Registration Dossier

- Oral: NOAEL = 9.7 and 10.1 mg/kg bw/day for males and females, respectively, rats, sub-chronic, 13 weeks, dietary, Bachmann 1993.

- Inhalation: NOAEL local >1050 mg/m3 and NOAEL systemic =99 mg/m3, rats, males and females, sub-acute, 4 weeks, Bernstein 1987.

- Dermal: NOAEL local >2000 mg/kg bw/day and NOAEL systemic = 200 mg/kg bw/day, rats, males and females, sub-acute, 4 weeks, Varney 1985.

Toxic effect type:

dose-dependent

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

26 Apr 1982 to 05 Aug 1982

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

1998

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: albino SPF

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Continuously

Dose / conc.:

80 mg/kg bw/day (nominal)

Remarks:

Low dose, dietary equivalent to 82.35 and 81.28 mg/kg bw/d for males and females, respectively.

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Mid dose, dietary equivalent to 256.84 and 252.61 mg/kg bw/d for males and females, respectively.

Dose / conc.:

800 mg/kg bw/day (nominal)

Remarks:

High dose, dietary equivalent to 825.53 and 806.15 mg/kg bw/d for males and females, respectively.

No. of animals per sex per dose:

20/sex/dose (+10/sex as recovery group for high dose only)

Control animals:

yes, plain diet

Dose descriptor:

NOAEL

Effect level:

80 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other:

Remarks:

Dietary equivalent to 82.35 and 81.28 mg/kg bw/day for males and females, respectively

Critical effects observed:

no

MORTALITY

Two females died in week 6 during blood sampling.

CLINICAL OBSERVATIONS

An increased incidence of alopecia was observed in both sexes at 800 mg/kg bw/day and females at 250 mg/kg bw/day. Diuresis and a soiled tail were noted in both sexes at 800 mg/kg bw/day. Clinical symptoms disappeared in the recovery group.

OPHTHALMOSCOPIC EXAMINATIONS

No ophthalmoscopic abnormalities were observed.

BODY WEIGHT

Body weight was decreased at 250 and 800 mg/kg bw/day in males (89 % and 79 % of control, respectively) and females (92 % and 79 % of control, respectively) in a dose-related manner. During the recovery phase body weight gain was similar to the body weight gain during treatment.

FOOD CONSUMPTION

Food consumption was decreased at 800 mg/kg bw/day in females (88 % of control). During recovery food consumption was not determined.

HAEMATOLOGY

Erythrocyte count was decreased in males at 800 mg/kg bw/day in week 6 of treatment (95 % of control) and in females at 250 mg/kg bw/day in week 6 and 14 (94-95 % of control) and at 800 mg/kg bw/day in week 14 (94 % of control). Haemoglobin was decreased at 800 mg/kg bw/day in males and females and at 250 mg/kg bw/day in females in week 6 and 14 (93-97 % of control). Packed cell volume was decreased in males at 800 mg/kg bw/day (93-95 % of control) in week 6 and 14 and in females at 250 and 800 mg/kg bw/day in week 14 only (93-95 % of control).

Reticulocyte count was increased in males at all dose levels (134-152 % of control) in week 6, which could reflect a regenerative response of the bone marrow. Thrombocytes were decreased in females at 250 and 800 mg/kg bw/day in week 6 (83-85 % of control). Since the thrombocyte count was already lower than the control group in females at 800 mg/kg bw/day before the start of the test, the change is not considered to be toxicologically relevant. Lymphocytes were increased in females at 250 and 800 mg/kg bw/day in week 14 and polymorphonuclear neutrophils were decreased in females at 250 and 800 mg/kg bw/day. Since the changes in lymphocytes and PMNs were not dose-related and values were within historical control range, the changes are not considered to be toxicologically relevant. A change in erythrocyte morphology was observed in all treated males and females in week 14 in the form of poikilocytosis. Anisocytosis was observed only at 250 and 800 mg/kg bw/day in males and 800 mg/kg bw/day in females in week 14. An increased incidence of Rouleau formation was found in all treated males. An increased incidence of poikilocytosis and Rouleau formation were still present in males after recovery.

CLINICAL CHEMISTRY

Alanine aminotransferase was increased in males at 800 mg/kg bw/day (128-139 % of control). Glutamate dehydrogenase was markedly increased in both sexes at 250 and 800 mg/kg bw/day (>500 % and >200 % of control, respectively). Increased ALAT and GLDH point to hepatocellular injury. Alkaline phosphatase was slightly increased at all dose levels compared to control in both sexes (120-138 % of control). Cholesterol was increased in males at 800 mg/kg bw/day (125-139 % of control) and females at 250 and 800 mg/kg bw/day (137-149 % of control). Bilirubin was increased at 800 mg/kg bw/day in week 14 (125 % of control). In females at 250 and 800 mg/kg bw/day bilirubin was also increased at the end of treatment (171-196 % of control). Increased ALP, cholesterol and bilirubin point to cholestasis. Plasma cholinesterase was increased in males at 250 and 800 mg/kg bw/day in week 6 (300-370 % of control) and at 250 mg/kg bw/day in week 14 (157% of control). In females cholinesterase was decreased at 800 mg/kg bw/day in week 6 and 14 (62-63 % of control). Urea was increased in males (108 % of control) and decreased in females (91 % of control) at 800 mg/kg bw/day in week 6. Glucose was decreased at 800 mg/kg bw/day in females in week 6, but this was due to a rather high control value. Total protein was increased in all treated females, but this was due to low values in the control group and values were normal according to historical control values. Albumin was increased at 250 and 800 mg/kg bw/day in males and all females. Globulins were decreased in general and mainly alphaglobulin 3 and beta-globulin and gamma-globulin were decreased. Consequently, the A/G ratio was increased at all dose levels.

URINALYSIS

Urinalysis showed no treatment-related findings.

ORGAN WEIGHTS

Absolute and relative liver weights were increased at all dose levels in both sexes (absolute: 118-195 % of control; relative: 120-204 % of control), with a dosage-related trend and returned to normal weight at 800 mg/kg bw/day after the recovery period. Absolute and relative thyroid weight was increased in males at 800 mg/kg bw/day (132-141 % of control) and in all females (112-149 % of control, with a dosage-related trend). Male thyroid weight had returned to control values and female thyroid weight had diminished partly during recovery. Absolute and relative adrenal weight was decreased in all females. The increased relative adrenal and kidney weight in males at 800 mg/kg bw/day might be due to reduced body weight.

Absolute and relative spleen weight was decreased at 250 and 800 mg/kg bw/day in females. Absolute and relative lung weight was decreased at 800 mg/kg bw/day in females. Relative uterus weight was decreased at 800 mg/kg bw/day. Absolute and relative prostate weight was increased at 800 mg/kg bw/day.

GROSS PATHOLOGY

Macroscopic examination revealed no treatment-related findings.

