Registration Dossier
Registration Dossier
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EC number: 443-870-0 | CAS number: 163520-33-0
Endpoint summary
Administrative data
Description of key information
Oral, OECD 453, rat, chronic: systemic NOAEL (males) = 8 mg/kg bw/day; systemic NOAEL (females) = 12 mg/kg bw/day
Dermal, OECD 410, rat, subacute: systemic NOAEL = 1000 mg/kg bw/day, local NOAEL = 10 mg/kg bw/day = 0.5 mg/cm²/day
Key value for chemical safety assessment
- Toxic effect type:
- dose-dependent
Repeated dose toxicity: via oral route - systemic effects
Link to relevant study records
- Endpoint:
- chronic toxicity: oral
- Remarks:
- combined chronic toxicity and carcinogenicity study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 14 Nov 1996 - 27 Nov 1998
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
- Version / remarks:
- Current version adopted in 2018
- Deviations:
- yes
- Remarks:
- Thyroid and uterus weights were not measured.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
- Version / remarks:
- Guideline in place during study conduct: adopted in 1981
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)
- Version / remarks:
- Adoption not stated
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: EPA OPP 83-5 (Combined Chronic Toxicity/Oncogenicity Study)
- Version / remarks:
- Adopted in 1984
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: JMAFF (Combined Chronic Toxicty/Oncogenicty Study)
- Version / remarks:
- adopted in 1985
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Remarks:
- CRL:CD (IGS) BR
- Details on species / strain selection:
- The rat was chosen as the test system according to regulatory guidelines.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River UK Ltd., Margate, UK
- Age at study initiation: 33 days
- Body weight at receipt (measured for 10% of the animals): male: 55 - 79 g, female: 55 - 84 g
- Fasting periods during study: overnight during urine collection
- Housing: group housed, 5 animals per cage by sex and dose group, in suspended polycarbonate cages with grid floor
- Diet: powdered laboratory rodent diet, Modified SQC Expanded Ground Rat and Mouse Maintenance Diet No. 1 (Special Diet Services Ltd., Witham, UK), ad libitum
- Water: tap water in drinking water quality, ad libitum
- Acclimation period: 15 days
DETAILS OF FOOD AND WATER QUALITY: At their respective levels in the diet and water, none of the contaminants known or expected to be present was considered likely to influence the outcome of the study.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 45 - 65
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 14 Nov 1996 To: 13 - 27 Nov 1998 - Route of administration:
- oral: feed
- Details on route of administration:
- The dietary route was selected as a major potential route of exposure to man.
- Vehicle:
- other: powdered laboratory rodent diet
- Remarks:
- Modified SQC Expanded Ground Rat and Mouse Maintenance Diet No. 1
- Details on oral exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly by two identical mixes.
- Mixing appropriate amounts: For the highest dose level, the required amount of test material and a small amount of diet were mixed. This mix was then blended with the remaining diet. Subsequent dietary concentrations were prepared by serial dilution. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Aliquots of freshly prepared test diets were taken were taken weekly from all concentration. Samples from Weeks 1, 4, 6, 11, 14, 23, 34, 47, 71, 83, 95 and 106 (all concentrations) were analysed. The results were within the range +10% to -15% of nominal.
- Duration of treatment / exposure:
- 52 weeks = 12 months (interim sacrifice)
104 weeks = 24 months (terminal sacrifice) - Frequency of treatment:
- Continuously
- Dose / conc.:
- 20 ppm
- Remarks:
- actual test substance intake: males: 0.8 mg/kg bw/day, females: 1.1 mg/kg bw/day, combined sexes: 1.0 mg/kg bw/day
- Dose / conc.:
- 200 ppm
- Remarks:
- actual test substance intake: males: 8 mg/kg bw/day, females: 12 mg/kg bw/day, combined sexes: 10.0 mg/kg bw/day
- Dose / conc.:
- 2 000 ppm
- Remarks:
- actual test substance intake: males: 84 mg/kg bw/day, females: 118 mg/kg bw/day, combined sexes: 101 mg/kg bw/day
- Dose / conc.:
- 4 000 ppm
- Remarks:
- actual test substance intake: males: 171 mg/kg bw/day, females: 249 mg/kg bw/day, combined sexes: 210 mg/kg bw/day
- No. of animals per sex per dose:
- 20 (interim sacrifice)
50 (terminal sacrifice) - Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale: Dose levels were based on the results of an earlier 90-day dietary rat study (M-141124-01-1).
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observation for abnormal behavior, including neuro-muscular coordination and physical appearance were done each morning and as well in the afternoon on Mondays to Fridays (except on holidays).
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once weekly prior to weighing.
BODY WEIGHT: Yes
- Time schedule for examinations: 10% of the animals upon receipt. Each animal at randomisation, at the start of treatment, at weekly intervals to Week 14, every second week thereafter and at necropsy.
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- For each sex, the weekly food consumption of each cage of animals was measured at weekly intervals for the first 13 weeks of treatment and approx. every 4 weeks thereafter.
- Compound intake calculated from the consumption and body weight data: Yes
FOOD EFFICIENCY: Yes
- Body weight gain in % calculated from the consumption and body weight gain data
WATER CONSUMPTION: Yes
- Time schedule for examinations: Weekly for each cage of the numerical last 20 animals from each group over 5-day periods (Monday to Friday) during Weeks 9, 17, 33 and 49.
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment in all animals and prior to the interim (Month 12) and terminal kill (Month 24) in the control and high dose group.
HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 3, 6, 12, 18 and 24 months of treatment.
- Anaesthetic used for blood collection: Yes (ether anaesthesia (Months 3, 6 and 12) or isoflurane anaesthesia (Months 18 and 24))
- Animals fasted: Yes
- How many animals: First 10 surviving animals/sex/dose group with the lowest identification number.
