Registration Dossier

Administrative data

Description of key information

OECD TG 407, oral (diet), rat, GLP-conform: NOAEL (females) = 346 mg/kg bw; NOAEL (males) = 1057 mg/kg bw/d

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:
June - July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
03 Oct 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No. of test material: Ra-He 2014-054
- Purity: 92.3 wt-%
- Purity test date: Apr 26 - Jun 21, 2016
- Date of production: Oct 2014
- Expiration Date: Oct 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient (room temperature)
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: For each concentration, the test substance was weighted out and mixed with a small amount of food. In order to obtain the desired concentrations, these premixes were added to the corresponding amounts of food, depending on test group. Mixing was carried out for about 10 minutes in a laboratory mixer.

FORM AS APPLIED IN THE TEST (if different from that of starting material): mixed with food
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is a frequently used laboratory animal, and there is comprehensive experience with this animal species.
Moreover, the rat has been proposed as a suitable animal species by the OECD and the EPA for this type of study.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 41 - 43 days
- Weight at study initiation: males: 160.2 g (mean); females: 123.6 g (mean)
- Housing: 5 animals/cage
- Diet: ad libitum; ground Kliba maintenance diet mouse-rat “GLP” (supplied by Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: 9 days

DETAILS OF FOOD AND WATER QUALITY: The drinking water was regularly assayed for chemical contaminants as well as for the presence of microorganisms. On the basis of the analytical findings the drinking water was found to be suitable. The supplier assayed the food used in the study for chemical and microbiological contaminants. On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants, the diet was found to be suitable.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%):45 - 65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 13 Jun 2017 To: 23 Jul 2017
Route of administration:
oral: feed
Details on route of administration:
The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): not specified
- Mixing appropriate amounts with (Type of food): ground Kliba maintenance diet mouse-rat “GLP” (supplied by Provimi Kliba SA, Kaiseraugst, Switzerland)
- Storage temperature of food: not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity was verified in 3 samples in the highest and lowest concentrations (used as a concentration control at the same time) before the start of the administration period.
Additional concentration control analyses were verified in one sample of the mid concentration. The samples were analysed via Quantitative LC/ESI-MS with evaluation by the external standard method.

Averaged recovery rates were in a range of 98 - 117%.
Duration of treatment / exposure:
4 weeks (28 days)
Frequency of treatment:
continuously
Dose / conc.:
12 000 ppm
Remarks:
= 1057.1 mg/kg bw/d in males and 1083.2 mg/kg bw/d in females (actual dose recieved);
group 3
Dose / conc.:
4 000 ppm
Remarks:
= 337.7 mg/kg bw/d in males and 346.4 mg/kg bw/d in females (actual dose recieved);
group 2
Dose / conc.:
1 000 ppm
Remarks:
= 85.6 mg/kg bw/d in males and 90.0 mg/kg bw/d in females (actual dose recieved);
group 1
No. of animals per sex per dose:
5
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The doses were selected based in the results of a 14-days range finding study.
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes (for any signs of morbidity, mortality, abnormal clinical signs)
- Time schedule: at least once daily (on working days twice daily)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals (in the morning)
- Examined parameters: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size (further parameters as required)

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period (to randomize the animals), study day 0 (start of the administration period) and thereafter once a week

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: weekly

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily visual inspection of the water bottles for any changes in volume

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination (study day 29)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: all
- Parameters checked in table 1 were examined.
- Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination (study day 29)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: all
- Parameters checked in table 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: study day 25
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (during stay in the metabolism cages)
- Parameters checked in table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes; functional observational battery and motor activity assessment (see "OTHER")

OTHER:
Functional observational battery (FOB): study day 26 (males), 27 (females)
- The functional observational battery was carried out in all animals once at the end of the administration period (generally in the morning).
- The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensory motor tests as well as reflex tests. The observations were performed at random.
- Examined parameters:
• Home cage observations (about 10-30 seconds): posture, tremors, convulsions, abnormal movements, gait, other findings
• Open field observations: behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/ pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/ stereotypes, gait, activity/ arousal level, feces excreted within 2 minutes (appearance/ consistency), urine excreted within 2 minutes (amount/ color), rearing within 2 minutes, other findings
• Sensory motor tests/ reflex tests: reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (auditory startle response), coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings

Motor activity assessment:
- measured from 14:00 h onwards on the same day as the FOB was performed
- The number of beam interrupts was counted over 12 intervals for 5 minutes per interval.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 4, Organ weights)