HISTOPATHOLOGY

Microscopic examination of the liver revealed an increased incidence of mainly centrilobular hypertrophy at 250 and 800 mg/kg bw/day in both sexes in a dose-related manner. Also an increased incidence of mainly centrilobular glycogen depletion was observed at all dose levels, which became marked at 250 mg/kg bw/day and higher. An increased incidence of interstitial/perivascular infiltrates, enlarged nuclei of tubular cells with iron-positive granulation in the kidney was observed at 800 mg/kg bw/day in both sexes. The incidence of hyaline casts was slightly increased at 800 mg/kg bw/day in both sexes.

The number of animals with the majority of thyroid follicles in the active stage was increased at 250 and 800 mg/kg bw/day. The study results indicate that the liver, kidney and thyroid are potential targets of test substance in rats.

Table 1: Overview of results

Dose (mg/kg bw/day)	0	0	80	80	250	250	800	800	recovery	recovery	dr
	m	f	m	f	m	f	m	f	m	f
Mortality	none
Clinical signs
alopecia	0	1	3	1	2	4	7	14
diuresis	0	0	0	0	0	0	3	11
soiled tail	0	0	0	0	0	0	17	14
Body Weight					dc	d	dc	dc			m/f
Food consumption								d	Not performed
Ophthalmoscopy	no treatment-related findings
Haematology
RBC						dc²	dc¹	dc³			f
haemoglobin						dc²	dc²	dc²			f
PCV						dc³	dc²	dc³			f
reticulocytes			ic¹		ic¹		i¹
prothrombin time							ic³
thrombocytes						dc¹		dc¹
lymphocytes						ic³		ic³
Polymorphonu clear neutrophiles						dc³		dc³
Clin. Chemistry
ALAT							ic²
GLDH					ic²	ic²	ic²	i²
ALP			ic³	ic³	ic¹	ic²	ic²	ic³
ChE					ic²		ic¹	dc²
Urea							ic¹	dc¹
bilirubin					dc¹	ic³	dc¹	ic³
cholesterol						ic²	ic³	ic²
glucose							ic²	dc¹
total protein				ic³		ic²		ic²
albumin				ic³	ic²	ic²	ic²	ic²
alpha-globulin 1					dc²		ic²	dc¹
alpha-globulin 2				dc³	dc¹	dc¹	dc²	dc¹
alpha-globulin 3			dc¹		dc²	dc³	dc²	dc³
beta-globulin			dc³	dc³	dc²	dc²	dc²	dc²
gamma-globulin			dc¹		dc²	dc³	dc¹	dc²
A/G ratio			i	ic³	ic²	ic²	d²	ic²
							ic²
Urine analysis	no treatment-related findings
Organ weights⁴
thyroid				i^a, ic^r		i^a, ic^r	i^a, ic^r	i^a, ic^r			f
adrenal				di^a, dc^r	i^a, ic^r	di^a, dc^r	ic^r	di^a, dc^r			f
prostate							i^a, ic^r
kidney
liver			i^a, ic^r	i^a, ic^r	i^a, ic^r	i^a, ic^r	i^a, ic^r	i^a, ic^r			m/f
spleen						d^a, dc^r		d^a, dc^r
lungs								d^a, dc^r
uterus								dc^r
Pathology
Macroscopy	no treatment-related findings
Microscopy
liver: centrilobular hypertrophy	0/20	0/20	0/20	0/20	6/20	14/20	19/20	20/20	1/10	1/10	m/f
liver: centrilobular glycogen depletion	1/20	1/20	2/20	3/20	6/20	17/20	13/20	20/20	1/10	3/10	m/f
Kidney: interstitial/ peri- vascular infiltrates	6/20	0/20	-⁵	-⁵	-⁵	-⁵	11/20	3/20	6/10	2/10
Kidney: enlarged nuclei tubular cells and granulation	2/20	0/20	-⁵	-⁵	-⁵	-⁵	6/20	2/20	2/10	1/10
Kidney: hyaline casts	2/20	0/20	-⁵	-⁵	-⁵	-⁵	5/20	1/10	3/10	2/10
Thyroid: majority follicles in active stage	1/20	0/20	0/20	1/20	1/20	6/20	3/19	7/19	1/10	0/10	m/f

dr dose related

dc/ic statistically significantly decreased/increased compared to the controls

d/i decreased/increased, but not statistically significantly compared to the controls

a/r absolute/relative

1 week 6

2 week 6 and 14

3 week 14

4 no statistical analysis performed on absolute organ weights

5 kidney tissue was not examined at 80 and 250 mg/kg bw/d

Conclusions:

The NOAEL is set at 80 mg/kg bw/day corresponding to an achieved dose of 82.35 mg/kg bw/day for males and 81.28 mg/kg bw/day for females.

Executive summary:

A 13-week feeding study was performed in the groups of 20 male and 20 female albino rat using dietary administration of the test substance in the feed at nominal concentrations of 0, 80, 250, 800 mg/kg bw/day (achieved concentrations 82.35, 256.84 and 825.53 mg/kg bw/day for males and 81.28, 252.61 and 806.15 mg/kg bw/day for females). This study was performed according to OECD 408 and in compliance with GLP. An additional group of 10 10 animals was included in the high dose group designated as "recovery animals". After termination of the 13-week treatment period these animals were placed on standard diet and observed for another weeks for reversibility or late occurrence of effects. All animals were observed daily for moribundity or mortalities and weekly to record clinical signs. Body weights and food consumption were determined weekly. Ophthalmoscopic examination was performed in all control animals and animals treated at the high dose in week 12. Blood samples for clinical-chemical were taken from the first ten animals in each group in week 6 and 14. Samples for haematological examinations the last ten animals in week 0, 6 and 14. Urine was collected in week 5 and 11 from the same animals which examined for blood chemistry. Food samples were analysed to determine the concentration and stability of the test substance before (fresh diet preparations) and at the end (residues in the food hopper) of feeding week 2 and 12. At sacrifice, animals were examined macroscopically and organ weights were recorded. Organs and tissues were collected and prepared for histopathological evaluation.

Two rats from the high dose killed accidentally in week 6 during bleeding for laboratory examinations but there was no indication that treatment was the initial cause of death. Effects were induced by the test substance correlated with the applied dose levels there was evidence that the majority of these abnormalities were reversible if animals were withdrawn from treatment. At the high dose (800 mg/kg bw/day) body weight development was markedly reduced in both sexes and clinical symptoms included alopecia, diuresis and a stained tail. Haematology parameters in a small number of males and females included decreased RBC counts and PCV in week 6. This was evident in one female only after 13 weeks of treatment. PT times were prolonged for some male animals at terminal examination, all other parameters of haematology were within normal biological limits. A small number of animals increased activities of GLDH were and AP were observed in week 6 and at termination of treatment. In both sexes, dysproteinaemia was evident at interim and final examinations whereas increased bilirubin levels were measured at final examination only. All other clinical chemical parameters were within normal biological limits. Urinalysis gave no evidence for a dose related effect. Animals of both sexes exhibited increased relative weight of the liver and the thyroid gland. Some males were also found with decreased weights of the prostate and slightly increased weights of the kidneys. Some females had decreased weights of the lung, uterus, spleen and adrenal glands. Histopathological examination revealed hypertrophy of mainly centrilobular liver cells which were associated with glycogen depletion. There was also evidence for an increased activity of the thyroid gland, especially in females. Few animals were also found with minor changes in the kidney.

