Registration Dossier

Administrative data

Description of key information

A 28-day study (OECD 407 and Eu Method B.7, GLP) was conducted (Stepan, 2000). Five Sprague-Dawley rats per sex per dose were exposed by gavage to 15, 150, and 1000 mg/kg/day of the active ingredient. 15 mg/kg/day was reported as NOEL based on liver, blood and spleen effects.

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:
From September 17, 1999 to June 30, 2000
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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK)
- Age at study initiation: approximately five to seven weeks old.
- Weight at study initiation: the males weighed 139 to 173 g, and the females weighed 127 to 155 g
- Housing: The animals were housed in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: nine days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 ºC
- Humidity (%): 55 ± 15%
- Air changes (per hr): approximately fifteen changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light and twelve hours darkness
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was prepared as a solution in distilled water. The analytical results indicate that the prepared formulations were within ±10 % of the nominal concentration. The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.

VEHICLE
- Concentration in vehicle: 3.68, 36.8, and 245 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test substance was determined by gas chromatography (GC) using an external standard technique. Samples were taken from the top, middle and bottom of the container. Sampling was performed in triplicate. The test material formulations were sampled and analysed initially and then after storage at approximately +4ºC in the dark for fourteen days.
Duration of treatment / exposure:
Twenty-eight consecutive days.
Frequency of treatment:
The test material was administered daily.
Dose / conc.:
18.4 mg/kg bw/day (nominal)
Remarks:
15 mg/kg/day of active ingredient
Dose / conc.:
184 mg/kg bw/day (nominal)
Remarks:
150 mg/kg/day of active ingredient
Dose / conc.:
1 227 mg/kg bw/day (nominal)
Remarks:
1000 mg/kg/day of active ingredient
No. of animals per sex per dose:
5 females and 5 males per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
A range-finding toxicity study in Sprague-Dawley rats was performed. Six animals, 3 males and 3 females, were included in the study. The test material was administered daily, for fourteen consecutive days, by gavage. Control animals were treated in an identical manner with 5 ml/kg/day of distilled water. The studied dose was 1250 mg/kg/day. The level doses were defined based on the results of necropsy data, bodyweights and clinical observations.
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before dosing and one and five hours after dosing during the working week. Animals were observed immediately before dosing and one hour after dosing at weekends.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 and on Days 7, 14, 21 and 28. Bodyweights were also recorded at terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: Food consumption was recorded for each cage group at weekly intervals throughout the study.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the study
- Animals fasted: No
- How many animals: investigations were performed on all surviving animals from each test and control group at the end of the study (Day 28)
- Parameters checked: see tables below.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Battery of functions tested: sensory activity, grip strength and motor activity.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS: Yes. The following organs removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation: adrenals, kidneys, testes, brain, liver, thymus, epididymides, ovaries, heart, spleen.

HISTOPATHOLOGY: Yes. Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin: adrenals, aorta (thoracic), bone and bone marrow (femur including stifle joint), brain (including cerebrum, cerebelum and pons), caecum, colon, duodenum, epididymides, eyes, gross lesions, heart, ileum, jejunum, kidneys, liver, lungs (with bronchi), lymph nodes (cervical and mesenteric), muscle (skeletal), oesophagus, ovaries, pancreas, pituitary, prostate, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical), spleen, stomach, testes, thymus, thyroid/parathyroid, trachea, urinary bladder, uterus.
Statistics:
Data were assessed for dose response relationships by linear regression analysis followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous pairwise comparisons were conducted using Dunnetts's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney "U" test.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: Animals treated with 1000 mg/kg/day began to show clinically observable signs of toxicity from Day 3. Symptoms gradually worsened and became more prevalent during weeks 1 and 2 before stabilising during the second half the study. Signs seen in most animals included fur loss, noisy respiration, increased salivation, red/brown staining of the fur, wet fur and hunched posture. The only observations seen in animals from the 150 or 15 mg/kg/day dose groups were incidents of fur loss, which is a common finding in group housed rats and in isolation is not toxicologically significant and a single incident of increased salivation entirely associated with the dosing procedure and not indicative of toxicity. One female treated with 1000 mg/kg/day was found dead on Day 12 of the study.

BODY WEIGHT AND WEIGHT GAIN: Males and females treated with 1000 mg/kg/day showed a substantially reduced bodyweight gain during week 1 of the study only. In subsequent weeks bodyweight gain in these animals was similar to controls but a full recovery was not apparent in males with terminal bodyweight remaining notably lower than controls. No adverse effect on bodyweight development was detected for animals of either sex from the 150 or 15 mg/kg/day dose groups.

