-) .04(-) .04(-) .00(-)
2M 6 16.1 7.31 43 22 38 59 13.1 16 1.12(7) 14.72(92) .16(1) .00(0) .00(0)
7 16.8 7.39 45 22.7 37 61 11.9 14.4 1.49(12) 10.66(86) .25(2) .00(0) .00(0)
8 16.6 7.26 44 22.9 38 61 12.7 13.9 .97(7) 12.79(92) .14(1) .00(0) .00(0)
9 15.9 6.68 43 23.8 37 65 11.6 16.4 1.48(9) 14.76(90) .16(1) .00(0) .00(0)
10 16.1 7.3 43 22.1 37 60 11.6 13.5 3.91(29) 9.31(69) .27(2) .00(0) .00(0)
mean:  16.3 7.19 44 22.7 37 61 12.2 14.44 1.79(12) 12.45(86) .20(1) .00(0) .00(0)
S.D.:  0.4 0.29 1 0.7 1 2 0.7 1.7 1.20(-) 2.43(-) .06(-) .00(-) .00(-)
1F 11 16.3 7.27 43 22.4 38 59 11.1 12 1.08(9) 10.68(89) .12(1) .12(1) .00(0)
12 16.5 7.31 44 22.6 38 60 11.1 10.8 1.08(10) 9.61(89) .11(1) .00(0) .00(0)
13 16.2 7.66 45 21.1 36 58 10.8 11.4 .68(6) 10.60(93) .11(1) .00(0) .00(0)
14 15.9 6.92 42 23 38 61 10.3 12.4 .87(7) 11.53(93) .00(0) .00(0) .00(0)
15 17.2 7.92 46 21.7 38 58 10.9 10.2 1.43(14) 8.57(84) .10(1) .10(1) .00(0)
mean:  16.4 7.42 44 22.2 38 59 10.8 11.36 1.03(9) 10.20(90) .09(1) .04(0) .00(0)
S.D.:  0.5 0.38 2 0.8 1 1 0.3 0.89 .28(-) 1.14(-) .05(-) .06(-) .00(-)
2F 16 16.1 7.43 44 21.7 37 59 10.8 23.3 3.73(16) 19.11(82) .23(1) .23(1) .00(0)
17 16.6 7.78 44 21.3 38 56 10.9 7.4 .89(12) 6.44(87) .00(0) .07(1) .00(0)
18 16.1 7.18 42 22.4 38 59 11.1 8 .88(11) 7.12(89) .00(0) .00(0) .00(0)
19 15.8 7.16 42 22.1 38 59 10.8 9.3 1.67(18) 7.53(81) .09(1) .00(0) .00(0)
20 15.8 6.91 43 22.9 37 62 11 7.3 1.46(20) 5.84(80) .00(0) .00(0) .00(0)
mean:  16.1 7.29 43 22.1 38 59 10.9 11.06 1.73(16) 9.21(83) .06(1) .06(1) .00(0)
S.D.:  0.3 0.33 1 0.6 1 2 0.1 6.89 1.17(-) 5.57(-) .10(-) .10(-) .00(-)

Table 7: Individual clinical chemistry data; occasion: week 3.