The majority of the treatment related effects were obviously reversible when animals from the high dose were returned to the normal diet for a 4-week recovery period. Most prominent features for recovery were re-growth of the coat, normalization of liver weights together with normalization of the liver histology and of clinical chemistry parameters.

The NOAEL is set at 80 mg/kg bw/day corresponding to an achieved dose of 82.35 mg/kg bw/day for males and 81.28 mg/kg bw/day for females.

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

9.7 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP compliant OECD TG 408 study

System:

other: hepatobiliary and thyroid gland

Organ:

liver

thyroid gland

Reference

Endpoint:: short-term repeated dose toxicity: inhalation
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 20 Apr 1987 to 17 Jun 1987
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Qualifier:: according to guideline
Guideline:: OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study
Version / remarks:: May 1981
GLP compliance:: yes
Limit test:: no
Species:: rat
Strain:: Wistar
Remarks:: KFM-Han
Sex:: male/female
Route of administration:: inhalation: aerosol
Type of inhalation exposure:: nose only
Vehicle:: other: ethanol
Mass median aerodynamic diameter (MMAD):: 3 µm
Geometric standard deviation (GSD):: 1.9
Duration of treatment / exposure:: 6 hours/day
Frequency of treatment:: 5 days/week, total of 21 exposures
Dose / conc.:: 10 mg/m³ air (nominal)
Remarks:: Group 2: Low dose, equivalent to 11 mg/m³ air (actual concentration)
Dose / conc.:: 100 mg/m³ air (nominal)
Remarks:: Group 3: Mid dose, equivalent to 99 mg/m³ air (actual concentration)
Dose / conc.:: 1 000 mg/m³ air (nominal)
Remarks:: Group 4: High dose, equivalent to 1050 mg/m³ air (actual concentration)
No. of animals per sex per dose:: 5
Control animals:: yes, concurrent no treatment
: Key result
Dose descriptor:: NOAEL
Remarks:: Local
Effect level:: > 1 000 mg/m³ air (nominal)
Based on:: test mat.
Sex:: male/female
Remarks on result:: other: no local effects observed. Equivalent to 1050 mg/m³ air (actual concentration)
: Key result
Dose descriptor:: NOAEL
Remarks:: Systemic
Effect level:: 100 mg/m³ air (nominal)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: body weight and weight gain; clinical biochemistry; food consumption and compound intake; organ weights and organ / body weight ratios
Remarks on result:: other: Equivalent to 99 mg/m³ air (actual concentration)
: Key result
Critical effects observed:: no
Conclusions:: Based on increased liver and kidney weights and clinical biochemical findings, the systemic NOAEL is set at 99 mg/m3. Since no adverse local effects were observed, the local NOAEL is set at >1050 mg/m3.
Executive summary:: The study was performed in accordance with OECD TG 412 and in compliance with GLP. Four groups of Wistar, KFM-Han rats, each containing 5 males and 5 females, inhaled 1.5, 11, 99 or 1050 mg/m3 test substance using ethanol as vehicle. The exposure was 6 hours per day for 5 days per week for 29 days. The mean particle size of the aerosol sampled which was 3 µm and the GSD 1.9 μm. No mortality occurred. No clinical or ocular abnormalities were observed. Body weight gain was decreased in males at 1050 mg/m3 (terminal body weight 93 % of control) at the end of the exposure period. Also a decrease in food consumption was observed in these males. At haematology, haemoglobin was slightly, but significantly decreased at 1050 mg/m3 in females (94 % of control). Clinical biochemistry investigations showed a dose-dependent and significant decrease in total bilirubin (72 % of control) and a dose-dependent and significant increase in potassium and chloride (122 % and 105 % of control, respectively) in the highest dosed males. Increased potassium may indicate kidney insufficiency. Calcium was slightly but significantly decreased in these males (97 % of control). In the females, urea was significantly decreased at the 99 mg/m3 dose (78 % of control) and sodium was slightly but significantly decreased at the highest dose (99 % of control). Albumin and total protein were both significantly increased at the highest dose level (111 % and 109 % of control, respectively). In the recovery groups, all values were normal again. Relative weights of the lungs, liver and testis were significantly increased in males of the highest dose group (116 %, 114 % and 116 %, respectively). In females of the highest dose group, absolute and relative liver weight were significantly increased (127 % and 134 %, respectively). Relative kidney weight was significantly increased (116 %) at the highest dose. Absolute and relative adrenals weight were significantly decreased (both 81 %) at 99 mg/m3. However, in the absence of any decreases at the high dose level this finding was considered not toxicologically relevant. Changes in relative testes and lung weight might be due to decreased terminal body weight. Macroscopically and microscopically no treatment-related effects were observed. Males of the control group and the 1050 mg/m3 dose group showed similar histopathological findings, namely bronchial associated lymphoid tissue hyperplasia (2/5 and 1/5, resp.) and interstitial pneumonitis (5/5 and 4/5, resp.) of the lungs, and mononuclear inflammatory cell foci (3/5 and 1/5, resp.) of the liver. Females of both the control group and the 1050 mg/m3 dose group showed nephrocalcinosis of the kidneys. Based on increased liver and kidney weights and clinical biochemical findings, the systemic NOAEL is set at 99 mg/m3. Since no adverse local effects were observed, the local NOAEL is set at >1050 mg/m3.

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

99 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP compliant OECD TG 412 study

System:

hepatobiliary

Organ:

liver

Reference

Endpoint:: short-term repeated dose toxicity: inhalation
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 20 Apr 1987 to 17 Jun 1987
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Qualifier:: according to guideline
Guideline:: OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study
Version / remarks:: May 1981
GLP compliance:: yes
Limit test:: no
Species:: rat
Strain:: Wistar
Remarks:: KFM-Han
Sex:: male/female
Route of administration:: inhalation: aerosol
Type of inhalation exposure:: nose only
Vehicle:: other: ethanol
Mass median aerodynamic diameter (MMAD):: 3 µm
Geometric standard deviation (GSD):: 1.9
Duration of treatment / exposure:: 6 hours/day
Frequency of treatment:: 5 days/week, total of 21 exposures
Dose / conc.:: 10 mg/m³ air (nominal)
Remarks:: Group 2: Low dose, equivalent to 11 mg/m³ air (actual concentration)
Dose / conc.:: 100 mg/m³ air (nominal)
Remarks:: Group 3: Mid dose, equivalent to 99 mg/m³ air (actual concentration)
Dose / conc.:: 1 000 mg/m³ air (nominal)
Remarks:: Group 4: High dose, equivalent to 1050 mg/m³ air (actual concentration)
No. of animals per sex per dose:: 5
Control animals:: yes, concurrent no treatment
: Key result
Dose descriptor:: NOAEL
Remarks:: Local
Effect level:: > 1 000 mg/m³ air (nominal)
Based on:: test mat.
Sex:: male/female
Remarks on result:: other: no local effects observed. Equivalent to 1050 mg/m³ air (actual concentration)
: Key result
Dose descriptor:: NOAEL
Remarks:: Systemic
Effect level:: 100 mg/m³ air (nominal)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: body weight and weight gain; clinical biochemistry; food consumption and compound intake; organ weights and organ / body weight ratios
Remarks on result:: other: Equivalent to 99 mg/m³ air (actual concentration)
: Key result
Critical effects observed:: no
Conclusions:: Based on increased liver and kidney weights and clinical biochemical findings, the systemic NOAEL is set at 99 mg/m3. Since no adverse local effects were observed, the local NOAEL is set at >1050 mg/m3.
Executive summary:: The study was performed in accordance with OECD TG 412 and in compliance with GLP. Four groups of Wistar, KFM-Han rats, each containing 5 males and 5 females, inhaled 1.5, 11, 99 or 1050 mg/m3 test substance using ethanol as vehicle. The exposure was 6 hours per day for 5 days per week for 29 days. The mean particle size of the aerosol sampled which was 3 µm and the GSD 1.9 μm. No mortality occurred. No clinical or ocular abnormalities were observed. Body weight gain was decreased in males at 1050 mg/m3 (terminal body weight 93 % of control) at the end of the exposure period. Also a decrease in food consumption was observed in these males. At haematology, haemoglobin was slightly, but significantly decreased at 1050 mg/m3 in females (94 % of control). Clinical biochemistry investigations showed a dose-dependent and significant decrease in total bilirubin (72 % of control) and a dose-dependent and significant increase in potassium and chloride (122 % and 105 % of control, respectively) in the highest dosed males. Increased potassium may indicate kidney insufficiency. Calcium was slightly but significantly decreased in these males (97 % of control). In the females, urea was significantly decreased at the 99 mg/m3 dose (78 % of control) and sodium was slightly but significantly decreased at the highest dose (99 % of control). Albumin and total protein were both significantly increased at the highest dose level (111 % and 109 % of control, respectively). In the recovery groups, all values were normal again. Relative weights of the lungs, liver and testis were significantly increased in males of the highest dose group (116 %, 114 % and 116 %, respectively). In females of the highest dose group, absolute and relative liver weight were significantly increased (127 % and 134 %, respectively). Relative kidney weight was significantly increased (116 %) at the highest dose. Absolute and relative adrenals weight were significantly decreased (both 81 %) at 99 mg/m3. However, in the absence of any decreases at the high dose level this finding was considered not toxicologically relevant. Changes in relative testes and lung weight might be due to decreased terminal body weight. Macroscopically and microscopically no treatment-related effects were observed. Males of the control group and the 1050 mg/m3 dose group showed similar histopathological findings, namely bronchial associated lymphoid tissue hyperplasia (2/5 and 1/5, resp.) and interstitial pneumonitis (5/5 and 4/5, resp.) of the lungs, and mononuclear inflammatory cell foci (3/5 and 1/5, resp.) of the liver. Females of both the control group and the 1050 mg/m3 dose group showed nephrocalcinosis of the kidneys. Based on increased liver and kidney weights and clinical biochemical findings, the systemic NOAEL is set at 99 mg/m3. Since no adverse local effects were observed, the local NOAEL is set at >1050 mg/m3.

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

1 050 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP compliant OECD TG 412 study

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

05 Mar 1985 to 27 Mar 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Version / remarks:

1981

Deviations:

yes

Remarks:

An occlusive application was used and no statistical analyses were performed.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 241 – 362 g
- Housing: exclusive rooms, single housed in suspended stainless steel mesh cages
- Diet: Rat and mouse Maintenance diet, ad libitum
- Water: filtered main water, ad libitum via water bottles
- Acclimation period: about 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 40 - 70
- Air changes (per hr): minimum 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 05 Mar 1985 To: 27 Mar 1985

Type of coverage:

occlusive

Vehicle:

corn oil

Details on exposure:

TEST SITE
- Area of exposure: non-abraded areas of skin
- % coverage: not less than 10 %

Duration of treatment / exposure:

21 days, 6 hours per day

Frequency of treatment:

Daily

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Remarks:

Group 2. Low dose

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Remarks:

Group 3. Mid dose

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

Group 4. High dose

No. of animals per sex per dose:

5

Control animals:

yes, concurrent no treatment

Details on study design:

- Rationale for animal assignment: Random

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: Daily
- Cage side observations: Viability/mortality (twice daily), detailed behavioural observation (once weekly),

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Daily, including skin irritation which was assessed according to the scale antioned in Any other information on material and method inlc tables.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule for examinations: No

HAEMATOLOGY:
- Time schedule for collection of blood: During the final week
- Anaesthetic used for blood collection: No
- Animals fasted: Yes (18 hours)
- How many animals: All animals
- Parameters: haemoglobin concentration(Hb), mean cell volume (MCV), red blood cell count (RBC) and indices: mean cell haemoglobin (MCH), packed cell volume (PCV), mean cell (haemoglobin concentration MCHC); total and differential white cell count (WBC) , platelet count.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: During the final week
- Animals fasted: Yes (18 hours)
- How many animals: All animals
- Parameters: glutamate oxaloacetate transaminase (GOT/AST), glutamate pyruvate transaminase (GPT/ALT), alkaline phosphatase (Alk.P), blood urea nitrogen (BUN), glucose, inorganic, bilirubin, creatinine, sodium, total protein (TP), potassium, albumin, calcium, albumin/globulin ratio.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Any other information on material and method inlc tables)
HISTOPATHOLOGY: Yes (see Any other information on material and method inlc tables)

Dose descriptor:

NOAEL

Remarks:

Local

Effect level:

> 2 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other:

Remarks:

No effects observed

Dose descriptor:

NOAEL

Remarks:

Systemic

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Critical effects observed:

no

ANALYSIS OF FORMULATIONS

The results obtained during the first week of the study were slightly lower than expected (83 - 88 % of nominal). The samples were analysed again and the results obtained were considered to be satisfactory (90 – 94 %) of normal). The values obtained at week 3 were also considered to be acceptable (94 – 96 %).

MORTALITY

There were no deaths during the study.

CLINICAL OBSERVATIONS

There were no treatment-related clinical changes.

SKIN IRRITATION

There was no evidence skin irritation at the application site in any of the treated animals.

BODY WEIGHT

There were no treatment-related changes in body weight gain

FOOD CONSUMPTION

Mean food consumptions of test and control groups remained closely comparable and there were no treatment-related changes.

HAEMATOLOGY

There were no haematological changes associated with treatment.

CLINICAL CHEMISTRY

Clinical chemistry parameters were unaffected by treatment.