- Parameters checked: Haematocrit (HCT), Haemoglobin (HB), Red blood celIs (RBC), Mean cell volume (MCV), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Platelets (PLT), Prothrombin time (PT), White blood cells (WBC), Neutrophils (NEUT), Lymphocytes (LYMP), Monocytes (MONO), Eosinophils (EOS), Basophils (BASO), Large unstained cells (LUC), Reticulocyte count (RET), and Activated partial thromoplastin time (APTT).
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 3, 6, 12, 18 and 24 months of treatment.
- Anaesthetic used for blood collection: Yes (ether anaesthesia (Months 3, 6 and 12) or isoflurane anaesthesia (Months 18 and 24))
- Animals fasted: Yes
- How many animals: First 10 surviving animals/sex/dose group with the lowest identification number.
- Parameters checked: Total protein (PROT), Albumin (ALB), Total globulin (GLOB), A/G ratio (A/G), Calcium (CA), Phosphate (PO4), Sodium (NA), Potassium (K), Urea (UREA), Creatinine (CREAT), Glucose (GLUC), Total cholesterol (CHOL), Total bilirubin (TBIL), Chloride (CL), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Alkaline phosphatase (AP), G-glutamyl transpeptidase (GGT), and Creatine kinase (CPK).
URINALYSIS: Yes
- Time schedule for collection of urine: After 3, 6, 12, 18 and 24 months of treatment.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- How many animals: First 10 surviving animals/sex/dose group with the lowest identification number.
- Parameters checked: Appearance (APP), pH (PH), Protein (PROT), Glucose (GLUC), Specific gravity (SG), Colour of Spun deposit (SDEP), Bacteria (BACT), Red blood cells (RBC), Epithelial cells (EPTH), Ketones (KET), Urobilinogen (UBIL), Bilirubin (BIL), Blood (BLD), Phosphate crystals (PO4), Urate crystals (URAT), White blood cells (WBC), Sperm (SPER), Volume (VOL), and Casts (CAST).
NEUROBEHAVIOURAL EXAMINATION: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
- Time schedule: After approx. 52 weeks the first 20 animals/sex/group (interim kill); the remaining animals after 104 weeks (terminal kill)
- Organ weights: Adrenals, brain, heart, kidneys, liver, ovaries, spleen, and testes with epididymides.
- List of organs: Adrenals, aorta (abdominal), bile duct*, brain, caecum, colon, diaphragm, duodenum, epididymides, eyes, femur and joint, Harderian gland, head, heart, ileum, jejunum, kidneys, lacrimal gland (exorbital), liver, lungs (inflated), lymph nodes (cervical and mesenteric), mammary gland, muscle (skeletal), nerves (optic and sciatic), oesophagus, oviducts, ovaries, pancreas, pituitary, prostate, rectum, salivary glands (submaxillary, sublingual and parotid), seminal vesicles, skin, spinal cord (3 levels), spleen, sternum, stomach, testes, thymus, thyroid (with parathyroids), tongue, trachea, urinary bladder, uterus, vagina, and any other tissue showing macroscopic abnormalities.
* Taken from animals killed from Week 62 of treatment onwards.
HISTOPATHOLOGY: Yes
Histopathology was conducted on organs/tissues indicated above from all animals. - Statistics:
- The significance of differences between control and treated groups was analysed by either parametric or non-parametric statistical tests as appropriate. A maximum 2-tailed probability value of 5% (p <0.05) was considered statistically significant.
The following convention has been used to indicate statistical significance:
* p <0.05
** p <0.01
*** p <0.001 - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- At 4000 ppm a slight increased incidence of urogenital staining for males from approx. Week 41 onwards (non-adverse).
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- There was no treatment-related effect on mortality during the course of the study.
The incidence of mortality after 12 months of treatment was 2/70, 1/70, 3/70, 1/70 and1/70 for males and 3/70, 5/70, 1/70, 2/70 and 5/70 for females, at 0, 20, 200, 2000 and 4000 ppm, respectively.
The incidence of mortality at 24 months was 23/50, 27/50, 24/50, 19/50 and 17/50 for males and 33/50, 28/50, 27/50, 34/50 and 31/50 for females, at 0, 20, 200, 2000 and 4000 ppm, respectively. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- At 4000 ppm, body weight gain of females was reduced by 8% (non-adverse). This was mainly due to a marked reduction during Week 1 (38%) although a trend was seen throughout the study. Males of the same group showed as well a reduction in Week 1 (19%) but body weight gain in general was unaffected (see Table 1 under "Any other information on results incl. tables").
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- At 4000 ppm food intakes of both sexes were reduced during Week 1 but reached the control level thereafter (see Table 2 and 3 under "Any other information on results incl. tables").
- Food efficiency:
- no effects observed
- Description (incidence and severity):
- At 4000 ppm food conversion efficiency of females were reduced by 31% during Week 1 but reached the control level thereafter.
- Water consumption and compound intake (if drinking water study):
- effects observed, treatment-related
- Description (incidence and severity):
- At 4000 ppm a slight (18 - 27%) but consistent increase in water consumption was seen throughout the study for females (see Table 4 under "Any other information on results incl. tables").
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- Single parameters of treated animals distributed through all dose levels were statistically significantly changed compared to control. However, all changes were without any dose-response dependence, therefore, they are of no toxicological relevance.
- Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Statistically significantly increased urea and creatinine levels were observed at several time points at 2000 and 4000 ppm in both sexes (for details see attached result tables under "Attached background material"). However, all values were within the physiological range of this rat strain.
- Urinalysis findings:
- effects observed, treatment-related
- Description (incidence and severity):
- At Month 3, 6 and 12 an increased incidence of ketones present in the urine was seen in males treated at 2000 and 4000 ppm and also at Month 18 in males given 4000 ppm (see Table 5 under "Any other information on results incl. tables").
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- At interim kill, relative liver weights were slightly increased for males (16%) and females (14%) treated at 4000 ppm. At 2000 ppm, relative liver weights were increased (8%) for males; however, this is considered not toxicologically relevant as there was no histopathological correlate at terminal kill.
