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Administrative data

Description of key information

Oral (28 days) NOAEL = 600 mg/kg bw/day, male/female rat, EU Method B.7, Lortie 2002

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 June 2002 to 9 August 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive,performed in accordance with valid guidelines and the study was conducted under GLP conditions.
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Chemical Substance Law, Notification No. 700 of the Environment Agency, No. 1039 of the Ministry of Health and welfare and No. 1014 of International Trade and Industry, Japan.
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: 186 - 215 g (males); 148 - 177 g (females)
- Housing: individually in suspended wire-mesh cages measuring 43.0 x 21.5 x 18.0 cm
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 50 ± 20 %
- Air changes (per hr): approximately 12 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: From 27 June 2002 to 9 August 2002
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was first ground to a fine powder before being suspended in the vehicle in order to achieve the concentrations of 10, 30 and 120 mg/mL. The suspensions were then homogenised with a magnetic stirrer. The test material preparations were made for up to 9 days and kept, protected from light, until administration.

STABILITY:
The stability of the solutions was checked. Samples of the 10 mg/mL and 120 mg/mL solutions were taken in duplicate just after preparation and again after 4 and 9 days storage at 4 °C and protected from light. they were analysed according to the method outlined below.

ADMINISTRATION:
The quantity of test material was adjusted according to the most recently recorded bodyweight.
A constant dosage volume of 5 mL/kg/day was used.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For each group, including the control, the preparations intended for use in weeks 1 and 4 were sampled for analyses.
The solution was stirred with a magnetic stirrer. 1 mL was diluted tenfold with water. Serial dilutions were carried out with water before 2-fold dilution in 50% v/v acetonitrile to achieve target concentrations of test material in the range 20 – 35 µg/mL. An aliquot of the test material solutions was analysed by HPLC/UV. A calibration curve was obtained by linear regression analysis of UV absorbance at 254 nm versus concentration of standard solutions for concentrations ranging from 1 - 40 μg/mL.

The regression analysis of the calibration data gave an equation as follows:
Y = a X + b

Where
Y – absorbance of test material
X = concentration of test material (μg/mL)
a = slope
b = intercept

The concentrations of test material were calculated using this equation.

Chromatographic conditions:
- Column: Lichrospher ODS2, 125 mm x 4.0 mm, particle size 5 µm
- Flow rate: 1.5 mL/min
- Temperature: 40 °C
- Injection volume: 30 µL
- Wavelength: 254 nm
- Retention time: 13.5 minutes
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 50, 150, 600 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 males and 10 females (0 and 600 mg/kg/day)
5 males and 5 females (50 and 150 mg/kg/day)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: doses were selected on the basis of a 7 day preliminary rangefinding study during which slightly lower body weight gain was noted in males given 150, 450 and 1000 mg/kg/day and slightly lower thymus weight was noted in females given 150, 450 and 1000 mg/kg/day. Consequently, it was considered that 1000 mg/kg/day would probably exceed the Maximum Tolerated Dose level and so 600 mg/kg/day was chosen as the high dose level.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were checked for mortality and morbidity at least twice a day during the treatment period and at least once a day during the observation period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical observations were performed daily; detailed clinical observations were performed once before the beginning of the treatment period and once weekly thereafter
- Parameters checked during the detailed clinical observations included (but were not limited to): changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity. Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes or bizarre behaviour were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: on the day before allocation into groups, on the first day of treatment and once a week until the end of the study

FOOD CONSUMPTION
- Food consumption for each animal was determined once a week, until the end of the study. Food consumption was calculated per animal and per day

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 28
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight period of at least 14 hours)
- How many animals: 5 males and 5 females of each sex
- Parameters checked included: erythrocytes, haemoglobin, mean cell volume, packed cell volume, mean cell haemoglobin concentration, mean cell haemoglobin, thrombocytes, leucocytes, differential white cell count, neutrophils, eosinophils, basophils, lymphocytes, monocytes, prothrombin time, activated partial thromboplastin time, fibrinogen

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 28
- Animals fasted: Yes (overnight period of at least 14 hours)
- How many animals: 5 males and 5 females of each sex
- Parameters checked included: sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total bilirubin, total proteins, albumin, albumin/globulin, cholesterol, triglycerides, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase

URINALYSIS: Yes
- Time schedule for collection of urine: day 28
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (overnight period of at least 14 hours)
- Parameters checked included: volume, pH, specific gravity, proteins, glucose, ketones, bilirubin, nitrites, blood, urobilinogen, cytology of sediment (leucocytes, erythrocytes, cylinders, magnesium ammonium phosphate crystals, calcium oxalate crystals, cells), appearance, colour

NEUROBEHAVIOURAL EXAMINATION: Yes
- Motor activity of all animals was measured once by automated infra-red sensor equipment over a 15 minute period at the end of the treatment period

FUNCTIONAL OBSERVATION BATTERY (FOB): Yes. This included a detailed clinical examination, measurement of reactivity to manipulation or to different stimuli and motor activity.
- Time schedule: Once at the end of the treatment period
- How many animals: All animals
- Parameters checked included: "touch escape"; in the hand: fur appearance, salivation, lachrymation, piloerection, exophthalmos, reactivity to handling, pupil size; in the standard arena (two minute recording): grooming, palberal closure, defecation, urination, tremors, twitches, convulsions, gait, arousal, posture, stereotypy, behaviour and breathing, ataxia, hypotonia.
- The following parameter measurements, reflexes and responses were recorded: touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay (and at the end of observation) rectal temperature.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- A complete macroscopic examination was performed on all animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

ORGAN WEIGHTS: Yes
- The body weight of all animals killed at the end of the treatment or treatment-free period was recorded before sacrifice and the following organ weights recroded: adrenals, brain, epididimydes, heart, kidneys, liver, ovaries, spleen, testes, thymus, thyroids with parathyroids.

HISTOPATHOLOGY: Yes
- Tissues examined included: any tissues with macroscopic lesions, adrenals, brain, caecum, colon, duodenum, epididymides, heart, ileium, jejenum, kidneys, liver, lungs with bronchi, lymph nodes (mandibular, mesenteric), ovaries, prostate, sciatic nerve, seminal vesicles, spinal cord (cervical, thoracic and lumbar), spleen, sternum with bone marrow, stomach with forestomach, testes, thymus, thyroids with parathyroids, urinary bladder, uterus (horns and cervix), vagina.
- All tissues were embedded in paraffin wax, sectioned at 4 microns and stained with hematoxylin-eosin.
Clinical signs:
no effects observed
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:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
None of the animals died during the study. The only treatment-related clinical sign consisted in ptyalism, observed in 1/10 males and in 5/10 females dosed at 600 mg/kg/day. However, this sign was not considered to represent an adverse effect. In addition, soft faeces were observed in 1/5 males given 150 mg/kg/day. This clinical sign, as well as area of hair loss, scattered hair, regurgitation and aggressive behaviour, was not dose-related, observed in a small number of animals, without the same trend in the two sexes and is commonly seen in the laboratory rat of this strain and age. Thus, it was not considered to be related to treatment with the test material.

BODY WEIGHT AND FOOD CONSUMPTION
When compared to controls, a slightly higher mean body weight gain was noted in all treated males and females given 600 mg/kg/day during the treatment period. This was related to a higher mean food consumption during the same period. Slightly higher mean body weight gain was noted during the treatment-free period in males previously given 600 mg/kg/day. As these differences were slight, not dose-related and without the same trend in the two sexes, they were not considered to be toxicologically relevant.

HAEMATOLOGY
When compared to control values, the only statistically significant differences noted at the end of the treatment period consisted in a slightly lower hemoglobin concentration in males given 150 mg/kg/day and slightly longer prothrombin time in females given 600 mg/kg/day. Since these differences were slight, not dose-related and without the same trend in the two sexes, they were not considered to be toxicologically relevant.

CLINICAL CHEMISTRY
When compared to control values, the only statistically significant differences noted at the end of the treatment period were as follows: slightly higher potassium and calcium levels in males given 150 mg/kg/day; slightly increased ASAT activity in males given 150 mg/kg/day; slightly lower glucose level in females given 150 mg/kg/day; and slightly lower cholesterol level in females given 150 and 600 mg/kg/day. Since these changes were slight, not dose-related and/or without the same trend in the two sexes, and since all the individual values were within or close to the range of historical background data for the test laboratory, they were considered to be of no toxicological importance.

URINALYSIS
No relevant differences from controls were noted in any quantitative or qualitative parameters of any treated group.

NEUROBEHAVIOUR AND FOB
There was no evidence of perturbation of either autonomic or physiological functions at any time or any of the dose-levels employed. There was no relevant changes in neurotoxicological parameters or motor activity in any treated animal.

