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Description of key information

In the key study (90d feeding in rat) the following observations were recorded: parameters of hematology and blood chemistry investigated at week 14 showed minimal variations in thrombocytes, sodium, inorganic phosphorus, protein, albumin and globulin concentrations in females of the high dose (10000 ppm). The toxicological significance of these findings is doubtful. A slight, but significant decrease of the liver weight in treated male rats of groups 3 and 4 (2000 and 10000 ppm) and a slight decrease of testes weight in treated male group 4. No correlate was identified in histological investigations and these effects were considered to be of no toxicological relevance. Males given 2000 and 10000 ppm additionally showed an increase of nonspecific, minimal inflammatory cell infiltration in the liver. The NOEL was derived at 400 ppm and the NOAEL at 10000 ppm (624 mg/kg).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 Jan 1983 - 25 Jan 1984
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
(adopted 1981)
Deviations:
no
GLP compliance:
yes
Remarks:
Ciba-Geigy Ltd., Basle/Switzerland, GU2 Toxicology
Limit test:
no
Species:
rat
Strain:
other: F3-hybrid of RII 1/Tif x RII 2/Tif
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Tif: RAIf (SPF) rats from Animal Production, CIBA-GEIGY LTD., 4332 Stein, Switzerland
- Age at study initiation: 6 weeks
- Weight at study initiation: 190-195 g (males), 173-175 g (females)
- Housing: 5 animals per cage (Makrolon Cage Type 4)
- Diet: Pelleted, certified standard diet Nafag No. 890 Tox, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-2
- Humidity (%): 55+/-10
- Air changes (per hr): 16-20
- Photoperiod: 12 hrs dark / 12 hrs light
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was weighed on a calibrated Mettler balance. The pulverised food was then homogeneously mixed with the appropriate concentrations of the compound and 30% water was added before pelleting to ensure the necessary pellet quality. The pellets were subsequently air dried.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the initiation of the study, pretest feed samples were analysed for concentration of test material. The same was undertaken periodically with the food batches applied during the test. The analysis were carried out in the Central Analytical Laboratories of CIBA-GEIGY LTD., Basle/Switzerland.
Duration of treatment / exposure:
90 days
Frequency of treatment:
continuous over 90 days
Dose / conc.:
400 ppm
Remarks:
Corresponding to 25 mg/kg bw in males and 27 mg/kg bw in females.
Dose / conc.:
2 000 ppm
Remarks:
Corresponding to 123 mg/kg bw in males and 127 mg/kg bw in females.
Dose / conc.:
10 000 ppm
Remarks:
Corresponding to 624 mg/kg bw in males and 667 mg/kg bw in females.
No. of animals per sex per dose:
20
Control animals:
yes, plain diet
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on working days (mortality); daily (symptoms)

BODY WEIGHT: Yes
- Time schedule for examinations: weekly (midweek)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- mean food conversion was calculated based on weekly food consumption and midweek body weight.

WATER CONSUMPTION: Yes
- Time schedule for examinations: monthly (week 3, 7, 11)

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at the beginning and towards the end of the treatment period (day -6 and day 86 of test)
- Dose groups that were examined: in control animals and in treated animals of the highest dose level

HEARING TEST: Yes
- Time schedule for examinations: at the beginning and towards the end of the treatment period (day -6 and day 86 of test)
- Dose groups that were examined: in control animals and in treated animals of the highest dose level

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period
- Anaesthetic used for blood collection: Yes (Ether anaesthesia)
- Animals fasted: Yes (20 hours before collection of blood)
- How many animals: 20 per sex and per group
- Following parameters were examined: RBC, HCT, Hb, MCV, PCV, MCH, PLT, WBC, Differential blood count, Reticulocytes, Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period
- Animals fasted: Yes (20 hours before collection of blood)
- How many animals: 20 per sex and per group
- Following parameters were examined: Glucose, Urea, Total Protein, Creatinine, Albumine, Globuline, A/G ratio, Sodium, Potassium, Chloride, Calcium, Phosphorus inorganic, GPT, GOT, AP, Total Cholesterol

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
At the end of the test period all control and test animals were bled under ether anaesthesia and subjected to detailed autopsy. Liver, adrenals, brain, thymus, heart, kidneys and gonads were weighed. The following organs and tissues were preserved in 10% neutral formalin:
skin, mammary area, spleen, mesenteric lymph node, axillary lymph node, sternum with bone marrow, femur with joint, skeletal muscle, trachea, lung, heart, aorta, submandibular salivary gland, liver, pancreas, oesophagus, stomach, small intestine, large intestine, kidney, urinary bladder, prostate, seminal vesicle, testis, epididymis, uterus, ovary, pituitary gland, adrenal gland, thyroid with parathyroid gland, thymus, peripheral nerve, brain, spinal cord, eye with optic nerve, orbital gland, extraorbital lacrimal gland, organs and tissues showing macroscopical changes.
Complete autopsy with tissue preservation was performed also in all animals which died during the test period.

