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Description of key information

The NOAEL of the test substance was 500 ppm in male (41 mg/kg bw/d) and female (42 mg/kg bw/d) Wistar rats after oral application for 28 days.

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:
2015-02-24 to 2015-11-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Experimental study according to guideline and GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: U.S. EPA Health Effects Test Guidelines. OPPTS 870.3050; Jul 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 42 ± 1 days
- Weight at study initiation: mean values fomr treatment groups: male 152.8-155.6, females 127.9-133.0
- Fasting period before study: no
- Housing: 5 animals per cage, in H-Temp polysulfonate cages type 2000P
- Diet: ad libitum, ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
The weighted test substance was mixed with food. Preperation was mixed once per week and stored in freezer for 4 days and 4 days at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses of the test-substance preparations were carried out in compliance with GLP. The stability of the test substance in the diet in a freezer for a period of 4 days and, thereafter, for 4 days at room temperature was proven before the start of the administration period. Homogeneity was verified in 3 samples in the highest and lowest concentration (was used as a concentration control at the same time) at the beginning of the study; additional concentration control analyses were done in the mid concentration. The samples were taken from the specific food containers.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
continuoulsy
Remarks:
Doses / Concentrations:
0, 150, 500 and 1500 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
132 (male) and 130 (female) mg/kg bw/d (= 1500 ppm)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
41 (male) and 42 (female) mg/kg bw/d (= 500 ppm)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
13 (male) and 14 (female) mg/kg bw/d (= 150 ppm)
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: In a previously performed test study (Supporting study) the test substance was administered via the diet to groups of 5 male and 5 female Wistar rats for 3 or 14 days at concentrations of 0, 3000 and 10000 ppm. At 10000 ppm, a reduction of food and water consumption as well as body weight loss was observed between study days 0 and 3. All animals were sacrificed in a moribund condition on study day 3. At 3000 ppm, a reduction of food and water consumption was also observed as well as an initial body weight loss between study days 0 to 3. After 14 days, body weights were significantly lower by -28 % in males and -16 % in females compared to control animals. Several hematology and clinical chemistry parameters were affected and absolute as well as relative liver weights were increased in both sexes. Therefore, based on these results the concentrations 1500, 500 and 150 ppm in the diet were selected for the present study. The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays.
- Cage side observations checked in table No. 1 were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was determined weekly over a period of 3 days and calculated as mean food consumption in grams per animal and day.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
Water consumption was determined weekly over a period of 3 days and calculated as mean water consumption in grams per animal and day.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after administration period
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, at least 16 hours
- How many animals: all
- Parameters checked in table No. 5 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after administration period
- Animals fasted: Yes, at least 16 hours
- How many animals: all
- Parameters checked in table No. 6 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: after administration period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, at least 16 hours
- Parameters checked in table No. 7 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the administration period
- Dose groups that were examined: all
- Parameters checked in tables No. 2,3,4 were examined.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, see table No. 8
HISTOPATHOLOGY: Yes, see table No. 9
Statistics:
Means and standard deviations of each test group were calculated. In addition other analysis were performed for some parameters.
- Body weight + change: A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means.
- rearing, grip strength forelimbs + hindlimbs, footsplay test, motor activity: Non-parametric one-way analysis using KRUSKALWALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON test (two-sided) for the equal medians.
- Blood parameters: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians. For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians.
- Urinalysis parameters (not pH, urine volume, specific gravity, color and turbidity): Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
- Urine pH, volume, specific gravity: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians.
- Pathology weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting pvalue was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No findings were observed in animals of any test group including the controls.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights of male and female animals (1500 ppm) were lower compared to the control group, significantly on study day 28 (-11 %) in males and on study day 21 (-10 %) in females. Mean body weight change values of male and female animals in (1500 ppm) were significantly lower over the entire study period, showing a maximum of -32 % on study day 7 in males and -34 % on study day 21 in females. In the other treatement groups (150 and 500 ppm) no effects were detected.

FOOD CONSUMPTION AND COMPOUND INTAKE
During numerous time intervals, food consumption values for male and female animals of test group 1-3 (150, 500 and 1500 ppm) were slightly lower when compared to the control group. However, these values reflected the normal range of biological variation inherent in the strain of rats used for this study.

WATER CONSUMPTION AND COMPOUND INTAKE
No test substance-related, adverse changes with regard to water consumption were observed.

HAEMATOLOGY
No treatment-related changes among hematological parameters were observed.

CLINICAL CHEMISTRY
No treatment-related, adverse changes among clinical chemistry parameters were observed.

URINALYSIS
No treatment-related, adverse changes among urinalysis parameters were observed.

NEUROBEHAVIOUR
No test substance-related effects were observed.

ORGAN WEIGHTS
Abolute organ weights: Females treated with 1500 ppm had statistically significant changes in liver (126 % ) and females treated with 500 ppm in spleen (113 %).
Relative organ weights: Statistically significant changes were seen in kidneys for all females (150 ppm: 109 %, 500 ppm: 111 %, 1500 ppm: 115 %) and in liver for males at 1500 ppm (122 %) and females (500 ppm: 108 %, 1500 ppm: 138 %).