At terminal kill, both absolute and relative kidney weights were decreased for males at 4000 ppm (12% and 9%, respectively) and at 2000 ppm (10% and 13%, respectively). Furthermore, relative liver weights were increased (8%) for males given 4000 ppm (see Table 6 under "Any other information on results incl. tables"). - Gross pathological findings:
- no effects observed
- Neuropathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- At interim and terminal kill, an increased incidence of centrilobular hepatocytic hypertrophy (minimal to slight) was seen in males receiving 4000 ppm.
At terminal kill, an increased incidence and severity of progressive nephropathy was observed in females treated at 4000 ppm (see Table 7 under "Any other information on results incl. tables"). - Histopathological findings: neoplastic:
- no effects observed
- Description (incidence and severity):
- Administration of the test substance did not result in any alterations to indicate a carcinogenic potential from animals killed after 52 or 104 weeks of treatment (for details see attached data tables under "Attached background material").
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 200 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- other: actual test substance intake: males: 8 mg/kg bw/day, females: 12 mg/kg bw/day, combined sexes: 10 mg/kg bw/day
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 2 000 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- organ weights and organ / body weight ratios
- urinalysis
- Remarks on result:
- other: actual test substance intake: males: 84 mg/kg bw/day, females: 118 mg/kg bw/day, combined sexes: 101 mg/kg bw/day
- Critical effects observed:
- no
- Conclusions:
- Under the conditions of the present study, after chronic oral exposure to the test substance, a LOAEL of 101 mg/kg bw/day for combined sexes was identified based on urinary/renal findings.
Reference
Table 1: Group mean body weights (g)
Weeks |
Dose level (ppm) |
|||||||||
Males |
Females |
|||||||||
0 |
20 |
200 |
2000 |
4000 |
0 |
20 |
200 |
2000 |
4000 |
|
0-14 |
347 (−) |
338 (97) |
338 (97) |
328 (95) |
316 (91) |
151 (−) |
151 (100) |
150 (99) |
146 (97) |
132 (87) |
14-28 |
108 (−) |
110 (102) |
107 (99) |
108 (100) |
107 (99) |
39 (−) |
45 (115) |
43 (110) |
37 (95) |
37 (95) |
28-52 |
99 (−) |
107 (108) |
99 (100) |
103 (104) |
88 (89) |
70 (−) |
80 (114) |
81 (116) |
72 (103) |
60 (86) |
52-78 |
69 (−) |
76 (110) |
60 (87) |
62 (90) |
61 (88) |
81 (−) |
85 (105) |
64 (79) |
66 (81) |
45 (56) |
0-104 |
606 (−) |
650 (107) |
618 (102) |
635 (105) |
593 (98) |
359 (−) |
385 (107) |
365 (102) |
345 (96) |
332 (92) |
() Percentage of control values
Table 2: Group mean food consumption (g/animal/day)
Weeks |
Dose level (ppm) |
|||||||||
Males |
Females |
|||||||||
0 |
20 |
200 |
2000 |
4000 |
0 |
20 |
200 |
2000 |
4000 |
|
1-12 |
27 (−) |
27 (100) |
27 (100) |
27 (100) |
26 (96) |
21 (−) |
21 (100) |
21 (100) |
21 (100) |
20 (95) |
13-26 |
26 (−) |
27 (104) |
26 (100) |
26 (100) |
26 (100) |
20 (−) |
22 (110) |
21 (105) |
22 (110) |
21 (105) |
30-51 |
27 (−) |
27 (100) |
27 (100) |
27 (100) |
27 (100) |
22 (−) |
23 (105) |
22 (100) |
23 (105) |
22 (100) |
54-74 |
27 (−) |
27 (100) |
27 (100) |
27 (100) |
26 (96) |
22 (−) |
24 (109) |
23 (105) |
23 (105) |
23 (105) |
79-103 |
26 (−) |
27 (104) |
26 (100) |
27 (104) |
27 (104) |
23 (−) |
24 (104) |
24 (104) |
24 (104) |
24 (104) |
1-103 |
27 (−) |
27 (100) |
27 (100) |
27 (100) |
26 (96) |
22 (−) |
22 (100) |
22 (100) |
22 (100) |
22 (100) |
() Percentage of control values
Table 3: Group mean test substance intake (mg/kg bw/day)
Weeks |
|
Dose level (ppm) |
|||
20 |
200 |
2000 |
4000 |
||
1-104 |
Males |
0.8 |
8 |
84 |
171 |
Females |
1.1 |
12 |
118 |
249 |
|
Combined sexes |
1.0 |
10 |
101 |
210 |
|
Table 4: Group mean water consumption (g/animal/day)
Week |
Dose level (ppm) |
|||||||||
Males |
Females |
|||||||||
0 |
20 |
200 |
2000 |
4000 |
0 |
20 |
200 |
2000 |
4000 |
|
9 |
35 (−) |
32 (91) |
32 (91) |
34 (97) |
35 (100) |
28 (−) |
28 (100) |
30 (107) |
31 (111) |
33 (118) |
17 |
31 (−) |
31 (100) |
29 (94) |
32 (103) |
33 (106) |
27 (−) |
29 (107) |
29 (107) |
28 (104) |
33 (122) |
33 |
29 (−) |
30 (103) |
28 (97) |
31 (107) |
32 (110) |
34 (−) |
31 (91) |
34 (100) |
30 (88) |
41 (121) |
49 |
28 (−) |
32 (114) |
31 (111) |
29 (104) |
33 (118) |
33 (−) |
35 (106) |
38 (115) |
31 (94) |
42 (127) |
() Percentage of control values
Table 5: Group incidence of ketones present in urine
Month |
Dose level (ppm) |
|||||||||
Males |
Females |
|||||||||
0 |
20 |
200 |
2000 |
4000 |
0 |
20 |
200 |
2000 |
4000 |
|
3 |
1/10 |
0/10 |
3/10 |
9/10 |
10/10 |
2/10 |
0/10 |
0/10 |
0/10 |
2/10 |
6 |
0/10 |
0/9 |
2/10 |
9/10 |
10/10 |
3/10 |
0/10 |
1/10 |
1/10 |
2/10 |
12 |
0/10 |
1/9 |
0/10 |
5/10 |
9/10 |
0/10 |
0/10 |
0/10 |
0/10 |
0/10 |
18 |
0/10 |
0/10 |
0/10 |
2/10 |
5/10 |
0/10 |
0/10 |
0/10 |
0/10 |
0/10 |
24 |
0/10 |
0/10 |
0/10 |
2/10 |
2/10 |
1/10 |
0/10 |
1/9 |
0/10 |
0/10 |
Table 6: Treatment-related effects on organ weights at interim and terminal kill
|
Dose level (ppm) |
|||||||||
Males |
Females |
|||||||||
0 |
20 |
200 |
2000 |
4000 |
0 |
20 |
200 |
2000 |
4000 |
|
Interim kill |
|
|||||||||