HISTOPATHOLOGY: Yes
After the fixation organ samples from each control and test rat were taken, embedded in paraplast, sectioned at 3-5 microns, stained with haematoxylin and eosin and subjected to microscopical examination.
Statistics:
For each time point and parameter a uni-variate statistical analysis was conducted. Due to the routine manner of the analysis system, parameter free methods were applied. Each treated group was compared to the control group in respect of dispersion and displacement (1). In addition a trend test (2) was applied considering all groups. Statistical analysis is performed to draw attention to distinct values. A statistically significant difference between two values does not necessarily imply biological relevance of that deviation and is not conclusive for a treatment related effect. Hence, the responsible scientist may not comment on statistically significant values lying within the physiological range and on the other hand may comment on statistically not significant values, which differ substantially from the expected normal values.
(1) Y. Lepage, Biometrika (1971) 58: pp. 213-217.
(2) H. R. Jonckheere, Biometrika (1954) 41: pp. 133-145
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical symptoms and no signs of local and/or systemic toxicity were observed.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No death which was related to the administration of the test compound occurred in the course of the study. One animal (male no 55, group 3) died during blood withdrawal at the end of the treatment period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean body weight gain of all treated male and female groups was similar to that of the respective control groups, except a marginally lower weight increase in treated male group 4 (10 000 ppm), which however was not significant and within a range of 10 % of the control value.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean food consumption of all treated male and female groups was similar to that of the respective control groups. Specific food consumption in relation to body weight in treated animals was similar to that of the respective control groups.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
The mean water consumption of all treated male and female groups was similar to that of the respective control groups.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmic inspections and hearing examinations performed before and towards the end of the administration period revealed no evidence of a reaction to the treatment.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematological investigations at week 14 showed a minimal increase in numbers of thrombocytes in females of group 4 (10000 ppm).
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Blood chemistry investigations revealed slightly lower sodium concentrations and higher concentrations of inorganic phosphorus in the females of group 4 (10000 ppm). The total protein concentration and the albumin concentration were noted to be marginally lower in the females of group 4 (10000 ppm). The globulin concentration of this group was slightly increased. The toxicological significance of these findings is doubtful.
Other parameters examined in hematology and blood chemistry achieving a level of statistical significance in their difference from control values were considered to have arisen fortuitously.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
A slight, but significant decrease of the liver weight was observed in treated male rats of groups 3 and 4 (2000 and 10000 ppm). A slight decrease of testes weight was noted in treated male group 4 (10000 ppm). Additional statistically significant differences in organ weights between treated and control groups were noted. Incidental, however statistically significant differences in organ weights between treated and control groups were noted. Since no systematic pattern emerged, these differences were attributed to spontaneous variation rather than to the treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopical changes were observed that were considered to be due to the administration of the test compound.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Apart from an increasing incidence of nonspecific, minimal inflammatory cell infiltration in the liver in male animals of treated groups 3 and 4 (2000 and 10000 ppm) no microscopical changes were observed that were considered to be due to the administration of the test compound.
Histopathological findings: neoplastic:
not examined
Dose descriptor:
NOEL
Effect level:
400 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: At the next higher dose level, slight decrease in the liver weight and an incidence of non-specific minimal inflammatory cell infiltration were recorded.
Dose descriptor:
NOAEL
Effect level:
624 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse effects at this dose level
Dose descriptor:
NOAEL
Effect level:
667 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effects at this dose level
Critical effects observed:
not specified

Table 1: Mean organ weights (g) and ratios (as a percentage of body- and brain weight) of male animals in test week 14.

Organs

Dose (ppm in food)

Trend

 

0

400

2000

10000

 

Body

457

460

450

435

 

Brain

2.40

2.41

2.45

2.44

 

Brain/body

0.53

0.53

0.55

0.56

 

Heart

1.37

1.29

1.28

1.31

 

Heart/body

0.3

0.28*

0.285

0.31

 

Heart/brain

56.99

53.50*

52.37*

53.56

 

Liver

14.97

16.11

13.92

12.36*

X

Liver/body

3.27

3.51*

3.10*

2.85*

X

Liver/brain

624.7

670.3

570.9*

506.8*

X

Kidneys

2.79

2.79

2.80

2.74

 

Kidneys/body

0.61

0.61

0.62

0.63

 

Kidneys/brain

116.1

115.9

114.3

112.4

 