ORGAN WEIGHTS

A slight increase in absolute and relative liver weights of about 19 per cent was observed in the group 4 animals when compared with the respective control value. This change was considered to be treatment-related. There were no other

changes in absolute or relative organ weights associated with treatment.

MACROSCOPIC NECROPSY FINDINGS

All rats had fur loss on the abdominal skin, accompanied in some cases by sores. The severity was similar in all groups. These abdominal skin lesions were probably associated with the occlusion technique. The only finding in the internal viscera was occasional cases of hydronephrosis in the right kidney. There were no findings of any unusual nature or incidence to suggest any gross toxic effect.

MICROSCOPIC PATHOLOGY FINDINGS

Minor changes such as low grade hypergranulosis and acanthosis were present in samples of skin from the site of application of the corn oil vehicle in group 1 rats.

Histopathological changes in the treated skin site of group 4 rats were similar to those in group 1. There was no evidence of increased irritation associated the with application of the test article in corn oil.

The most common finding in the liver of both control and treated rats was minor foci of leucocyte accumulation. minimal hepatocellular hypertrophy was probably present in a few animals, as suggested by the slightly increased liver weights but this was difficult to define with certainty against the normal background variations based on the histopathology of relatively few animals. There was no evidence of any degenerative changes in either the liver or kidney to suggest any systemic toxicity due to test article application.

Table 1 Overview of results

Dose (mg/kg bw/day)	0	0	20	20	200	200	2000	200	dr
	m	f	m	f	m	f	m	f
Mortality	none
Clinical signs	No treatment-related findings
Body Weight	No treatment-related findings
Food consumption	No treatment-related findings
Haematology	No treatment-related findings
Clin. Chemistry
ALAT						d		d
ASAT						d		d
Urinalysis	Not performed
Organ weights
liver					d^r		i^a,r	i^a,r
gonads					d^r		d^a,r
kidneys							d^a,r
Pathology
Macroscopy	no treatment-related findings
Microscopy
liver: hypertrophy	0/5	0/5	0/5	0/5	0/5	0/5	2/5	3/5
kidney: hydronephrosis	0/5	1/5	2/5	0/5	1/5	1/5	1/5	0/5

d/i decreased/increased, but not statistically significantly compared to the controls

a absolute organ weight

r relative organ weight

Conclusions:

Based on increased liver weights and concomitant liver hypertrophy of half of the test animals, the systemic NOAEL is set at 200 mg/kg bw/day. Since no adverse local effects were observed, the local NOAEL is set at >2000 mg/kg bw/day.

Executive summary:

An OECD TG 410-like study as performed in compliance with GLP to assess the subacute dermal toxicity. Groups of 5 male and 5 female Crl:CD(SD)BR rats were dermally exposed to 20, 200 and 2000 mg/kg test item under occlusive conditions for 21 days - 6 hours/day. An additional group was treated with the corn oil vehicle alone and acted as controls. Twice daily all animals were examined to detect any which were dead or moribund.All animals were examined once daily for clinical changes including skin irritation. Individual body weights were recorded on the first day of the test and at weekly intervals throughout the study. Individual food consumption was recorded at weekly intervals throughout the study. Individual blood samples were collected from the orbital sinus of all animals (after an 18 hours fast) during the final week of the study. Laboratory analysis (haematology and clinical chemistry) were performed on individual blood samples. The animals were killed by an intraperitoneal injection of pentobarbitone sodium solution and exsanguination, following about 19 hours without food. A full macroscopic examination was made.

No treatment-related local skin effects were observed. One control animal, four animals given 20 mg/kg and one animal given 200 mg/kg showed slight, mainly reversible erythema. No treatment-related effects were observed on mortality, clinical observations, body weight, food consumption and haematology. In clinical chemistry analyses, females given 200 and 2000 mg/kg showed decreased levels of ASAT (80 % and 82 % of control, respectively) and ALAT (65 and 60 % of control, respectively). These changes were not considered to be treatment-related, since decreases were observed and no dose-response relationship was noted. In both males and females of the 2000 mg/kg dose group, increased absolute liver weights (122 % of control) and relative liver weights (119 % of control) were noted. Male rats given 2000 mg/kg showed decreased absolute weights of the gonads (94 % of control) and kidneys (91 % of control). In male rats given 200 and 2000 mg/kg, decreased relative weights of the gonads (94 % and 92 % of control, respectively) and the kidneys (94 % and 89 % of control, respectively) were seen.

At necropsy, no treatment-related changes were observed. Histopathology showed mild renal hydronephrosis in the control group (1/10) and 20 mg/kg group (2/10). Mild to moderate renal hydronephrosis was seen in 2/10 rats of the 200 mg/kg dose group, and the symptom was severe in one animal of the highest dose group. Hypertrophy of the liver was observed in 5 rats of the highest dose group. In the absence of histopathological correlates at 200 mg/kg, the slight decreases in relative gonad and kidney weight at 200 mg/kg were not considered toxicologically relevant.

Based on increased liver weights and concomitant liver hypertrophy of half of the test animals, the systemic NOAEL is set at 200 mg/kg bw/day. Since no adverse local effects were observed, the local NOAEL is set at >2000 mg/kg bw/day.

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP compliant OECD TG 410-like study

System:

hepatobiliary

Organ:

liver

Repeated dose toxicity: oral

Sub-chronic toxicity oral study in rats, Bachmann 1993

In an OECD TG 408 study in compliance with GLP, the test substance was administered in the diet for 3 months at dietary levels of 0, 30, 150, 750, and 3000 ppm to a total of 140 Tif: RAIf (SPF) albino rats. In each dose group 10 animals per sex and group sacrificed at the end of the treatment period; 10 animals per sex in the control and high dose groups were kept for a 4-week recovery period before sacrifice. Corrected for the analytically determined test article concentrations in the diet, the mean daily intake of test substance was 2.17, 9.71, 45.1, and 199 mg/kg body weight in males, and 2.29, 10.1, 49.6, and 203 mg/kg body weight in females.

Treatment did not affect the appearance or behaviour of the rats. All rats survived the study. During the treatment period the mean body weight gain in males and females of group 5 (3000 ppm) was minimally to slightly lower than in the control group. The effect was reversible in males but not in females over the 4-week recovery period. A reduced food intake by females of group 5 (3000 ppm) became similar to that of the controls during the recovery period.

Food consumption ratios were similar between treated and control groups and no effects on water intake was observed. No evidence of ocular toxicity was detected. The treatment had no influence on the haematological profile. At the end of the treatment, plasma cholesterol levels were slightly increased in males and females of groups 4 and 5 (750, 3000 ppm). In addition, high dose males (3000 ppm) had minimally lower triglyceride levels, increased activities of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase, whereas high dose females had only minimally increased alkaline phosphatase activities. Furthermore, both males and females of the top dose group (3000 ppm) showed higher albumin to globulin ratios which in males resulted mainly from lower globulin levels and in females from higher albumin levels. All these alterations were reversible within the recovery period. Treatment had no influence on urine parameters. In the high dose group (3000 ppm), reduced carcass weights, higher absolute and relative mean liver weights, and higher relative thyroid weights in both sexes and higher absolute thyroid weights in females were observed at treatment end. Normal values were recorded at the end of the recovery period. Macroscopically, enlarged livers were seen in 2/10 female rats of group 4 and in 4/10 males and 7/10 females of group 5 (750 and 3000 ppm) at the end of the treatment period. Minimal to moderate hypertrophy of the hepatocytes was observed in females of group 4 and in males and females of group 5. Hypertrophy of the follicular epithelium of the thyroid gland in females of group 4 and in both sexes of group 5 was considered of secondary nature. Within 4 weeks of recovery, both the liver and the thyroid gland changes were fully reversible.