Relative liver weight (%) |
2.67 (−) |
2.76 (103) |
2.68 (100) |
2.88 (108) |
3.10 (116) |
2.78 (−) |
2.97 (107) |
2.77 (100) |
2.95 (106) |
3.16 (114) |
Terminal kill |
|
|||||||||
Absolute kidney weights (g) |
4.11 (−) |
4.18 (102) |
4.29 (104) |
3.68 (90) |
3.60 (88) |
No treatment-related effects |
||||
Relative kidney weight (%) |
0.53 (−) |
0.51 (96) |
0.56 (106) |
0.46 (87) |
0.48 (91) |
|||||
Relative liver weight (%) |
2.56 (−) |
2.54 (99) |
2.70 (105) |
2.53 (99) |
2.77 (108) |
|||||
() Percentage of control values
Table 7: Histopathological treatment-related effects at interim and terminal kill
|
Dose level (ppm) |
|||||||||
Males |
Females |
|||||||||
0 |
20 |
200 |
2000 |
4000 |
0 |
20 |
200 |
2000 |
4000 |
|
Interim kill |
||||||||||
Liver |
|
|||||||||
Centrilobular hepatocytic hypertrophy |
1/19 |
2/20 |
0/19 |
5/20 |
15/20 |
No treatment-related effects |
||||
Terminal kill |
|
|||||||||
Liver |
|
|||||||||
Centrilobular hepatocytic hypertrophy |
0/26 |
0/22 |
0/25 |
0/31 |
12/33 |
No treatment-related effects |
||||
Kidney |
|
|||||||||
Progressive nephropathy |
No treatment-related effects |
25/50 |
22/50 |
23/50 |
17/50 |
31/50 |
||||
Minimal |
16 |
13 |
16 |
12 |
11 |
|||||
Slight |
8 |
7 |
4 |
5 |
10 |
|||||
Moderate |
0 |
2 |
2 |
0 |
6 |
|||||
Severe |
1 |
0 |
1 |
0 |
4 |
|||||
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 8 mg/kg bw/day
- Study duration:
- chronic
- Experimental exposure time per week (hours/week):
- 168
- Species:
- rat
- Quality of whole database:
- The study was conducted according to OECD TG 453 and in compliance with GLP (RL1).
- System:
- urinary
- Organ:
- kidney
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
- Endpoint:
- short-term repeated dose toxicity: dermal
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 27 May - 25 Jun 1997
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
- Version / remarks:
- Adopted in 1981
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))
- Version / remarks:
- Adopted in 1992
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)
- Version / remarks:
- Adopted in 1984
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Remarks:
- CRL:CD (SD) BR
- Details on species / strain selection:
- The rat was chosen as the test system according to regulatory guidelines.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River UK Ltd., Margate, UK
- Age at receipt: approximately 42 days
- Body weight at receipt: males: 180 - 196 g, females: 188 - 207 g
- Housing: individually, in polycarbonate cages with mesh bases suspended over waste trays. The waste tray of each cage was lined with absorbent paper
- Diet: ground laboratory rodent diet, Modified SQC Expanded Rat and Mouse Maintenance Diet No. 1 (Special Diet Services Ltd., Witham, UK), ad libitum
- Water: tap water in drinking water quality, ad libitum
- Acclimation period: 6 days
DETAILS OF FOOD AND WATER QUALITY: At their respective levels in the diet and water, none of the contaminants known or expected to be present was considered likely to influence the outcome of the study.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 27 May 1997 To: 25 Jun 1997 - Type of coverage:
- occlusive
- Vehicle:
- other: 1% (w/v) methyl cellulose in water
- Details on exposure:
- TEST SITE
- Area of exposure:dorsal area of the trunk
- % coverage: 10%
- Type of wrap if used: the test substance was held in contact with the skin on a 2 x 2 cm pad of porous gauze pad backed by aluminium foil which was held in place over the treatment site by elastic bandage
- Time intervals for shavings or clippings: clipping was repeated as necessary during the study
REMOVAL OF TEST SUBSTANCE
- Washing (if done): residual test material was rinsed of with soap and water
- Time after start of exposure: 6 h
TEST MATERIAL
- Amount applied: 5 mL/kg bw, daily preparation of test suspension
- Concentration: 2, 20 and 200 mg/mL
- Constant concentration used: yes
VEHICLE
- Amount(s) applied (volume or weight with unit): 5 mL/kg bw
USE OF RESTRAINERS FOR PREVENTING INGESTION: no - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The samples were analysed by extraction of AE F122006 from the suspensions by shaking with acetonitrile + methanol (90 + 10, v/v). The concentration of the test substance in the extract was measured by HPLC using UV detection.The mean results for the test suspension samples analysed and prepared for dosing were within the range 89.5 to 122.3% of nominal at the time of preparation (specified range +20% to -20% of nominal).
- Duration of treatment / exposure:
- 28 days (males)
29 days (females) - Frequency of treatment:
- 5 days/week, excluding weekends
- Dose / conc.:
- 10 mg/kg bw/day
- Dose / conc.:
- 100 mg/kg bw/day
- Dose / conc.:
- 1 000 mg/kg bw/day
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Dose levels were selected on the basis of findings in an earlier dose ranging study conducted immediately prior to this study with 4 groups of 3 males dosed at 0, 250, 500 and 1000 mg test substance kg bw/day for 7 days, excluding the weekend. There were no treatment-related effects at 1000 mg/kg bw/day, equivalent to the accepted international regulatory limit dose, in this range-finding study.