Adrenals

0.068

0.070

0.066

0.068

 

Adrenals/body

0.0148

0.0152

0.0147

0.0157

 

Adrenals/brain

2.83

2.91

2.70

2.78

 

Thymus

0.41

0.43

0.44

0.46

 

Thymus/body

0.090

0.093

0.098

0.105

 

Thymus/brain

17.10

17.83

17.96

18.72

 

Gonads

3.82

3.53

3.62

3.43*

X

Gonads/body

0.84

0.78

0.81

0.79

 

Gonads/brain

159.2

147.2

148.3*

140.6*

X

 

*: Significant difference (location and/or dispersion) between control group and test group (sign. l. = 0.05)

X: Significant negative trend from control to highest dosage group (sign. L. = 0.1)


Table 2: Mean organ weights (g) and ratios (as a percentage of body- and brain weight) of female animals in test week 14.

Organs

Dose (ppm in food)

Trend

 

0

400

2000

10000

 

Body

302.3

312.4

315.6

298.7

 

Brain

2.33

2.33

2.36

2.32

 

Brain/body

0.774

0.749

0.757

0.780

 

Heart

0.972

0.974

0.970

0.916

 

Heart/body

0.322

0.313*

0.310*

0.308

 

Heart/brain

41.77

41.99

41.29

39.66

 

Liver

10.74

10.60

10.33

10.31

 

Liver/body

3.56

3.40

3.29*

3.45

 

Liver/brain

461.2

455.4

438.5

446.1

 

Kidneys

2.06

1.98

2.04

2.08

 

Kidneys/body

0.69

0.64*

0.65

0.70

 

Kidneys/brain

88.6

85.3

86.5

89.8

 

Adrenals

0.093

0.087

0.094

0.088

 

Adrenals/body

0.0308

0.0282

0.0300

0.0298

 

Adrenals/brain

3.99

3.76

4.00

3.82

 

Thymus

0.303

0.315

0.312

0.286

 

Thymus/body

0.101

0.101

0.099

0.096

 

Thymus/brain

13.08

13.59

13.33

12.38

 

Gonads

0.174

0.190

0.175

0.177

 

Gonads/body

0.058

0.062

0.056

0.060

 

Gonads/brain

7.49

8.20

7.42

7.66

 

*: Significant difference (location and/or dispersion) between control group and test group (sign. l. = 0.05)

Table 3: Summary of macroscopical findings in female and male animals.

 

Dose (ppm in food)

 

0

400

2000

10000

 

m

f

m

f

m

f

m

f

Animals examined macroscopically

20

20

20

20

20

20

20

20

Abdominal cavity nodule (<0.5 cm)

 

 

1

 

 

 

 

 

Body as a whole, no changes observed

20

19

18

19

19

20

18

18

Brain

Damaged during autopsy

malformation

 

 

1

 

 

 

1

 

 

 

 

 

Kidney

Fibrinous adhesion

Small

 

 

 

 

 

 

 

 

 

1

 

1

 

1

Ovary

Cyst (<1 cm)

 

 

 

 

1

 

 

 

 

Spleen

Fibrinous adhesion

small

 

 

 

 

 

 

 

 

1

 

1

Testis

Small bilateral

 

 

 

 

 

1

 

 

1

 

Thymus

Haemorrhage

 

 

1

 

 

 

 

 

 

Uterus

Dilatation

 

 

 

 

 

 

 

 

1

 

 

Executive summary:

In an oral subchronic study Tif : RAIf (SPF) rats (20 per sex and dose group) were dosed for 90 days with 400, 2000 and 10000 ppm test substance added to the diet (calculated mean daily intake: 25, 123 and 624 mg/kg bw for males and 27, 127 and 667 mg/kg bw for females). No deaths related to the administration of the test compound occurred in the course of the study. No clinical symptoms and no signs of local and/or systemic toxicity were observed. The mean body weight gain of all treated male and female groups was similar to that of the respective control groups, except a marginally lower weight increase in treated male group 4 (10000 ppm), which however was not significant and within the range of 10 % of the control value. The mean food consumption of all treated male and female groups was similar to that of the respective control groups. Hematological investigations at week 14 showed a minimal increase in numbers of thrombocytes in females of group 4 (10000 ppm). Blood chemistry investigations revealed slightly lower sodium concentrations and higher concentrations of inorganic phosphorus in the females of group 4 (10000 ppm). The total protein concentration and the albumin concentration were noted to be marginally lower in the females of group 4 (10000 ppm). The globulin concentration of this group was slightly increased. The toxicological significance of these findings is doubtful. Other parameters examined in hematology and blood chemistry achieving a level of statistical significance in their difference from control values were considered to have arisen fortuitously. A slight, but significant decrease of the liver weight was observed in treated male rats of groups 3 and 4 (2000 and 10000 ppm). A slight decrease of testes weight was noted in treated male group 4 (10000 ppm). Additional statistically significant differences in organ weights between treated and control groups were noted. Since no systematic pattern emerged, these differences were attributed to spontaneous variation rather than to the treatment. Apart from an increasing incidence of nonspecific, minimal inflammatory cell infiltration in the liver in male animals of treated groups 3 and 4 (2000 and 10000 ppm) no macroscopical or microscopical changes were observed that were considered to be due to the administration of the test compound. In conclusion a NOEL of 25 (males) and 27 (females) mg/kg bw per day was derived, based on increased incidence of inflammatory cell infiltration in the liver of mainly male animals. Since no specific local or systemic effects have been observed up to the highest dose tested, the NOAEL was raised to 624 (males) and 667 mg/kg bw (females).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
624 mg/kg bw/day
Study duration:
subchronic
Species:
rat