Group and gender Animal number GOT (Iu/l) Alk.P (Iu/l) Sodium (mEq/l) Potassium (mEq/l) Chloride (mEq/l) Calcium (mEq/l) Glucose (mg//dl) B.U.N. (mg/dl) Creatine (mg/dl) T.Bilirubin (mg/dl) Protein  A/G ratio I.Phos (mg/dl)
Total (g/dl) Albumin (g/dl)
1M 1 86 310 151 3.6 94 10.1 100 14 1.0 0.1 5.9 3.3 1.3 8.5
2 75 380 146 3.6 97 9.6 113 13 0.9 0.1 5.6 3.1 1.2 5.1
3 90 374 139 3.8 90 10.4 114 12 1.0 0.0 6.0 3.3 1.2 7.5
4 57 376 146 3.3 95 10.0 88 13 1.0 0.1 5.4 3.3 1.6 7.3
5 75 430 147 3.4 93 9.9 126 14 1.0 0.0 6.1 3.5 1.3 7.6
mean:  77 374 146 3.5 94 10.0 108 13 1.0 0.1 5.8 3.3 1.3 7.2
S.D.:  13 43 4 0.2 3 0.3 15 1 0.0 0.1 0.3 0.1 0.2 1.3
2M 6 65 367 146 3.4 94 11.0 108 13 1.4 0.1 5.9 3.2 1.2 7.7
7 71 390 147 3.8 93 10.5 112 21 1.1 0.0 6.2 3.5 1.3 7.4
8 58 327 148 3.4 95 10.3 126 19 1.1 0.0 6.0 3.4 1.3 7.3
9 80 401 149 3.6 101 11.0 127 16 1.1 0.0 6.2 3.6 1.4 7.1
10 77 492 151 3.2 99 10.7 92 17 1.1 0.1 6.2 3.5 1.3 6.3
mean:  70 395 148 3.5 96 10.7 113 17 1.2 0.0 6.1 3.4 1.3 7.2
S.D.:  9 61 2 0.2 3 0.3 14 3 0.1 0.1 0.1 0.2 0.1 0.5
1F 11 39 270 147 2.9 97 10.9 95 18 1.0 0.1 6.8 3.9 1.3 5.9
12 72 462 146 3.8 95 10.4 116 16 1.0 0.1 6.0 3.9 1.9 6.4
13 75 325 149 2.7 98 9.9 100 16 1.1 0.1 6.2 3.6 1.4 5.3
14 121 170 145 3.5 91 10.2 109 18 1.0 0.0 6.4 3.7 1.4 7.5
15 135 20 146 3.3 99 10.3 99 15 1.1 0.0 6.6 3.7 1.3 6.4
mean:  88 285 147 3.2 96 10.3 104 17 1.0 0.1 6.4 3.8 1.5 6.3
S.D.:  39 116 2 0.4 3 0.4 9 1 0.1 0.1 0.3 0.1 0.3 0.8
2F 16 75 248 142 3.4 98 10.4 110 15 1.1 0.0 6.3 3.6 1.3 6.2
17 95 299 146 3.4 95 10.1 114 18 1.2 0.0 6.3 3.6 1.3 6.1
18 80 381 142 3.2 97 10.0 114 15 1.2 0.1 6.5 3.6 1.2 5.8
19 82 227 145 3.5 94 9.5 99 22 1.2 0.0 6.3 3.5 1.2 5.9
20 88 348 149 3.1 93 10.5 121 17 1.1 0.0 6.4 3.8 1.5 5.7
mean:  84 301 145 3.3 95 10.1 112 17 1.2 0.0 6.4 3.6 1.3 5.9
S.D.:  8 65 3 0.2 2 0.4 8 3 0.1 0.0 0.1 0.1 0.1 0.2

Table 8: Individual organ weights of test animals.

Group and gender Animal number Body weight Brain Liver Heart Gonads Adrenals Kidneys
Left  Right Total Left  Right Total Left  Right Total
1M 1 341 - 14.15 - 1.589 1.636 3.225 0.028 0.033 0.061 1.27 1.30 2.57
2 319 - 11.71 - 1.364 1.395 2.759 0.034 0.031 0.065 0.98 0.98 1.96
4 353 - 13.89 - 1.658 1.681 3.339 0.041 0.033 0.074 1.31 1.26 2.58
5 295 - 12.23 - 1.601 1.601 3.202 0.022 0.027 0.049 1.11 1.18 2.29
mean: 327 - 12.99 - 1.553 1.578 3.131 0.031 0.031 0.062 1.17 1.18 2.35
S.D.: 25.6 - 1.206 - 0.13 0.126 0.255 0.008 0.003 0.01 0.53 0.142 0.291
2M 6 336 - 13.76 - 1.469 1.453 2.922 0.036 0.028 0.064 1.29 1.24 2.52
7 293 - 10.19 - 1.741 1.764 3.505 0.032 0.027 0.059 1.08 1.10 2.18
8 337 - 16.94 - 1.552 1.550 3.102 0.029 0.037 0.066 1.30 1.37 2.67
9 358 - 13.89 - 1.645 1.634 3.279 0.033 0.037 0.070 1.38 1.43 2.81
10 303 - 11.54 - 1.633 1.611 3.244 0.036 0.041 0.077 1.07 1.03 2.10
mean: 325 - 13.26 - 1.608 1.602 3.210 0.033 0.034 0.067 1.22 1.23 2.46
S.D.: 26.7 - 2.577 - 0.103 0.114 0.217 0.003 0.006 0.007 0.14 0.171 0.308
1F 11 216 - 9.14 - 0.034 0.034 0.068 0.033 0.033 0.066 0.76 0.80 1.56
12 191 - 7.57 - 0.030 0.029 0.059 0.025 0.027 0.052 0.67 0.70 1.36
13 206 - 7.62 - 0.040 0.039 0.079 0.037 0.034 0.071 0.70 0.73 1.43
14 224 - 10.07 - 0.030 0.028 0.058 0.038 0.036 0.074 0.83 0.86 1.69
15 186 - 7.14 - 0.028 0.028 0.056 0.036 0.034 0.070 0.66 0.73 1.39
mean: 205 - 8.31 - 0.032 0.032 0.064 0.034 0.033 0.067 0.72 0.76 1.49
S.D.: 16.1 - 1.243 - 0.005 0.005 0.010 0.005 0.003 0.009 0.072 0.066 0.137
2F 16 212 - 9.57 - 0.031 0.027 0.058 0.029 0.026 0.055 0.76 0.76 1.52
17 202 - 7.33 - 0.027 0.029 0.056 0.040 0.039 0.079 0.62 0.67 1.29
18 188 - 6.64 - 0.024 0.018 0.042 0.031 0.022 0.053 0.66 0.71 1.37
19 180 - 6.31 - 0.022 0.027 0.049 0.031 0.031 0.062 0.62 0.69 1.32
20 214 - 8.32 - 0.027 0.031 0.058 0.032 0.034 0.066 0.71 0.75 1.46
mean: 199 - 7.63 - 0.026 0.026 0.053 0.033 0.030 0.063 0.67 0.72 1.39
S.D.: 14.9 - 1.329 - 0.003 0.005 0.007 0.004 0.007 0.010 0.060 0.037 0.097