Under the conditions of this test, treatment with test substance resulted in reduced food intake and depressed body weight gain in high dose animals. Liver changes at the higher dose levels were substantiated by increased weight, hepatocellular hypertrophy and changes to plasma levels for proteins, cholesterol, and liver enzymes. Hypertrophy of thyroid follicular epithelium was considered of secondary nature. This slight liver toxicity accompanied adaptive changes of a functional nature, indicative of metabolic load or hormonal adjustment, were completely reversible upon cessation of treatment. It can be inferred from the observations during the above study, that a "no observable effect level" for test substance when offered to rats continuously in their food over a period of 3 months is 150 ppm, corresponding to a mean daily intake of 9.7 mg/kg body weight in males and 10.1 mg/kg body weight in females.

Hence, the study results indicate that the liver and thyroid are potential targets of test substance in rats. The NOAEL is set at 150 mg/kg food (equal to 9.71 mg/kg bw for males and 10.1 mg/kg bw for females) based on effects on the liver and thyroid.

Sub-acute toxicity oral study in rats, Suter 1986

The test substance was administrated via gavage for 28 days to male and female Wistar (KFM-han) rats at concentrations of 10, 50, 200 or 1000 mg/kg bw/day (equivalent to 6.8, 48.5, 138, 790 mg/kg bw/day after correction for measured test substance concentration). This study was performed according to OECD TG 407 and performed under GLP. Each animal was observed twice daily for any sign of toxicity or clinical symptoms. The consumption was once during the acclimation period and weekly thereafter using an on-line electronic recording system. The body weight of each animal was recorded once during the acclimation period, twice during the week of treatment and weekly thereafter. All animals were examined for ocular at 4 weeks of treatment. Blood samples for haematology and clinical biochemistry were collected from all animals after 28 days. Urine was collected over an 18-hour period into a specimen vial using a metabolism cage after 28 days of treatment. Organ weights were recorded at scheduled necropsy and tissues were prepared for histopathological examinations.

No mortality was observed. No treatment-related clinical signs were seen. No treatment-related change in body weight or food consumption was noted. No effect on ophthalmoscopy was observed. Erythrocyte count and haemoglobin were significantly decreased at 1000 mg/kg in males (95 and 96 % of control, respectively) and females (91 and 94 % of control, respectively). Haematocrit was slightly, but significantly decreased at 1000 mg/kg in females (95 % of control). Platelets were significantly increased at 1000 mg/kg in males (114 % of control). Thromboplastin time and pro-thromboplastin time were significantly increased at 1000 mg/kg in males (108 and 121 of control). Thromboplastin time was significantly reduced in all treated females, but since no dose-response relationship was present, this is not considered to be toxicologically relevant.

Glucose was increased at 1000 mg/kg in males and females (110 and 115 % of control). Urea was significantly increased at 1000 mg/kg in males (120 % of control). Lactate dehydrogenase was significantly increased at 1000 mg/kg in males (119 % of control). Alkaline phosphatase was significantly increased at 1000 mg/kg in males and females (148 and 155 % of control) and at 200 mg/kg in females (142 % of control). Calcium was significantly increased at 1000 mg/kg in males and females (103 and 108 % of control). Chloride was significantly decreased at 200 and 1000 mg/kg in females (97-98 % of control). Albumin was significantly increased at 1000 mg/kg in males and females (115 and 122 % of control). Albumin/globulin ratio was increased at 50, 200 and 1000 mg/kg in both sexes with a dose-response relationship (109-121 % of control for males and 111-120 % of control for females). The significantly increased specific gravity in urinalysis at 1000 mg/kg in males was due to one male and not considered to be toxicologically relevant. Absolute and relative liver weight was increased in all treated animals in a dose-related manner (a/r: 113- 183 %/108-187 % of control for males; 122-203 %/110-198 % of control for females). The slight increase in liver weight at 10 mg/kg without any other effects is considered not adverse. The slightly, but significantly increased relative heart and kidney weights at 1000 mg/kg in males were considered to be related to a slight decrease in body weight. Macroscopic examination showed no treatment-related effects. All animals at 1000 mg/kg bw/day showed hepatocellular hypertrophy. The cytoplasm showed a rather homogeneous structure and slight to severe eosinophilia. Electron microscopy of hepatocytes revealed marked proliferation and hypertrophy of peroxisomes and a moderate proliferation of smooth endoplasmic reticulum. An increased incidence of follicular hyperplasia of the thyroid was observed at 1000 mg/kg bw/day in both sexes.

The minor changes in haematology and clinical biochemistry parameters in group 5 animals (1000 mg/kg bw/day) and dose-related hepatomegaly are considered to be adaptive and not indicative of any toxic effect. Therefore, the NOAEL is set at 200 mg/kg bw/day (corresponding to 138 mg/kg bw/day).

Sub-chronic toxicity oral study in rats, Buser 1983

A 13-week feeding study was performed in the groups of 20 male and 20 female albino rat using dietary administration of the test substance in the feed at nominal concentrations of 0, 80, 250, 800 mg/kg bw/day (achieved concentrations 82.35, 256.84 and 825.53 mg/kg bw/day for males and 81.28, 252.61 and 806.15 mg/kg bw/day for females). This study was performed according to OECD 408 and in compliance with GLP. An additional group of 10 10 animals was included in the high dose group designated as "recovery animals". After termination of the 13-week treatment period these animals were placed on standard diet and observed for another weeks for reversibility or late occurrence of effects. All animals were observed daily for moribundity or mortalities and weekly to record clinical signs. Body weights and food consumption were determined weekly. Ophthalmoscopic examination was performed in all control animals and animals treated at the high dose in week 12. Blood samples for clinical-chemical were taken from the first ten animals in each group in week 6 and 14. Samples for haematological examinations the last ten animals in week 0, 6 and 14. Urine was collected in week 5 and 11 from the same animals which examined for blood chemistry. Food samples were analysed to determine the concentration and stability of the test substance before (fresh diet preparations) and at the end (residues in the food hopper) of feeding week 2 and 12. At sacrifice, animals were examined macroscopically and organ weights were recorded. Organs and tissues were collected and prepared for histopathological evaluation.