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 test article’s toxicity upon repeated oral administration was assessed in a subacute 28 day and a subchronic 90 day study performed in rats.

The 90 day study was in accordance to OECD TG 408 (adopted 1981) and in compliance with GLP. Twenty Tif:RAIf (SPF) rats per sex and dose group were dosed with test substance added to the diet at 400, 2000, 10000 ppm (calculated mean daily intake: 25, 123 and 624 mg/kg body weight for males and 27, 127 and 667 mg/kg body weight for females). No deaths related to compound administration occurred in the course of the study. No clinical symptoms and no signs of local and systemic toxicity were observed. No effects were observed on mean body weight gain (except a marginally lower but statistically insignificant weight increase in treated male group 4 (10 000 ppm), mean food consumption and ophthalmic parameters. Parameters of hematology and blood chemistry investigated at week 14 showed minimal variations in thrombocytes, sodium concentrations, inorganic phosphorus concentrations, protein concentration, albumin concentration and globulin concentration in females of the high dose (10000 ppm). The toxicological significance of these findings is doubtful. Other parameters examined in hematology and blood chemistry achieving a level of statistical significance in their difference from control values were considered to have arisen fortuitously. A slight, but significant decrease of liver weights were observed in treated male rats of groups 3 and 4 (2000 and 10000 ppm). Although this effect is statistically significant, the toxicological relevance is equivocal. No correlation was found in histological examinations that can explain this organ weight loss. Furthermore, this effect was only observed in male animals. Therefore, this slight liver weight loss was considered as non adverse. Additionally, a slight decrease of testes weight was noted in treated males of group 4 (10000 ppm). The statistical significance of this weight loss, however, is mainly triggered by one individual animal which displayed a reduced testes weight of -65% compared to controls. In addition, there were no histopathological effects noted for the testes in any dose group. Therefore, this weight loss is considered of no toxicological relevance. Additional statistically significant differences in organ weights between treated and control groups were noted. Since no systemic pattern emerged, these differences were attributed to spontaneous variation rather than to the treatment. Apart from an increasing incidence of nonspecific, minimal inflammatory cell infiltration in the liver in male animals of treated groups 3 and 4 (2000 and 10 000 ppm) no macroscopical or microscopical changes were observed that were considered to be due to the administration of the test compound. In conclusion, based on the described effects, a NOEL of 400 ppm was derived (25 mg/kg body weight for males, 27 mg/kg body weight for females). Since no specific systemic effects have been observed up to the highest dose group tested, the NOAEL was established at 624 (males) and 667 mg/kg body weight (females).

In a supporting subacute 28 day feeding study, groups of male and female Tif:RAIf (SPF) rats were treated with 1000, 3000, 10000 ppm test substance in food. The study was similar to the OECD TG 407 but had several limitations. No hematology, clinical chemistry and histopathology were performed, thus limiting the informative value of the study. No death occurred during the course of the study. No clinical symptoms and no signs of local and/or systemic toxicity were observed. The mean body weight gain of all treated male and female groups was similar to that of the respective controls. The mean food consumption of all treated male and female groups was similar to that of the respective control groups. Specific food consumption in relation to body weight - addressed as food conversion ratio - of treated animals was similar to the control ratios. Macroscopical examination at autopsy revealed no evidence of a reaction to the treatment. Based on these results it can be inferred that a "no observable effect level" for the test article when offered to rats continuously in their feed over a period of 28 days is above 10000 ppm, corresponding to a mean daily intake of 1013 mg/kg bodyweight for males and 936 mg/kg bodyweight for females.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the present data, classification for repeated dose oral toxicity is not warranted under Regulation (EC) No.1272/2008.