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed each morning for abnormal behaviour, including neuro-muscular coordination and physical appearance. Animals were also observed in the afternoon on Mondays to Fridays except on public and Company holidays.
DETAILED CLINICAL OBSERVATIONS: No
DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: daily
BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed at randomisation, at the start of treatment, weekly thereafter and at
necropsy.
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
FOOD EFFICIENCY: No
WATER CONSUMPTION: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 29
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No
- How many animals: 5 per sex and dose
- Parameters examined: Haematocrit (HCT), White blood cells (WBC), Haemoglobin (HB), Neutrophils (NEUT), Red blood cells (RBC), Lymphocytes (LYMP), Mean corpuscular volume (MCV), Monocytes (MONO), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Eosinophils (EOS), Basophils (BASO), Large unstained cells (LUC), Platelets (PLT), Reticulocyte count (RET), Prothrombin time (PT), and Activated partial thromboplastin time (APTT).
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29
- Animals fasted: No
- How many animals: 5 per sex and dose
- Parameters examined: Total protein (PROT), Albumin (ALB), Total globulin (GLOB), A/G ratio (A/G), Calcium (CA), Phosphate (PO4), Sodium (Na), Potassium (K), Urea (UREA), Creatinine (CREAT), Glucose (GLUC), Total cholesterol (CHOL), Total bilirubin (TBIL), Chloride (Cl), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Alkaline phosphatase (AP), G-glutamyl transpeptidase (GGT), and Creatine kinase (CPK).
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes, organ weights of kidneys, adrenals, liver and testes were recorded. Following tissues were fixed in 10% neutral buffered formalin: kidneys, liver, ovaries, pinnae, skin (treated and untreated), testes, adrenals, and any other tissue showing macroscopic abnormalities.
HISTOPATHOLOGY: Yes, of kidney and liver sections and treated and untreated skin. - Statistics:
- The significance of differences between control and treated groups was analysed by either parametric or non-parametric statistical tests as appropriate. A maximum 2-tailed probability value of 5% (p <0.05) was considered statistically significant.
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- Test substance related clinical signs other than local dermal irritation were not evident.
- Dermal irritation:
- effects observed, treatment-related
- Description (incidence and severity):
- At 1000 mg/kg bw/day, slight dermal irritation, as indicated by skin reddening at the application site, was seen in all animals from Week 2 till study termination. Slight sloughing, defined as shedding of the superficial layer of skin at the application site, was seen in 3/5 males and 3/5
females from Week 2 until Week 3 and 4, respectively.
At 100 mg/kg bw/day, slight dermal irritation was seen in 2/5 females from Day 4 and 11 to Day 6 and 19, respectively. - Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- No treatment-related effects were observed. At the 1000 mg/kg bw/day dose level a mild decrease in cholesterol levels was observed in female animals compared with concurrent control, but this decrease was within the normal physiological range of control data.
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- 100 mg/kg bw/day: scabs at the application site in 2/5 female rats
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- At 1000 mg/kg bw/day, 3/5 males and 5/5 females had mononuclear cell infiltration of the dermis to a moderate degree.
At 100 mg/kg bw/day, 2/5 males and 3/5 females had moderate cell infiltration.
At 10 mg/kg bw/day, 1/5 males had a mild infiltration. Because a similar lesion was observed in a control female this is considered not to be induced by application of the test compound. - Histopathological findings: neoplastic:
- not examined
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- systemic
- Effect level:
- 1 000 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no effects observed
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- local
- Effect level:
- 10 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no cell infiltration observed at doses <100 mg/kg bw/day
- Remarks on result:
- other: 0.5 mg/cm²/day, calculated based on a body weight of 200 g and a patch area of 4 cm²
- Key result
- Dose descriptor:
- LOAEL
- Remarks:
- local
- Effect level:
- 100 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- gross pathology
- histopathology: non-neoplastic
- Key result
- Critical effects observed:
- no
Reference
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 1 000 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- The study was conducted according to OECD TG 410 and in compliance with GLP (RL1).
Repeated dose toxicity: dermal - local effects
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: dermal
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 27 May - 25 Jun 1997
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
- Version / remarks:
- Adopted in 1981
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))
- Version / remarks:
- Adopted in 1992
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)
- Version / remarks:
- Adopted in 1984
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Remarks:
- CRL:CD (SD) BR
- Details on species / strain selection:
- The rat was chosen as the test system according to regulatory guidelines.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River UK Ltd., Margate, UK
- Age at receipt: approximately 42 days
- Body weight at receipt: males: 180 - 196 g, females: 188 - 207 g
- Housing: individually, in polycarbonate cages with mesh bases suspended over waste trays. The waste tray of each cage was lined with absorbent paper
- Diet: ground laboratory rodent diet, Modified SQC Expanded Rat and Mouse Maintenance Diet No. 1 (Special Diet Services Ltd., Witham, UK), ad libitum
- Water: tap water in drinking water quality, ad libitum
- Acclimation period: 6 days
DETAILS OF FOOD AND WATER QUALITY: At their respective levels in the diet and water, none of the contaminants known or expected to be present was considered likely to influence the outcome of the study.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 27 May 1997 To: 25 Jun 1997 - Type of coverage:
- occlusive
- Vehicle:
- other: 1% (w/v) methyl cellulose in water
- Details on exposure:
- TEST SITE
- Area of exposure:dorsal area of the trunk
- % coverage: 10%
- Type of wrap if used: the test substance was held in contact with the skin on a 2 x 2 cm pad of porous gauze pad backed by aluminium foil which was held in place over the treatment site by elastic bandage
- Time intervals for shavings or clippings: clipping was repeated as necessary during the study
REMOVAL OF TEST SUBSTANCE
- Washing (if done): residual test material was rinsed of with soap and water
- Time after start of exposure: 6 h
TEST MATERIAL
- Amount applied: 5 mL/kg bw, daily preparation of test suspension
- Concentration: 2, 20 and 200 mg/mL
- Constant concentration used: yes
VEHICLE
- Amount(s) applied (volume or weight with unit): 5 mL/kg bw
USE OF RESTRAINERS FOR PREVENTING INGESTION: no - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The samples were analysed by extraction of AE F122006 from the suspensions by shaking with acetonitrile + methanol (90 + 10, v/v). The concentration of the test substance in the extract was measured by HPLC using UV detection.The mean results for the test suspension samples analysed and prepared for dosing were within the range 89.5 to 122.3% of nominal at the time of preparation (specified range +20% to -20% of nominal).