ORGAN WEIGHTS
A few differences in organ weights (% variation from control group) were observed between treated and control animals. However, they were minor and/or not dose-related, did not show the same trend in the two sexes and/or there were variations in the individual values between treated and control groups and/or within the same group. They were thus considered to be of no toxicological significance.

GROSS PATHOLOGY
The few macroscopic post-mortem observation noted were not dose-related and are commonly recorded spontaneously in the untreated laboratory rat of this strain and age and were thus considered to be without toxicological significance.

HISTOPATHOLOGY: NON-NEOPLASTIC
No treatment-related microscopic findings were observed.
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Critical effects observed:
not specified

Chemical analysis of the dosage forms

The results of the analyses demonstrated a satisfactory stability of the two dosage forms prepared (10 and 120 mg/mL) over a 9 -day period at 4 °C (protected from light). Throughout the study, a satisfactory agreement was observed between the nominal and actual concentrations of the test material in the administered dosage forms since the deviations from nominal concentrations were in an aceptable range (± 4 %).

Table 1: Hematology results (mean values)

Parameter

Males

Females

0 mg/kg/day

50 mg/kg/day

150 mg/kg/day

600 mg/kg/day

0 mg/kg/day

50 mg/kg/day

150 mg/kg/day

600 mg/kg/day

WBC (g/L)

9.66

11.03

9.21

10.87

9.66

6.88

6.96

8.52

RBC (T/L)

8.04

7.70

7.81

7.81

7.26

7.28

7.59

7.23

HB (g/dL)

15.4

15.0

14.8*

15.1

14.2

14.1

14.9

14.4

PCV (L/L)

0.45

0.45

0.44

0.44

0.40

0.40

0.43

0.41

MCV (fL)

56.4

57.8

56.1

56.9

55.5

55.3

56.4

56.6

MCH (pg)

19.2

19.5

19.0

19.4

19.6

19.4

19.6

19.9

MCHC (g/dL)

34.1

33.7

33.8

34.1

35.2

35.1

34.8

35.1

PLAT (g/L)

1120

1059

986

1217

937

961

1088

956

N (g/L)

0.99

1.39

1.74

1.19

1.35

0.73

0.91

1.03

E (g/L)

0.08

0.10

0.10

0.08

0.31

0.10

0.11

0.15

B (g/L)

0.02

0.03

0.02

0.02

0.02

0.01

0.01

0.01

L (g/L)

8.29

9.21

7.12

9.35

7.77

5.85

5.72

7.14

M (g/L)

0.28

0.31

0.24

0.23

0.23

0.20

0.21

0.19

PT (s)

14.8

14.4

15.1

16.0

13.0

13.4

13.7

14.5*

APIT (s)

26.9

24.3

25.4

29.2

15.3

16.5

16.8

17.2

FIB (g/L)

2.94

2.95

3.21

3.11

2.54

2.28

2.50

2.32

* p < 0.05

Table 2: Blood chemistry results (mean values)

Parameter

Males

Females

0 mg/kg/day

50 mg/kg/day

150 mg/kg/day

600 mg/kg/day

0 mg/kg/day

50 mg/kg/day

150 mg/kg/day

600 mg/kg/day

Na+ (mmol/L)

140.8

140.6

140.9

141.4

138.8

139.1

140.3

139.9

K+ (mmol/L)

3.92

4.06

4.48*

3.98

4.19

3.85

4.27

4.29

Cl- (mmol/L)

101.5

101.6

102.1

102.2

101.6

102.3

103.1

102.6

Ca2+ (mmol/L)

2.75

2.82

2.81*

2.81

2.73

2.70

2.74

2.71

Inorg. P (mmol/L)

2.79

2.83

2.88

2.71

2.41

2.28

2.68

2.44

Gluc. (mmol/L)

6.91

6.37

7.13

6.64

6.56

5.91

5.52*

5.72

Urea (mmol/L)

4.2

3.9

3.9

3.9

4.3

5.0

4.8

5.0

Creat. (µmol/L)

33

33

32

32

35

40

39

38

Prot. (g/L)

62

62

63

64

65

64

62

62

ALB (g/L)

38

38

38

39

41

42

40

40

A/G

1.58

1.60

1.54

1.50

1.75

1.83

1.80

1.84

Tot. BIL (µmol/L)

2

2

1

2

2

2

2

2

Chol. (mmol/L)

1.5

1.3

1.2

1.4

1.8

1.6

1.3**

1.3**

Trig. (mmol/L)