HISTOPATHOLOGY: Yes (see table 4)
Statistics:
Means and standard deviations were calculated. In addition, the following statistical analyses were carried out: see table 5
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Mean body weight change values were significantly lower in male animals of test group 3 (12000 ppm) from study day 0 to 14 with the deviation of -13% and not statistically significantly decreased between study days 0 and 28 (-14.7%). For these minor alterations of the body weight change without a correlating significant finding in the body weight itself it could not be excluded to be treatment-related. However, these minor alterations of body weight change were assessed as non-adverse.
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:
effects observed, treatment-related
Description (incidence and severity):
- At the end of the administration period, in females of test group 3 (12000 ppm), hemoglobin and hematocrit values were significantly decreased. The values were marginally below historical control ranges (females, hemoglobin 8.1-9.1 mmol/L, hematocrit 0.377-0.412 L/L)

- In males of test group 2 (4000 ppm) hemoglobin values were significantly decreased and large unstained cell (LUC) counts were significantly increased. However, both parameters were not dose-dependently changed. Therefore, these alterations were regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- At the end of the administration period in females of test group 3 (12000 ppm) total bile acid levels were significantly increased (regarded as due to the high amount of fatty acids in the compound which increases the turn-around of bile acids for absorption of these components). This is the only relevantly changed parameter among females of test group 3. Therefore, the increased total bile acid levels were regarded as treatment-related, but non-adverse.
- In males of test group 3 total bile acid levels were contrarily lower (not statistically significantly) compared to controls.

- In females of test groups 1, 2 and 3 (1000, 4000 and 12000 ppm), cholesterol levels were significantly higher compared to controls. However, all means were within the historical control ranges (females, cholesterol 1.05-1.65 mmol/L). Therefore, this alteration was regarded as incidental and not treatment-related.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, without a dose-response relationship or occurred in single animals only, these observations were considered as incidental.

Regarding the overall motor activity, in interval 10 of males in test group 1, 2, and 3 (1000, 4000, and 12000 ppm) a significantly lower number of beam interruptions were determined without a dose dependency. Overall, the shape of the habitation curves was comparable in all dose groups to the current control. Therefore, the finding in interval 10 of males in test groups 1-3 were considered as treatment-related but not adverse.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
- significantly decreased kidney weights (94%) in male animals of test group 1 and significantly increased heart weights in female animals of test groups 1 (108%) and 2 (110%) (regarded as incidental, since there was no dose-response relationship and only the relative weight parameters were affected)
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- 2 male animals of test group 3 (12000 ppm) showed a minimal centrilobular hepatocellular hypertrophy (regarded as treatment-related but not adverse, since the hypertrophy was only minimal and there were no corresponding liver weight changes nor deviating clinical chemistry parameters observed)
- In contrast to male animals of test groups 0 to 2, animals of test group 3 did not show a periportal fat storage (regarded as related to the slightly decreased terminal body weight in test group 3 males; therefore regarded as treatment-related but not adverse)

- Male animals of test groups 2 and 3 showed a minimally increased severity of inflammatory cell infiltrates composed of polymorph nuclear leukocytes and lymphocytes that were distributed throughout the submucosal layer of the glandular stomach (regarded as treatment-related but not adverse, since there were no additional degenerating changes in the mucosal or submucosal layer observed)
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Dose descriptor:
NOAEL
Remarks:
(= 4000 ppm)
Effect level:
346.4 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
haematology
Dose descriptor:
NOAEL
Remarks:
(= 12000 ppm)
Effect level:
1 057.1 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse effects observed up to and including the highest dose group
Critical effects observed:
yes
Lowest effective dose / conc.:
1 083.2 mg/kg bw/day (actual dose received)
System:
haematopoietic
Organ:
blood
Treatment related:
yes
Dose response relationship:
yes

The various analyses confirmed

·     the stability of the test-substance preparations for a period of 32 days,

·     the homogeneous distribution of the test substance in the vehicle,

·     the correctness of the prepared concentrations.

Table 6: Altered relative organ weights

 

Male animals

Female animals

Test group (ppm)

1

(1000)

2

(4000)

3

(12000)

1

(1000)

2

(4000)

3

(12000)

Kidneys

94%*

95%

100%

 

 

 

Heart

 

 

 

108%**

110%**

105%

Table 7: Summary - changes body weights (males)

 

0 / M

1 / M

2 / M

3 / M

0 ppm

1000 ppm

4000 ppm

12000 ppm

d 0 -> 7

Mean

40.6 n

41.7

42.4

36.1

 

S.d.