- Duration of treatment / exposure:
- 28 days (males)
29 days (females) - Frequency of treatment:
- 5 days/week, excluding weekends
- Dose / conc.:
- 10 mg/kg bw/day
- Dose / conc.:
- 100 mg/kg bw/day
- Dose / conc.:
- 1 000 mg/kg bw/day
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Dose levels were selected on the basis of findings in an earlier dose ranging study conducted immediately prior to this study with 4 groups of 3 males dosed at 0, 250, 500 and 1000 mg test substance kg bw/day for 7 days, excluding the weekend. There were no treatment-related effects at 1000 mg/kg bw/day, equivalent to the accepted international regulatory limit dose, in this range-finding study.
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed each morning for abnormal behaviour, including neuro-muscular coordination and physical appearance. Animals were also observed in the afternoon on Mondays to Fridays except on public and Company holidays.
DETAILED CLINICAL OBSERVATIONS: No
DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: daily
BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed at randomisation, at the start of treatment, weekly thereafter and at
necropsy.
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
FOOD EFFICIENCY: No
WATER CONSUMPTION: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 29
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No
- How many animals: 5 per sex and dose
- Parameters examined: Haematocrit (HCT), White blood cells (WBC), Haemoglobin (HB), Neutrophils (NEUT), Red blood cells (RBC), Lymphocytes (LYMP), Mean corpuscular volume (MCV), Monocytes (MONO), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Eosinophils (EOS), Basophils (BASO), Large unstained cells (LUC), Platelets (PLT), Reticulocyte count (RET), Prothrombin time (PT), and Activated partial thromboplastin time (APTT).
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29
- Animals fasted: No
- How many animals: 5 per sex and dose
- Parameters examined: Total protein (PROT), Albumin (ALB), Total globulin (GLOB), A/G ratio (A/G), Calcium (CA), Phosphate (PO4), Sodium (Na), Potassium (K), Urea (UREA), Creatinine (CREAT), Glucose (GLUC), Total cholesterol (CHOL), Total bilirubin (TBIL), Chloride (Cl), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Alkaline phosphatase (AP), G-glutamyl transpeptidase (GGT), and Creatine kinase (CPK).
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes, organ weights of kidneys, adrenals, liver and testes were recorded. Following tissues were fixed in 10% neutral buffered formalin: kidneys, liver, ovaries, pinnae, skin (treated and untreated), testes, adrenals, and any other tissue showing macroscopic abnormalities.
HISTOPATHOLOGY: Yes, of kidney and liver sections and treated and untreated skin. - Statistics:
- The significance of differences between control and treated groups was analysed by either parametric or non-parametric statistical tests as appropriate. A maximum 2-tailed probability value of 5% (p <0.05) was considered statistically significant.
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- Test substance related clinical signs other than local dermal irritation were not evident.
- Dermal irritation:
- effects observed, treatment-related
- Description (incidence and severity):
- At 1000 mg/kg bw/day, slight dermal irritation, as indicated by skin reddening at the application site, was seen in all animals from Week 2 till study termination. Slight sloughing, defined as shedding of the superficial layer of skin at the application site, was seen in 3/5 males and 3/5
females from Week 2 until Week 3 and 4, respectively.
At 100 mg/kg bw/day, slight dermal irritation was seen in 2/5 females from Day 4 and 11 to Day 6 and 19, respectively. - Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- No treatment-related effects were observed. At the 1000 mg/kg bw/day dose level a mild decrease in cholesterol levels was observed in female animals compared with concurrent control, but this decrease was within the normal physiological range of control data.
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- 100 mg/kg bw/day: scabs at the application site in 2/5 female rats
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- At 1000 mg/kg bw/day, 3/5 males and 5/5 females had mononuclear cell infiltration of the dermis to a moderate degree.
At 100 mg/kg bw/day, 2/5 males and 3/5 females had moderate cell infiltration.
At 10 mg/kg bw/day, 1/5 males had a mild infiltration. Because a similar lesion was observed in a control female this is considered not to be induced by application of the test compound. - Histopathological findings: neoplastic:
- not examined
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- systemic
- Effect level:
- 1 000 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no effects observed
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- local
- Effect level:
- 10 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no cell infiltration observed at doses <100 mg/kg bw/day
- Remarks on result:
- other: 0.5 mg/cm²/day, calculated based on a body weight of 200 g and a patch area of 4 cm²
- Key result
- Dose descriptor:
- LOAEL
- Remarks:
- local
- Effect level:
- 100 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- gross pathology
- histopathology: non-neoplastic
- Key result
- Critical effects observed:
- no
Reference
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 0.5 mg/cm²
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- The study was conducted according to OECD TG 410 and in compliance with GLP (RL1).
Additional information
Reliable subacute, subchronic and chronic studies with dietary administration of the test substance have been conducted in mice, rats and dogs.