0.70

0.94

0.86

0.77

0.37

0.35

0.28

0.31

ALP (IU/L)

323

358

326

371

211

257

239

230

ASAT (IU/L)

46

51

80**

53

50

55

56

64

ALAT (IU/L)

13

13

13

15

10

12

11

8

* p < 0.05

** p < 0.01

Conclusions:
Under the conditions of the study the No Observed Adverse Effect Level (NOAEL) of the test material was determined to be 600 mg/kg/day. The study is considered to be reliable, relevant and adequate for risk assessment and classification and labelling purposes.
Executive summary:

The oral repeat dose toxicity of the test material was determined in accordance with standardised guideline EU Method B.7. Groups of five male and five female rats were dosed orally by gavage with 0 (control), 50, 150 or 600 mg/kg bw/day of the test material for 28 consecutive days. Following completion of the 28 day treatment period five males and five females dosed at 0 and 600 mg/kg/day were observed for a two week period. During the study, the animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded once a week. Once a week, detailed clinical observations were made and a functional observation battery performed at the end of the treatment period. Hematological, blood biochemical and urinalysis investigations were performed on 5 male and 5 female animals of each dose group at the end of week 4. On completion of the study, animals were subjected to a macroscopic post-mortem examination and specified organs were weighed and preserved. Microscopic examination was performed on selected tissues from animals of the control and 600 mg/kg/day groups.

Dosing preparations were analysed for achieved concentration, homogeneity and stability and were considered to be satisfactory. During the study none of the animals died. There were no clinical signs that were related to toxicity of the test material during the study. Ptyalism (which was not considered to represent an adverse effect) was observed in 1/10 males and 5/10 females given 600 mg/kg/day. Detailed clinical observation, functional observation battery and motor activity investigation revealed no relevant changes in treated animals from controls. No relevant differences were noted between control and treated animals in body weight or food consumption. No test material related changes were noted at the end of the treatment period following haematology, blood biochemistry and urinalysis investigations. No abnormalities were noted at gross pathology and no relevant differences in organ weights were noted in any treated group compared to controls. Microscopic examination of tissues revealed no treatment-related toxicity.

Under the conditions of the study the No Observed Adverse Effect Level of the test material was determined to be 600 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
600 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, performed in accordance with valid guidelines and the study was conducted under GLP conditions. The study was assigned a reliability score of 1 in accordance with the criteria of Klimisch (1997) and was considered suitable as an accurate reflection of the test material.

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

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The oral repeat dose toxicity of the test material was determined in accordance with standardised guideline EU Method B.7. Groups of five male and five female rats were dosed orally by gavage with 0 (control), 50, 150 or 600 mg/kg bw/day of the test material for 28 consecutive days. Following completion of the 28 day treatment period five males and five females dosed at 0 and 600 mg/kg/day were observed for a two week period. During the study, the animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded once a week. Once a week, detailed clinical observations were made and a functional observation battery performed at the end of the treatment period. Hematological, blood biochemical and urinalysis investigations were performed on 5 male and 5 female animals of each dose group at the end of week 4. On completion of the study, animals were subjected to a macroscopic post-mortem examination and specified organs were weighed and preserved. Microscopic examination was performed on selected tissues from animals of the control and 600 mg/kg/day groups.

Dosing preparations were analysed for achieved concentration, homogeneity and stability and were considered to be satisfactory. During the study none of the animals died. There were no clinical signs that were related to toxicity of the test material during the study. Ptyalism (which was not considered to represent an adverse effect) was observed in 1/10 males and 5/10 females given 600 mg/kg/day. Detailed clinical observation, functional observation battery and motor activity investigation revealed no relevant changes in treated animals from controls. No relevant differences were noted between control and treated animals in body weight or food consumption. No test material related changes were noted at the end of the treatment period following haematology, blood biochemistry and urinalysis investigations. No abnormalities were noted at gross pathology and no relevant differences in organ weights were noted in any treated group compared to controls. Microscopic examination of tissues revealed no treatment-related toxicity.

Under the conditions of the study the No Observed Adverse Effect Level (NOAEL) of the test material was determined to be 600 mg/kg/day.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only one study is available. The study was considered sufficiently reliable to address the repeated dose toxicity endpoint.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the test material does not require classification for specific organ toxicity, repeated dose. The effects observed in the main study are not considered to be toxicologically significant and do not indicate any signs of organ dysfunction.