Two rats from the high dose killed accidentally in week 6 during bleeding for laboratory examinations but there was no indication that treatment was the initial cause of death. Effects were induced by the test substance correlated with the applied dose levels there was evidence that the majority of these abnormalities were reversible if animals were withdrawn from treatment. At the high dose (800 mg/kg bw/day) body weight development was markedly reduced in both sexes and clinical symptoms included alopecia, diuresis and a stained tail. Haematology parameters in a small number of males and females included decreased RBC counts and PCV in week 6. This was evident in one female only after 13 weeks of treatment. PT times were prolonged for some male animals at terminal examination, all other parameters of haematology were within normal biological limits. A small number of animals increased activities of GLDH were and AP were observed in week 6 and at termination of treatment. In both sexes, dysproteinaemia was evident at interim and final examinations whereas increased bilirubin levels were measured at final examination only. All other clinical chemical parameters were within normal biological limits. Urinalysis gave no evidence for a dose related effect. Animals of both sexes exhibited increased relative weight of the liver and the thyroid gland. Some males were also found with decreased weights of the prostate and slightly increased weights of the kidneys. Some females had decreased weights of the lung, uterus, spleen and adrenal glands. Histopathological examination revealed hypertrophy of mainly centrilobular liver cells which were associated with glycogen depletion. There was also evidence for an increased activity of the thyroid gland, especially in females. Few animals were also found with minor changes in the kidney.

The majority of the treatment related effects were obviously reversible when animals from the high dose were returned to the normal diet for a 4-week recovery period. Most prominent features for recovery were re-growth of the coat, normalization of liver weights together with normalization of the liver histology and of clinical chemistry parameters.

The NOAEL is set at 80 mg/kg bw/day corresponding to an achieved dose of 82.35 mg/kg bw/day for males and 81.28 mg/kg bw/day for females.

Sub-chronic toxicity oral study in mice, Camponovo 1983

The objective of this study was to assess the potential toxicity of the substance when administered orally via feed admix to groups of 16 male and 16 female albino mice at dose levels of 0, 100, 300 and 900 mg/kg bw/day (dietary equivalent to 0, 101.3, 302.4, and 921.5 mg/kg bw/day for males and 0, 102.6, 309.7 and 923.2 mg/kg bw/day for females), for 7 days a week for 13 weeks, in order to define a dose range for a carcinogenicity study. Body weight, feed consumption and signs of toxicity were recorded weekly. Ophthalmoscopic examination and urinalysis were carried out once towards the end of the study. Haematological, as well as clinical investigations, were performed immediately prior to the terminal sacrifice. Post-mortem examinations comprised full autopsy, organ weight determinations and histological examinations.

One male in the control group died during ophthalmoscopic examination. No treatment-related effects were observed for clinical signs, body weights, ophthalmoscopy, urinalysis and gross pathology. White blood cell count and polymorphonuclear neutrophils were decreased at 900 mg/kg bw/day in males. This is probably due to some individual, high values for the control and low dose group. Thrombocytes were decreased at 900 mg/kg bw/day in females. Alkaline phosphatase was significantly increased in males at 900 mg/kg bw/day (196 % of control). Glucose was increased in all groups of treated females (114-117 % of control). Since, the change is only small and no dose-response relationship is present, the change in glucose is considered to be of no toxicological significance. A disturbance in plasma protein-related values was observed in both sexes. Albumin was increased at 100 and 900 mg/kg bw/day (111-113 % of control) in males and in all groups of treated females (112-117 % of control). Alpha-2-globulin was decreased at 900 mg/kg bw/day in males (78 % of control) and females (83 % of control). Alpha-1-globulin, alpha-3-globulin and beta-globulin were decreased at 300 and 900 mg/kg bw/day in females (63-72 %, 72-73 % and 84 % of control, respectively). The A/G ratio was increased at 100 and 900 mg/kg bw/day in males (125-132 % of control) and in all females (124-151 % of control). Only the changes in alpha-1-globulin and A/G ratio in females were clearly dose-related. Absolute and relative liver weights were increased at 900 mg/kg bw/day in males (absolute: 117 % of control; relative: 125 % of control) and at 300 and 900 mg/kg bw/day in females (absolute: 122-136 % of control; relative: 124-138 % of control). The change in the liver weight of females was dose related. Histopathology showed fatty changes in the liver in all groups, which were predominantly centrilobular in the control group and predominantly periportal at 300 and 900 mg/kg bw/day. An increased incidence of reduced glycogen content in hepatocytes was observed in all treated groups, which became more severe at higher doses. Multinucleated hepatocytes were observed with increased incidence at all dose levels, with a dosage-related trend.

The presence of multinucleated hepatocytes, more frequently found in treated animals, is considered to reflect an adaptive change of the livers rather than a toxic effect of the test compound, none of the clinical chemical parameters examined was indicative for a liver damage. Histological findings gave no evidence for organ specific toxicity.

It is concluded that under the conditions of this study the dose of 100 mg/kg bw/day does not induce any adverse effect in the mouse. Therefore, the NOAEL is set at 100 mg/kg bw/day (dietary equivalent to 101.3 and 102.6 mg/kg bw/day for males and females, respectively.

Sub-chronic toxicity oral study in Beagle dogs, Keller-Rupp 1988

In an OECD TG 452 study in compliance with GLP, the test substance was given orally via gelatine capsules to groups of 4 male and 4 female Beagle dogs at daily of 25, 80 and 260 mg/kg bw/day. Control animals were given empty gelatine capsules. The animals were treated 7 days a week for at least 52 consecutive weeks. Clinical observations were made until termination of the study. Individual body weights were recorded before treatment, on day 0 (first day of treatment), 1 and 2 and at weekly intervals thereafter. The amount of feed consumed by the 4 dogs of each cage was measured on one or two days per week for the whole duration of the study. Due to technical reasons, individual caging was not possible. However, the situation on food consumption by each dog was carefully observed. The dogs were daily controlled and inspected with regard to their general state of health. A detailed inspection of the dogs’ behaviour, vivacity, sings of injury, sings of sickness and abnormality, appearance of the fur and skin, skin turgor, discharge from eye and nose, nature of excretes and soiled body regions (anus, genitals), was performed once weekly. All lesions or symptoms deviating from normal were recorded for each individual dog. Mortality was recorded continuously. About 15-30 minutes prior to the examination a mydriatic was instilled in each eye of the dogs. The examinations were subsequently performed using a “Keeler” dualite ophthalmoscope at week 52. Haematological and clinical chemistry examinations were performed at week 2, 6, 13, 26, 39 and 52. Urinalysis was performed at week 5, 25 and 50. Necropsy was performed immediately following euthanasia. At necropsy, tissues were collected from each dog and prepared for histological examination.