2.1

1.8

4.8

3.6

 

N

5

5

5

5

 

Deviation Vs Control

 

2.6

4.3

-11.1

d 0 -> 14

Mean

83.4 n

81.8

85.4

72.5 *

 

S.d.

5.3

6.2

7.2

6.3

 

N

5

5

5

5

 

Deviation Vs Control

 

-1.9

2.5

-13.0

d 0 -> 21

Mean

113.9 n

110.7

117.1

96.9

 

S.d.

8.7

12.6

10.4

9.9

 

N

5

5

5

5

 

Deviation Vs Control

 

-2.8

2.8

-14.9

d 0 -> 28

Mean

135.4 n

129.6

134.2

115.4

 

S.d.

11.7

13.6

13.0

12.2

 

N

5

5

5

5

 

Deviation Vs Control

 

-4.3

-0.9

-14.7

Table 8: Excerpt of the motor activity measurements (males)

M A L E S

 

 

Interv. 9 Beam

Interr.

Interv.10Beam

Interr.

 

Interv.11 Beam

Interr.

Interv.12 Beam

Interr.

Interv.1-12Sum

Interr.

0 PPM

 

 

 

 

 

 

 

M55.6

145.6

 

93.2

42.6

2926.6

 

SD77.2

165.2

 

123.9

34.5

385.8

 

N5

5

 

5

5

5

 

1000 PPM

 

 

 

 

 

 

 

M36.0

18.8

**

142.0

98.6

2994.6

 

SD50.6

15.0

 

144.1

172.5

787.8

 

N5

5

 

5

5

5

 

4000 PPM

 

 

 

 

 

 

 

M10.2

12.0

**

122.0

103.2

2614.2

 

SD9.9

13.8

 

203.9

152.0

618.6

 

N5

5

 

5

5

5

 

12000 PPM

 

 

 

 

 

 

 

M16.2

12.6

**

23.0

32.8

2635.4

 

SD23.8

13.0

 

21.7

52.8

417.9

 

N5

5

 

5

5

5

Kruskal-Wallis + Wilcoxon-tests (two-sided): * p<=0.05; ** p<=0.01 (Statistical unit = Animal)

Table 9: Excerpt of the red blood cell and coagulation parameters (females)

 

0 / F

1 / F

2 / F

3 / F

0 ppm

1000 ppm

4000 ppm

12000 ppm

RBC

Mean

7.37 k

7.28

7.29

7.09

[tera/L]

S.d.

0.27

0.20

0.16

0.23

day 32

N

5

5

5

                       5

 

Median

7.34

7.39

7.24

6.98

 

Deviation Vs Control [%]

 

-1.17

-1.09

-3.77

HGB

Mean

8.5 v

8.4

8.3

8.0 *

[mmol/L]

S.d.

0.3

0.3

0.2

0.2

day 32

N

5

5

5

5

 

Median

8.6

8.4

8.3

8.0

 

Deviation Vs Control [%]

 

-1.9

-2.1

-5.9

HCT

Mean

0.397 v

0.389

0.388

0.373 **

[L/L]

S.d.

0.012

0.010

0.007

0.009

day 32

N

5

5

5

5

 

Median

0.404

0.393

0.388

0.374

 

Deviation Vs Control [%]

 

-1.966

-2.319

-6.099

MCV

Mean

53.9 k

53.4

53.2

52.6

[fL]

S.d.

1.1

0.4

0.6

                   2.3

day 32

N

5

5

5

5

 

Median

54.1

53.6

53.1

52.6

 

Deviation Vs Control [%]

 

-0.9

-1.3

-2.4

MCH

Mean

1.16 k

1.15

1.14

1.14

[fmol]

S.d.

0.04

0.01

0.01

0.05

day 32

N

5

5

5

5

 

Median

1.16

1.15

1.14

1.15

 

Deviation Vs Control [%]

 

-1.03

-1.55

-2.07

MCHC

Mean

21.50 k

21.51

21.45

21.58

[mmol/L]

S.d.

0.31

0.42

0.29

0.35

day 32

N

5

5

5

5

 

Median

21.40

21.43

21.52

21.63

 

Deviation Vs Control [%]

 

0.06

-0.21

0.40

RETA

Mean

162.6 k

154.3

168.2

181.2

[giga/L]

S.d.