FOOD CONSUMPTION: Males and females treated with 1000 mg/kg/day showed reduced food consumption and reduced food efficiency during the first week of the study only. No adverse effects were detected in animals of either sex treated with 150 or 15 mg/kg/day.

WATER CONSUMPTION: Water consumption measurement, initiated on Day 15, revealed a substantial increase for animals of either sex treated with 1000 mg/kg/day, which persisted until the end of the study. There was no convincing effect on water consumption in animals from the 150 or 15 mg/kg/day dose groups.

HAEMATOLOGY: Haematological changes consistent with haemolytic anaemia were detected in animals of either sex treated with 1000 or 150 mg/kg/day. The condition was dose related in severity and involved statistically significant reductions in erythrocyte count, haemoglobin, haematocrit and mean corpuscular haemoglobin and increases in mean corpuscular volume, mean corpuscular haemoglobin and reticulocyte count. The majority of 1000 mg/kg/day reticulocyte values were outside the normally expected range for rats of the strain and age used. Males and females from the 1000 mg/kg/day dose group also showed a statistically significant increase in neutrophil numbers, but not in the total leucocyte count, together with a slightly elevated prothrombin time. Animals of either sex treated with 15 mg/kg/day showed no toxicologically or statistically significant haematological changes.

CLINICAL CHEMISTRY: Males and females treated with 1000 mg/kg/day showed statistically significant elevations in plasma aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) including individual values outside the normally expected range for rats of the strain and age used, particularly for ALAT. The group mean ALAT concentration for males from the 150 mg/kg/day dose group was also significantly elevated and there was a slight, but not statistically significant increase in this and ASAT levels for animals of either sex at this dose level. Plasma bilirubin levels were increased for animals of either sex from the 1000 mg/kg/day dose group but statistical significance was confined to the males.

FUNCTIONAL OBSERVATIONS: Weekly behavioural assessments supported the clinical observations recorded during the study with incidents of tiptoe gait, hunched posture and noisy respiration seen at each observation time for animals treated with 1000 mg/kg/day. There were no treatment-related changes in the functional performance parameters measured. Males treated with 1000 mg/kg/day showed an enhanced starle reflex compared with controls. The percentage peak response was statistically significantly elevated and the percentage average response and RMS response were also higher than controls, although the differences did not achieve statistical significance.

ORGAN WEIGHTS: Males and females treated with 1000 mg/kg/day showed a statistically significant increase in spleen weight, both absolute and relative to bodyweight, compared with controls. Many of the individual values were outside the normally expected range for rats of the strain and age used and the effect extended to either sex from the 150 mg/kg/day dose group, although statistical significance was not always achieved at this dose level. A statistically significantly higher relative testes weight for 1000 mg/kg/day males was considered to result from the lower terminal bodyweight for these animals and was not indicative of a target organ effect. Reductions in absolute liver and kidney weight for 1000 mg/kg/day males were not reflected in the relative weights and were considered to be of no toxicological importance. There were no toxicologically significant effects on organ weight for animals of either sex treated with 15 mg/kg/day.

HISTOPATHOLOGY: The following treatment-related changes were observed:
Liver: centrilobular hepatocyte enlargement was observed for three males and scattered deposits of pigment were seen in a few animals of either sex dosed at 1000 mg/kg/day. One female dosed at 150 mg/kg/day similarly demonstrated hepatic pigment accumulation. The accumulated pigment was Perl´s positive and therefore probably haemosiderin.

Spleen: increased severities of extramedullary haemopoiesis and pigment accumulation were observed in relation to treatment for rats of either sex dosed at 1000 or 150 mg/kg/day. Female rats receiving 15 mg/kg/day demonstrated increased severities of splenic pigment accumulation but no alteration in extramedullary haemopoiesis compared with control animals. The accumulated pigment was Perl´s positive and therefore probably haemosiderin.

Kidneys: an increased severity of pigment accumulation was observed in the renal tubular epithelium of animals of either sex dosed at 1000 mg/kg/day. The accumulated pigment was Perl´s positive and therefore probably haemosiderin.

Urinary bladder: hyperplasia of the transitional cell epithelial lining of the urinary bladder was seen for males and females dosed at 1000 mg/kg/day.