No treatment-related effects were observed for mortality, clinical sings, ophthalmoscopic examinations, urinalysis, gross pathology and histopathology. Significantly decreased body weights were noted at 260 mg/kg bw/day in males during the whole study (60% of control at the end of the study). Males and females at 260 mg/kg bw/day hardly gained any weight. Total body weight gains were lower in all groups of treated males (63, 61 and 10% of control) and in females at 260 mg/kg bw/day (13% of control). Food consumption was reported to be decreased at 260 mg/kg bw/day in males (86% of control), with no tabulated data being presented. Deviations observed in haematological parameters were incidental and not considered to be related to treatment. Alkaline phosphatase was significantly increased at 260 mg/kg bw/day in males in week 26 and 39 (199-206% of control) and was constant during the study at 260 mg/kg bw/day, whilst values in the control group declined as is normal for young adult dogs. Reduced inorganic phosphate levels were observed in week 52 in both sexes at 260 mg/kg/day and in males at 80 mg/kg bw/day. Other deviations were incidental and not considered to be toxicologically relevant. Increased relative weights of kidney (in females), heart (in males) and testes at 260 mg/kg bw/day is considered to be related to the reduced body weight compared to control. Absolute adrenal weight was significantly decreased at 80 and 260 mg/kg bw/day (66-80% of control) in males which was also attributed to reduced terminal body weights. Relative liver weights were higher in all groups of treated males and females, when compared with controls (111-126% of control for males and 131-157% of control for females).

Minor signs most probably due to reduced feed ingestion and adaptation phenomena caused by overloading of the liver with the foreign test substance became overt in male and female dogs of the high dose group (260 mg/kg/d) and consisted in a stagnation of the body weight gain (both sexes), reduced feed intake (males), diminished plasma concentration of inorganic phosphorus at week 52 (both sexes), increased relative liver weights (both sexes) and decreased absolute adrenal weights (males only).

Under the conditions of this study, the NOAEL is set at 25 mg/kg bw/day.

Repeated dose toxicity: inhalation

4 weeks, sub-acute toxicity inhalation study in rats, Bernstein, 1987

The study was performed in accordance with OECD TG 412 and in compliance with GLP. Four groups of Wistar, KFM-Han rats, each containing 5 males and 5 females, inhaled 1.5, 11, 99 or 1050 mg/m3 test substance using ethanol as vehicle. The exposure was 6 hours per day for 5 days per week for 29 days. The mean particle size of the aerosol sampled which was 3 µm and the GSD 1.9 μm. No mortality occurred. No clinical or ocular abnormalities were observed. Body weight gain was decreased in males at 1050 mg/m3 (terminal body weight 93 % of control) at the end of the exposure period. Also a decrease in food consumption was observed in these males. At haematology, haemoglobin was slightly, but significantly decreased at 1050 mg/m3 in females (94 % of control). Clinical biochemistry investigations showed a dose-dependent and significant decrease in total bilirubin (72 % of control) and a dose-dependent and significant increase in potassium and chloride (122 % and 105 % of control, respectively) in the highest dosed males. Increased potassium may indicate kidney insufficiency. Calcium was slightly but significantly decreased in these males (97 % of control). In the females, urea was significantly decreased at the 99 mg/m3 dose (78 % of control) and sodium was slightly but significantly decreased at the highest dose (99 % of control). Albumin and total protein were both significantly increased at the highest dose level (111 % and 109 % of control, respectively). In the recovery groups, all values were normal again. Relative weights of the lungs, liver and testis were significantly increased in males of the highest dose group (116 %, 114 % and 116 %, respectively). In females of the highest dose group, absolute and relative liver weight were significantly increased (127 % and 134 %, respectively). Relative kidney weight was significantly increased (116 %) at the highest dose. Absolute and relative adrenals weight were significantly decreased (both 81 %) at 99 mg/m3. However, in the absence of any decreases at the high dose level this finding was considered not toxicologically relevant. Changes in relative testes and lung weight might be due to decreased terminal body weight. Macroscopically and microscopically no treatment-related effects were observed. Males of the control group and the 1050 mg/m3 dose group showed similar histopathological findings, namely bronchial associated lymphoid tissue hyperplasia (2/5 and 1/5, resp.) and interstitial pneumonitis (5/5 and 4/5, resp.) of the lungs, and mononuclear inflammatory cell foci (3/5 and 1/5, resp.) of the liver. Females of both the control group and the 1050 mg/m3 dose group showed nephrocalcinosis of the kidneys. Based on increased liver and kidney weights and clinical biochemical findings, the systemic NOAEL is set at 99 mg/m3. Since no adverse local effects were observed, the local NOAEL is set at >1050 mg/m3.

Repeated dose toxicity: dermal

4 weeks, sub-acute toxicity dermal study in rats, Varney 1985

An OECD TG 410-like study as performed in compliance with GLP to assess the subacute dermal toxicity. Groups of 5 male and 5 female Crl:CD(SD)BR rats were dermally exposed to 20, 200 and 2000 mg/kg test item under occlusive conditions for 21 days - 6 hours/day. An additional group was treated with the corn oil vehicle alone and acted as controls. Twice daily all animals were examined to detect any which were dead or moribund. All animals were examined once daily for clinical changes including skin irritation. Individual body weights were recorded on the first day of the test and at weekly intervals throughout the study. Individual food consumption was recorded at weekly intervals throughout the study. Individual blood samples were collected from the orbital sinus of all animals (after an 18 hours fast) during the final week of the study. Laboratory analysis (haematology and clinical chemistry) were performed on individual blood samples. The animals were killed by an intraperitoneal injection of pentobarbitone sodium solution and exsanguination, following about 19 hours without food. A full macroscopic examination was made.

No treatment-related local skin effects were observed. One control animal, four animals given 20 mg/kg and one animal given 200 mg/kg showed slight, mainly reversible erythema. No treatment-related effects were observed on mortality, clinical observations, body weight, food consumption and haematology. In clinical chemistry analyses, females given 200 and 2000 mg/kg showed decreased levels of ASAT (80 % and 82 % of control, respectively) and ALAT (65 and 60 % of control, respectively). These changes were not considered to be treatment-related, since decreases were observed and no dose-response relationship was noted. In both males and females of the 2000 mg/kg dose group, increased absolute liver weights (122 % of control) and relative liver weights (119 % of control) were noted. Male rats given 2000 mg/kg showed decreased absolute weights of the gonads (94 % of control) and kidneys (91 % of control). In male rats given 200 and 2000 mg/kg, decreased relative weights of the gonads (94 % and 92 % of control, respectively) and the kidneys (94 % and 89 % of control, respectively) were seen.

At necropsy, no treatment-related changes were observed. Histopathology showed mild renal hydronephrosis in the control group (1/10) and 20 mg/kg group (2/10). Mild to moderate renal hydronephrosis was seen in 2/10 rats of the 200 mg/kg dose group, and the symptom was severe in one animal of the highest dose group. Hypertrophy of the liver was observed in 5 rats of the highest dose group. In the absence of histopathological correlates at 200 mg/kg, the slight decreases in relative gonad and kidney weight at 200 mg/kg were not considered toxicologically relevant.

Based on increased liver weights and concomitant liver hypertrophy of half of the test animals, the systemic NOAEL is set at 200 mg/kg bw/day. Since no adverse local effects were observed, the local NOAEL is set at >2000 mg/kg bw/day.

Based on the available information classification upon repeated exposure is not warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.