Subacute studies
In a 28 day-study, the test substance was administered via the diet to 5 mice/sex/group at dose levels of 2500, 5000 and 10000 ppm (equivalent to 507, 1101 and 1806 mg/kg bw/day for the combined sexes). The study was performed similar to OECD TG 407. However, the objective of this study was to characterise the toxicity of the test substance in mice for the selection of suitable dose levels for a subsequent 90-day dietary repeated dose toxicity study in this species. Therefore, detailed clinical observations and several organ weights were not assessed and histopathological examinations were performed on a limited range of tissues only. The LOAEL was 507 mg/kg bw/day (actual test substance intake: males: 424 mg/kg bw/day, females: 590 mg/kg bw/day) (M-147436-01-1, 1998). The target organs in this study were heart, liver and kidneys and were all affected at the LOAEL (lowest dose tested). The observed adverse effects were: increased absolute and relative liver weights in both sexes, histopathological changes in the heart (focal sub-endocardial necrosis and degeneration (2/5 males and 1/5 females)), and juxta-glomerular hyperplasia of the kidneys (4/5 males and 4/5 females).
A second 28 day-study was conducted in dogs, administering 40, 400 and 1250 ppm (corresponding to 1.9, 17.4 and 49.4 mg/kg bw/day for combined sexes) via the diet to 2 animals/sex/group. The study was performed similar to OECD 409 and the NOAEL was 1.9 mg/kg bw/day (M-147707-02-1, 1998). The LOAEL (17.4 mg/kg bw/day) was based on a the increased incidence and/or severity of vacuolation of the renal cortical distal tubules in both sexes.
Subchronic studies
In a 90-day study performed similar to OECD TG 408, 10 mice/sex/group were administered 13, 125, 1250 and 2500 ppm (corresponding to 2.5, 22.9, 223 and 484 mg/kg bw/day for the combined sexes) via the diet. The NO(A)EL was 22.9 mg/kg bw/day (M-142011-01-1, 1997). The effect level was set based on decreased absolute kidney weights as well as centrilobular hepatocyte enlargement and vacuolation with increased fat deposition. The effects were only slight at this dose level but showed a dose-dependent tendency.
In a 90-day study performed similar to OECD TG 408 using the same study design as for mice, 20, 200, 2000 and 4000 ppm (corresponding to 1.6, 15.3, 153 and 303 mg/kg bw/day for combined sexes) was administered to 10 or 20 rats/sex/group in the low and intermediate dose group or the control and high dose group, respectively. At the highest dose level only adaptive hepatic effects were observed. The NOAEL was set at 303 mg/kg bw/day (M-141124-01-1, 1996).
The third sub-chronic study was performed in dogs similar to OECD TG 409. The animals were administered dose levels of 25, 125 and 1000 ppm (corresponding to 1.3, 6.3 and 49.6 mg/kg bw/day for combined sexes). Here, administration of 6.3 mg/kg bw/day led to fat deposits in the collecting ducts of the kidneys of males and females. Consequently, the NO(A)EL was 1.3 mg/kg bw/day (M-142680-01-1, 1997).
Chronic studies
Chronic toxicity of the test substance was assessed after exposure for 12 months in beagle dogs (M-184636-01-1). In accordance with OECD TG 452, 4 animals/sex/group received dietary dose levels of 20, 100 and 700 ppm (corresponding to 0.7, 3.5 and 24 mg/kg bw/day actual test substance intake for combined sexes). Controls received untreated diet. Animals were observed daily for clinical signs. Body weight was recorded weekly and food consumption was measured daily. Detailed clinical examinations and ophthalmoscopy were carried out pre-treatment and prior to necropsy. Electrocardiography investigations were carried out pre-treatment and at 3, 6 and 12 months of treatment. Haematology and clinical chemistry investigations were conducted prior to treatment and at 3, 6 and 12 months of treatment. Urinalysis parameters were measured at 3 and 6 months of treatment and at study termination. At necropsy, the weights of selected organs were recorded and, subsequently a range of tissues was examined histopathologically. At 20 and 100 ppm, no treatment-related effects were observed. At 700 ppm, microscopic examinations showed an increase in the incidence and severity of vacuolation in the straight tubule of the kidney. Hence, the kidney was identified as the target organ of test substance-toxicity and the NO(A)EL was set to 100 ppm, equating to an actual test substance intake of 3.3 mg/kg bw/day in males and 3.6 mg/kg bw/day in females.
In a combined chronic toxicity/carcinogenicity study (M-191414-01-1, 1999), 70 rats/sex/dose received the test substance via the diet at dose levels of 20, 200, 2000 and 4000 ppm (corresponding to 1.0, 10, 101 and 210 mg/kg bw/day for combined sexes) for 104 weeks (2 years). After 52 weeks, 20 animals of each group were subjected to an interim necropsy. Organ weights of these animals were recorded and tissues microscopically examined. The remaining animals continued treatment until termination of the study after 104 weeks. All animals were observed daily for clinical signs and palpated weekly for tissue masses. Body weight was recorded at the start of treatment, at weekly intervals to Week 14, and every second week thereafter until necropsy. Food consumption was monitored at weekly intervals for the first 13 weeks of treatment and approx. every 4 weeks thereafter. Water consumption was measured during Month 3, 5, 9 and 12. Haematology, clinical chemistry and urinalysis were carried out at 3, 6, 12, 18 and 24 months of treatment. Ophthalmoscopy was performed prior to treatment and at 12 and 24 months of treatment. At terminal necropsy, the weights of selected organs were recorded and a full list of tissues from all animals was examined histopathologically.
At 20 and 200 ppm, no treatment-related effects were observed. At 2000 ppm, an increased incidence in the presence of ketones in the urine of male animals was observed after 3, 6 and 12 months of treatment. Absolute and relative kidney weights were decreased in males at terminal sacrifice (10 and 13%, respectively). At interim kill only, relative liver weights were slightly increased (8%) in males, however, without a histopathological correlate at terminal kill. Therefore, this finding was considered adaptive and not toxicologically relevant at this dose level.