Stomach: acanthosis and hyperkeratosis, occasionally with associated subepithelial inflammatory cell infiltrates, were observed in relation to treatment in the forestomach of animals of either sex receiving 1000 mg/kg/day of the test material.
Dose descriptor:
NOEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: see "Remarks"
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
other: hepatobiliary, immune system, urinary system, haematopoietic
Organ:
bladder
blood
kidney
liver
spleen
stomach
Treatment related:
yes
Dose response relationship:
no

Group mean weekly bodyweights and standard deviation (Tables 1 and 2)

 

Table 1: Males

Dose Level of Active Ingredient

mg/kg/day

Number of Animals

BODYWEIGHT(g)AT DAY

 

1

7

14

21

28

0 (Control)

5

mean

160

207

257

303

328

sd

12

17

18

20

22

15

5

mean

162

206

264

309

337

sd

8

8

11

10

13

150

5

mean

155

201

245

297

324

sd

10

11

13

18

20

1000

5

mean

150

159

202

235

265

sd

6

14

18

25

29

 

Table 2: Females

Dose Level of Active Ingredient

mg/kg/day

Number of Animals

BODYWEIGHT (g) AT DAY

 

1

7

14

21

28

0 (Control)

3

mean

143

1B0

205

222

231

sd

6

9

7

7

10

15

5

mean

139

168

193

209

218

sd

10

12

14

16

17

150

5

mean

146

179

204

228

240

sd

6

7

8

10

11

1000

5/4*

mean

142

159

180

205

220

sd

4

5

13

11

9

*Animal found dead Day 12

Group mean haematological values and standard deviation (Tables 3, 4, 5 and 6)

Table 3: Males

 

Dose Level of Active Ingredient

mg/kg/day

Number of Animals

 

Hb

RBC

Hrt

MCH

MCV

MCHC

WBC

 

g/dl

1012/l

%

pg

fl

g/dl

109/l

150

5

mean

*13.9

***6.42

41.2

*21.7

**64.2

***33. 8

10.7

sd

0.4

0.19

0.9

0.7

1.8

0.8

0.9

1000

5

mean

***12.0

***5.38

**37.1

**22.2

**69.0

***32.3

11.3

sd

0.6

0.34

2.2

0.6

0.7

1.0

2.5

Table 4: Males

Dose Level of Active Ingredient

mg/kg/day

Number of Animals

DIFFERENTIAL (109/l)

CT

PLT

APTT

Retics

 

Neut

l ymph

Mono

fcos

Bas

sees

109/l

(sees)

%

150

5

mean Sd

2.29

0.82

8.36

0.76

0.00 0.00

0.09

0.10

0.00

0.00

25.1 0.6

1089 96

11.2 1.3

**5.9

0.8

1000

5

mean

sd

**3.85

7.30

0. 06

0.07

0.00

**26.5

1012

11.1

*9.9

1.20

1.47

0.05

0.07

0 .00

1. 1

71

1.2

2.1

 

Table 5: Females

Dose Level of Active Ingredient

mg/kg/day

Number of Animals

 

Hb

 

 

RBC

Hrt

MCH

MCV

MCHC

WBC

 

 

 

g/dl

1012/l

%

pg

fl

g/dl

109/l

150

 

5

mean sd

13.3

0.6

**6.06

0.27

37.7

1.4

**21.9

0.4

***62.2

0.5

***35.3

0.5

8.0

2.6

1000

4

mean sd

**12.1

0.7

***5.13

0.46

35.9

2.5

***23.7 0.8

***70.1

1.9

***33.8

0.4

9.2

2.3

 

Table 6: Females

Dose Level of Active Ingredient

mg/kg/day

Number of Animals

DIFFERENTIAL (109/l)

CT

PLT

APTT

Retics

 

Neut

l ymph

Mono

fcos

Bas

sees

109/l

(sees)

%

150

5

mean Sd

1.87

1.00

5.96

1.57

0.00

0.00

0.13

0.14

0.00

0.00

24.8

1.3

11.09

7.3

10.8

2.1

**5.6

0.5

1000

5

mean

sd

**3.05

1. 09

6.05

1.67

0.04 0.05

0.09

0.02

0.00

0.00

26.0

1.6

949

227

11.4

1.5

*13.1

1.4

*= significantly different from control group p<0.05

**= significantly different from control group p<0.01

***= significantly different from control group<0.001

 

Group mean chemical values and standard deviations (Tables 7 and 8)

 

Table 7: Males

Dose Level of Active Ingredient

mg/kg/day

Number of Animals

 

ASAT

(IU/1)

ALAT

(IU/1)

Bill

(mg/dl)

150

5

mean

102

*96

0.13

sd

15

14

0.02

1000

5

mean

**114

**124

**0.20

sd

14

27

0.05

 

Table 8: Females

Dose Level of Active Ingredient

mg/kg/day

Number of Animals

 

ASAT

(IU/1)

ALAT

(IU/1)