GROSS PATHOLOGY
All findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
In the liver of male animals of test group 3 (1500 ppm) all animals revealed a minimal to slight increase of karyocytomegaly in the intermediate zone (between the periportal and centrilobular area). There was an increase in nuclear size and cell size of hepatocytes. The karyocytomegaly was
regarded to be treatment-related and as the meaning of this finding was not clear an adverse effect could not be excluded.
Females of the same test group revealed a minimal to slight centrilobular hypertrophy. These findings were regarded to be treatment-related but not adverse. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Dose descriptor:
NOAEL
Effect level:
41 - 42 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Body weight, organ weights (liver); histopathology (liver) / equivalent to 500 ppm
Critical effects observed:
not specified

Results

 

Absolute organ weights

When compared to control group 0 (set to 100%), the mean absolute weights of following organs were significantly changed (statistically significant changes printed in bold):

 

Male animals

Female animals

Test group (ppm)

1

(150)

2

(500)

3

(1500)

1

(150)

2

(500)

3

(1500)

Terminal body weight

100%

95%

88%*

96%

99%

92%

Liver

 

 

 

95%

107%

126%**

Spleen

 

 

 

101%

113%*

96%

*p ≤ 0.05; **p ≤ 0.01

 

Relative organ weight

When compared to control group 0 (set to 100%), the mean relative weights of following organs were significantly changed (statistically significant changes printed in bold):

 

Male animals

Female animals

Test group (ppm)

1

(150)

2

(500)

3

(1500)

1

(150)

2

(500)

3

(1500)

Liver

 

 

 

109%**

111%**

115%**

Spleen

98%

99%

122%*

100%

108%*

138%**

*p ≤ 0.05; **p ≤ 0.01

 

Histopathology

Treatment-related findings were observed in the liver of male and females with incidences and grading according to the table below:

Liver

Male animals

Female animals

Test group (ppm)

0

(0)

1

(150)

2

(500)

3

(1500)

0

(0)

1

(150)

2

(500)

3

(1500)

No. of animals

5

5

5

5

5

5

5

5

Karyocytomegaly,

intermediate

0

0

0

5

 

 

 

 

Grade 1

 

 

 

4

 

 

 

 

Grade 2

 

 

 

1

 

 

 

 

Karyocytomegaly,

intermediate

0

0

0

0

0

0

0

5

Grade 1

 

 

 

 

 

 

 

4

Grade 2

 

 

 

 

 

 

 

1

 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
41 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Experimental study according to guideline and GLP.

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

Repeated dose toxicity

oral

A oral repeated dose toxicity study was conducted according to OECD 407 in Wistar rats. The test substance was administered via the diet to groups of 5 male and 5 female rats at concentrations of 0, 150, 500 and 1500 ppm over a period of 4 weeks. The actual ingest of the test substance was 13 (male) and 14 (female) mg/kg bw/d for 150 ppm, 41 (male) and 42 (female) mg/kg bw/d for 500 ppm and 132 (male) and 130 (female) mg/kg bw/d for 1500 ppm. Clinical examinations revealed treatment-related, adverse signs of systemic toxicity in male and female animals at a concentration of 1500 ppm taking impaired body weight development into account. Body weight values in both sexes were lower during the entire administration period, with a maximum of -11 % in males (study day 28, significantly) and -10 % in females (study day 21, significantly). Body weight change values in both sexes were significantly decreased with a maximum of -32 % in males (study day 7) and -34 % in females (study day 21). No clinical findings of concern were observed for male and female animals treated with 150 and 500 ppm. No treatment-related, adverse effects were observed in all does groups for clinical pathology. Pathology showed significant decrease in the terminal body weight (-12 %) in males treated with 1500 ppm. It was regarded to be treatment-related and adverse. In addition, the non-significantly altered terminal body weight of female animals (-8 %) was assumed to be a sign of toxicity as the body weight effects became more obvious during clinical examinations. In the liver, males treated with1500 ppm revealed karyocytomegaly and females of the same test group showed a centrilobular hypertrophy. The centrilobular hypertrophy findings were regarded to be treatment-related, and for females, as no other histopathologic findings in the liver were observed in addition and also hepatic parameters in clinical pathology did not reveal any changes of concern, these effects were regarded to be not adverse. In males the karyocytomegaly was regarded to be treatment-related and as the meaning of this finding was not clear an adverse effect could not be excluded. Analysis of relative organ weights additionally showed statistically significant, but dose independent, changes in kidneys for all females (150 ppm: 109 %, 500 ppm: 111 %, 1500 ppm: 115 %). All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. Therefore it was concluded that the test substance administered via the diet to male and female Wistar rats for 4 weeks caused test substance-related, adverse signs of toxicity only at a concentration of 1500 ppm. Under the conditions of the present study the no observed adverse effect level (NOAEL) was determined to be 500 ppm in male (41 mg/kg bw/d) and female (42 mg/kg bw/d) Wistar rats.

 

In a 14-day dose range finding study the test substance was orally administered to rats. 5 male and female animals each received 10000 ppm or 3000 ppm of the test substance in the diet. All animals treated with 10000 ppm were sacrificed moribund on study day 3. All animals showed a reduced body weight and reduced food and water consumption. While no gross pathological changes were observed in the animals killed after 3 days (10000 ppm), an increase in liver weight was detected in animals killed after 14 days of treatment (3000 ppm). In those animals blood was analysed and an increase in γ-glutamyltransferase (both sexes) and alanine aminotransferase activities (males) was detected. In addition reticulocyte, platelet counts, total protein, albumin and globulin levels were reduced in males. In females an increase in total white blood cell and absolute lymphocyte counts was detected. As clear signs of toxicity were observed, the concentrations in the diet for the subsequent 28-day study in male and female Wistar rats were set to 0, 150, 500 and 1500 ppm.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The study is considered reliable.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

Repeated dose toxicity

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

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008, as amended for the seventh time in Regulation (EU) No 2015/1221. As a result the substance is considered not to be classified.

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