Table 9: Group incidence: necropsy - all animals.

Tissue/observation Group no.: 1 2 1 2
Sex: M M F F
Abdominal cavity: gelatinous 1 0 0 0
Bladder: distension 1 0 0 0
Heart: pale area  1 0 0 0
Kidney: enlarged 1 0 0 0
Kidney: hydronephrosis 1 1 0 1
Prostate: red 1 0 0 0
Skin subcutis: sore 1 5 4 2
Skin subcutis: alopecia 4 5 5 5
Skin treated: sore 0 0 0 1
Tail: abnormal colour 0 5 0 5

Ureter: distension

 1 0 0 0
Number of animals examined 5 5 5 5

Table 10: Group incidence: histopathology - all animals.

Tissue/observation Group no.: 1 2 1 2
Sex: M M F F
Abdominal cavity: lesion 1 0 0 0
Adrenal hypertrophy 1 0 0 0
Bladder: cyctitis 1 0 0 0
Heart: myocarditis 1 0 0 0
Kidney: casts 1 0 0 0
Kidney: hydronephrosis 1 1 0 1
Kidney: Tubular regeneration 1 2 0 0
Kidney: mineralisation 0 0 1 3
Kidney: hyaline droplets 3 3 0 0
Liver: congestion 1 0 0 0
Liver: leucocyte foci 4 4 2 5
Prostate: prostatitis 1 0 0 0
Skin subcutis: acanthosis 4 5 5 5
Skin treated: not remarkable 0 0 1 0

Skin treated: erosion

 0 1 0 1
Skin treated: acanthosis 5 5 4 5
Skin untreated: not remarkable 0 0 4 3
Skin untreated: acanthosis 4 5 1 2
Tail: abnormal colour 0 5 0 5
Ureter: distension 1 0 0 0

Number of animals examined

 5 5 5 5

Applicant's summary and conclusion

Conclusions:
NOAEL (dermal, rat, 28 days) ≥ 1000 mg/kg bw/day
Executive summary:

The repeated dose toxicity of the test item was evaluated in a subacute, 28-day dermal study using a method comparable to the OECD Guideline 410 (1981). 5 male and 5 female Sprague-Dawley rats were exposed to 1000 mg/kg bw/day of test item to the shaved, dorsal region for 6 hours/day for 28 days, after which they were sacrificed. An additional 5 male and 5 female rats were administered the vehicle alone (4.0 ml/kg bw/day bi-distilled water) and monitored similarly in parallel to the test group. Mortality, skin irritation and clinical signs were monitored daily; body weight and food consumption were determined weekly. Blood samples were taken at 3 weeks and analysed for haematological and clinical chemistry parameters. Upon termination, necropsy, organ weight and histological examination were performed.

One control animal died spontaneously on day 22 due to a urinary tract obstruction. No change in clinical observations, body weight gains or food intakes related to treatment of the test item were observed. Skin irritation at the treatment site occurred in some animals exposed to 1000 mg/kg bw/day and in no controls: slight redness was observed in one female (days 2 to 4) and one male (days 23 to 28); slightly increased redness (grade 2) of the treatment site was observed in two females (days 22 to 29 in one and days 11 to 26 in the other); and very slight oedema was observed in one female (days 11 and 12). This did not indicate systemic toxicity. No significant changes in haematological and clinical chemistry parameters were observed. Brown skin discolouration to the tail was observed consistently. Histopathic examination of the treated skin site demonstrated erosions in 1 male and 1 female test animal. No consistent histopathological findings were observed which would suggest systemic toxicity due to test item application.

Based on the results observed, the NOEL for the substance in male and female rats is equal to or greated than 1000 mg/kg bw/day.