Bill

(mg/dl)

1000

4

mean

*108

*118

0 17

sd

14

36

0.02

*= significantly different from control group p<0.05

**= significantly different from control group p<0.01

 

Group mean organ weights and standard deviations (Tables 9 and 10)

Table 9: Males

Dose Level of Active Ingredient

mg/kg/day

Number of Animals

BODYWEIGHT(g) AT TERMINAL KILL

ORGAN WEIGHT (g)

 

 

 Kidneys          Liver          Spleen

150

5

mean

323

2.2380

12.2605

**0.9822

sd

21

0.1800

1.3449

0.1775

1000

5

mean

269

**1.8176

*10.6267

*0.9212

sd

26

0.1702

1.1522

0.0837

 

Table 10: Females

Dose Level of Active Ingredient

mg/kg/day

Number of Animals

BODYWEIGHT(g) AT TERMINAL KILL

ORGAN WEIGHT (g)

Spleen

150

5

mean

235

0.6926

sd

13

0.0841

1000

4

mean

221

*0.7741

sd

8

0.1586

Conclusions:
The "No Observed Effect Level" (NOEL) was considered to be 15 mg/kg/day.
Executive summary:

The Repeated dose 28-Day Oral toxicity Study in Rodents for the test substance was performed in Sprague-Dawley rats.The test material was administered by gavage to three groups, each of five male and five female, for up to twenty-eight consecutive days, at dose levels of 15, 150 and 1000 mg/kg/day (incorporating a correction factor for test substance purity). A control group of five males and five females was dosed with vehicle alone (distilled water). Clinical signs, functional observations, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Clinical signs of toxicity, functional incidents as hunched posture, sensory reactivity, reduced food consumption, increase of water consumption and reduced bodyweight gain were observed at dose of 1000 mg/kg/day. In the blood chemistry, elevations in plasma aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT) and bilirubin was noted at the dose of 1000 mg/kg/day; ASAT and ALAT were also elevated for 150 mg/kg/day animals. Haematologycal changes as haemolytic anaemia, significant elevation in neutrophil numbers, and slightly elevated ptothrobin time were observed at doses of 1000 and 150 mg/kg/day, as well as, a statistically significant increase in spleen weight and treatment-related changes inliver, spleen, kidneys, urinary bladder and stomach.

Oral administration of the test substance to rats for a period of up to twenty-eight consecutive days at dose levels of up to 1000 mg/kg/day resulted in toxicologically significant effects at dose levels of 1000 and 150 mg/kg/day. No such effects were demonstrated in animals of either sex treated with 15 mg/kg/day and the "No Observed Effect Level" (NOEL) was, therefore, considered to be 15 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
15 mg/kg bw/day
Study duration:
subacute
Species:
rat
System:
haematopoietic
Organ:
blood
liver
spleen

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 Repeated dose 28-Day Oral toxicity Study in Rodents for the test substance was performed inSprague-Dawley rats.Thetest material was administeredby gavage to three groups, each of five male and five female, for up to twenty-eight consecutivedays, at dose levels of 15, 150 and 1000 mg/kg/day (incorporating a correction factor for test substance purity). A control group of five males and five females was dosed with vehicle alone (distilled water).Clinical signs, functional observations, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study.All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Clinical signs of toxicity, functional incidents as hunched posture, sensory reactivity, reduced food consumption, increase of water consumption and reduced bodyweight gain were observed at dose of 1000 mg/kg/day.In the blood chemistry, elevations in plasmaaspartate aminotransferase (ASAT), alanine aminotransferase (ALAT) and bilirubin was noted atthe dose of1000 mg/kg/day;ASAT and ALAT were also elevated for 150 mg/kg/day animals.Haematologycal changes as haemolytic anaemia, significant elevation in neutrophil numbers, and slightly elevated ptothrobin time were observed at doses of 1000 and 150 mg/kg/day, as well as, a statistically significant increase in spleen weight and treatment-related changes inliver, spleen, kidneys, urinary bladder and stomach.

Oral administration of the testsubstanceto rats for a period of up to twenty-eight consecutive days at dose levels of up to 1000 mg/kg/day resulted in toxicologically significant effects at dose levels of 1000 and 150 mg/kg/day. No such effects were demonstrated in animals of either sex treated with 15 mg/kg/day and the "No Observed Effect Level" (NOEL) was, therefore, considered to be 15 mg/kg/day.

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 1272/2008. Based on the criteria laid down in Regulation (EC) No. 1272/2008, as amended for the second time in Directive EC 286/2011, the test substance is considered to be classified as STOT RE Cat. 2 (H373).