At 4000 ppm, palatability of the diet affected body weight gain, food consumption and food efficiency during the first week of treatment in both sexes. All parameters recovered to a great extend during the study compared with control. An increased incidence in the presence of ketones in the urine of male animals was observed after 3, 6 and 12 months of treatment. At interim kill, relative liver weights were slightly increased (16 and 14% for males and females, respectively) in both sexes compared to control. Increased relative liver weights (8%) were also observed for male animals at terminal kill. Absolute and relative kidney weights were decreased (12 and 9%, respectively) for males at terminal kill. An increased incidence of centrilobular hepatocytic hypertrophy in the liver of male animals was observed at both examination time points (after 12 and 24 months of treatment). An increase in the incidence and severity of progressive nephropathy in the kidneys was observed after 24 months of treatment in female animals.
In summary, the examinations revealed an increased incidence of ketones in the urine as well as decreased absolute and relative kidney weights in male animals starting at 2000 ppm. Hence, the NO(A)EL was set at 200 ppm, corresponding to an actual test substance intake of 8 mg/kg bw/day for male animals and 12 mg/kg bw/day for female animals (M-191414-01-1, 1999).
All NO(A)ELs and LO(A)ELs of oral repeated dose toxicity studies including toxicity studies on reproduction are displayed in Table 1. The endpoint summaries for the latter studies can be found under the respective endpoint.
Table 1: NOAELs and LOAELs for oral (dietary) repeated dose toxicity studies (actual test substance intake for both sexes combined)
Species |
Duration (weeks) |
NO(A)EL (mg/kg bw/day) |
LO(A)EL (mg/kg bw/day) |
Reference |
Mouse |
4 |
<507 |
507 |
1998 M-147436-01-1 |
Dog |
4 |
1.9 |
17.4 |
1998 M-147707-02-1 |
Mouse |
13 |
22.9 |
223 |
1997 M-142011-01-1 |
Rat |
13 |
303 |
> 303 |
1996 M-141124-01-1 |
Dog |
13 |
1.3 |
6.3 |
1997 M-142680-01-1 |
Dog |
56 |
3.5 |
24 |
1999 M-184636-01-1 |
Rat |
52/104 |
10 |
101 |
1999 M-191414-01-1 |
Rat (gavage) |
2 |
120 |
1000 |
1996 M-142033-01-1 |
Rabbit (gavage) |
2 |
50 |
500 |
1998 M-180231-01-1 |
Rat |
19 |
15.5 |
306.5 |
1999 M-185452-01-1 |
Table 1 shows that dogs are the most sensitive species among all species tested, providing the lowest NOAELs in subacute, subchronic and chronic studies. As dog studies are less covered from a statistical point of view due to the amount of animals used, the combined chronic toxicity/carcinogenicity study with rats is considered as well. Taken into account that the allometric scaling factor for the rat is 4 and for the dog it is 1.4, both NOAELs are the same (10/4 = 2.5 mg/kg bw/day and 3.5/1.4 = 2.5 mg/kg bw/day, respectively).
Hence, for a conservative approach, the chronic rat study with the longest duration, highest statistical power and lowest NOAEL was chosen as key, and the NOAEL of this study was chosen for the risk assessment.
For the test
substance, there is no current harmonized classification for STOT RE. An
evaluation of the adversity of the kidney effects, which were observed
in some studies, was performed in order to conclude on a STOT RE Cat. 2
self-classification.
Based on the results of the long-term and 90-day studies in rats, a STOT
RE classification is not warranted. In the long-term rat study, the
kidney effects of a chronic progressive nephropathy (CPN) were only
minimal and are regarded as a slight exacerbation of a normal
age-related background finding of older rats and not as a specific toxic
effect on kidneys. CPN is not regarded as relevant to humans according
to literature, and the effect dose and the NOAEL are far above the
guidance value of 25 mg/kg bw/day for category 2 STOT RE. In addition,
the kidney results in the 28-day and 2-generation rat study would not
warrant a STOT RE classification.
In the 12-month dog study, the minimal to moderate vacuolation of the
straight tubules and the incidences of lipid content do not justify a
STOT RE classification, based on the low severity and the fact that
fatty change is a normal variation in Beagle dogs. Therefore, this
normal variation was only exacerbated in the 12-month and 28-day dog
studies, but not a specific adverse pathological effect. An isolated
finding of mortality in the 90-day dog study does not warrant STOT RE
classification.
Regarding the studies in mice, a slightly increased mortality in the
18-month study does not warrant STOT RE classification since this
occurred only at a dose above the STOT RE equivalent guidance value of
25 mg/kg bw/day. In addition, the kidney weight effects in the 28- and
90-day mice studies do not justify a classification, since they occurred
at doses above the equivalent guidance values.
Overall, the kidney findings do not warrant STOT RE classification of
the test substance, based on severity and relevance to human health.
For the dermal route, one subacute study was conducted in rats. In a 28 day-study, the test substance was administered to 5 animals/sex/group at dose levels of 10, 100 and 1000 mg/kg bw/day. The study was performed in accordance with OECD TG 410. At 1000 mg/kg bw/day, slight dermal irritation, as indicated by skin reddening at the application site, was seen in all animals from Week 2 till study termination. Slight sloughing, defined as shedding of the superficial layer of skin at the application site, was seen in 3/5 males and 3/5 females from Week 2 until Week 3 and 4, respectively. Microscopic examinations revealed that 3/5 males and 5/5 females had mononuclear cell infiltration of the dermis to a moderate degree.
At 100 mg/kg bw/day, slight dermal irritation was seen in 2/5 females from Day 4 and 11 to Day 6 and 19, respectively. Microscopic examinations revealed that 2/5 males and 3/5 females had moderate cell infiltration.
At 10 mg/kg bw/day, no treatment-related effects were observed.
The NOAEL for systemic toxicity was set at 1000 mg/kg bw/day and the NOAEL for local toxicity was set 10 mg/kg bw/day, equating to 0.5 mg/cm²/day (calculated as follows: 10 mg/kg bw*0.2 kg bw rat/4 cm²) (M-184633-01-1, 1999).
Justification for classification or non-classification
The available data on repeated dose toxicity of the test substance
do not meet the criteria for classification according to Regulation (EC)
1272/2008, and are therefore conclusive but not sufficient for
classification.
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