22.9

34.0

48.2

41.4

day 32

N

5

5

5

5

 

Median

156.3

167.1

159.5

197.1

 

Deviation Vs Control [%]

 

-5.1

3.5

11.4

PLT

Mean

724 k

745

767

767

[giga/L]

S.d.

50

108

92

63

day 32

N

5

5

5

5

 

Median

710

708

818

776

 

Deviation Vs Control [%]

 

3

6

6

HQT

Mean

38.2 k

36.1

37.3

36.1

[sec]

S.d.

2.1

1.1

1.5

1.2

day 32

N

5

5

5

5

 

Median

38.7

35.9

37.1

35.8

 

Deviation Vs Control [%]

 

-5.5

-2.4

-5.5

 Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics k=KRUSKALL-WALLIS; v=KRUSKALL-WALLIS-WILCOX

Table 10: Histopathology - treatment-related findings

 

Male animals

Test group (ppm)

0

(0)

1

(1000)

2

(4000)

3

(12000)

No. of animals

5

5

5

5

Liver

 

 

 

 

Hypertrophy, centrilobular

0

0

0

2

·        Grade1

 

 

 

2

Fatty change, periportal

3

2

3

0

·        Grade1

3

2

3

 

Glandular stomach

 

 

 

 

Inflammatory cell infiltrates

5

5

5

5

·        Grade1

5

5

2

3

·        Grade2

 

 

3

2

Conclusions:
Under the conditions of the present study the no observed adverse effect level (NOAEL) for this test substance was 4000 ppm in female (346 mg/kg bw/d) and 12000 ppm in male (1057 mg/kg bw/d) Wistar rats.
Executive summary:

The test substance was administered via the diet to groups of 5 male and 5 female Wistar rats at concentrations of 0 ppm (test group 0), 1000 ppm (test group 1), 4000 ppm (test group 2) and 12000 ppm (test group 3) in the diet over a period of 4 weeks.

Food consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. Beside this, a functional observational battery (FOB) as well as measurement of motor activity (MA) was carried out at the end of the administration period. Clinicochemical and hematological examinations were performed towards the end of the administration period. After the administration period all rats were sacrificed and assessed by gross pathology, followed by histopathological examinations.

The following test substance-related, relevant findings were noted:

Test group 3: 12000 ppm (1057 mg/kg bw/d in males and 1083 mg/kg bw/d in females)

Clinical Examinations:

• No test substance-related, adverse effects

Clinical Pathology:

• Decreased hemoglobin and hematocrit values in females

Pathology:

• No test substance-related, adverse effects

Test group 2: 4000 ppm (338 mg/kg bw/d in males and 346 mg/kg bw/d in females)

Clinical Examinations, Clinical Pathology, and Pathology:

• No test substance-related, adverse effects

Test group 1: 1000 ppm (86 mg/kg bw/d in males and 90 mg/kg bw/d in females)

Clinical Examinations, Clinical Pathology, and Pathology:

• No test substance-related, adverse effects

The administration of the test substance via the diet to male and female Wistar rats for 4 weeks caused treatment-related signs of systemic toxicity at the concentrations of 12000 ppm in the diet of female Wistar rats (1083 mg/kg bw/d) manifested in an anemia, but not in males.

Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) for this test substance was 4000 ppm (346 mg/kg bw/d) in female and 12000 ppm (1057 mg/kg bw/d) in male Wistar rats.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
346 mg/kg bw/day
Study duration:
subacute
Species:
rat
System:
haematopoietic

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

In a subacute repeated dose toxicity study according to OECD guideline 407 the test substance was administered via the diet to groups of 5 male and 5 female Wistar rats at concentrations of 0 ppm (test group 0), 1000 ppm (test group 1), 4000 ppm (test group 2) and 12000 ppm (test group 3) in the diet over a period of 4 weeks.

Food consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. Beside this, a functional observational battery (FOB) as well as measurement of motor activity (MA) was carried out at the end of the administration period. Clinicochemical and hematological examinations were performed towards the end of the administration period. After the administration period all rats were sacrificed and assessed by gross pathology, followed by histopathological examinations.

The administration of the test substance via the diet to male and female Wistar rats for 4 weeks caused treatment-related signs of systemic toxicity at the concentrations of 12000 ppm in the diet of female Wistar rats (1083 mg/kg bw/d) manifested in an anemia, but not in males.

Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) for this test substance was 4000 ppm (346 mg/kg bw/d) in female and 12000 ppm (1057 mg/kg bw/d) in male Wistar rats.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.