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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 13, 2006 to March 13, 2006 (experimental period)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Guideline study, well performed and reported according to good scientific standards. There were minor deviations from the study plan as follows: Functional tests: the high sensitivity of the motility meter was chosen to evaluate slight movements instead of active moving, and the low sensitivity to evaluate active moving instead of slight movements. Histopathology: The organs of all animals of group 4 (animal nos. 61-80) were examined histopathologically instead of the animals nos. 60-80 as stated in the Study Plan. The animal no. 60 did not belong to group 4. These minor deviations were caused by typing errors in the Study plan and had no influence on the validity of the study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): TPI 1618
- Substance type: Formaldehade releaser
- Physical state: Clear, colourless solution
- Analytical purity: Releasable formaldehyde: 46.1%
- Composition of test material, percentage of components: Reaction product of urea, etyleneglycol and formaldehyde. Therefore the purity is not exactly known
- Density: 1.256 mg/mL
- Purity test date: November 30, 2005
- Lot/batch No.: 1100968
- Stability under test conditions: The test item was prepared daily freshly for administration. Prior to dosing, the dosing solutions were left to stand for 30 min at room temperature
- Storage: test item stored at room temperature, protected from light.
- Other: Samples of dosing solutions were stored deep frozen for possible further analyses upon stability, homogeneity and concentration

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Age at study initiation : males 35 days, females 36 days (at 1st dosing)
- Weight at study initiation: males 128.9-158.9 g; females 118.1-140.6g
- Fasting period before study: no
- Housing: Single in Macrolon Typ III cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +-3
- Humidity (%): 55 +-15
- Photoperiod: 12 hrs dark /12 hrs light

IN-LIFE DATES: From December 13, 2006 to March 13, 2007

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Tap water was used to mimic normal use of employ
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The correct concentrations were monitored trough the preparation protocols.
The test item is a formaldehyde generator. Hence it is neither technically possible nor does it make any scientific sense to determine the concentartion of dosing solutions.
Duration of treatment / exposure:
Treatment was performed troughout 90 days
Frequency of treatment:
Once daily as oral gavage
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 150 or 500 mg/kg bw
Basis:
other: doses applied via gavage
No. of animals per sex per dose:
10 per sex and dose group (1 control and three dose groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected following the results of a preliminary dose range finding study over 28 days
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily;
- Cage side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory system, somatomotor activity and behaviour patterns

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to study start and weekly thereafter at 1, 2, 4, 8 and 24 hours post dosing. Standard arena observations included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity.

BODY WEIGHT: Yes
- Time schedule for examinations: once on the day of initiation of treatment and weekly thereafter

FOOD CONSUMPTION : Yes
- Food consumption for each animal determined, total feed input and left over were recorded once a week
FOOD EFFICIENCY:
No data

WATER CONSUMPTION AND COMPOUND INTAKE No
- Monitored daily by visual appraisal

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once before commencement of treatment and prior to terminal sacrifice
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of study
- Anaesthetic used for blood collection: Yes (light ether anesthesia)
- Animals fasted: yes (overnight)
- How many animals: all study animals
- Parameters examined: haemoglobin content, erythrocytes, leucocytes, differential blood count (relative and absolute), reticulocytes, platelets, haematocrit, thromboplastin time, activated partial thromboplastin time, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of study
- Anaesthetic used for blood collection: yes (light ether anesthesia)
- Animals fasted: yes (overnight)
- How many animals: all study animals
- Parameters examined: albumin, bilirubin (total), cholesterol (total),creatinine, glucose, protein (total), urea, calcium, chloride, potassium, sodium, alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 13
- Dose groups that were examined: all
- Battery of functions tested: righting reflex, body temperature, salivation, startle response, respiration, mouth breathing, urination, convulsions, piloerection, diarrhea, pupil size, pupil response, lacrimation, impaired gait, stereotypy, toe pinch, tail pinch, wire manoeuvre, hind leg splay, positional passivity, tremors, positive geotropism, limb rotation and auditory function

Sacrifice and pathology:
GROSS PATHOLOGY: Yes (all animals examined)

HISTOPATHOLOGY: Yes
Organs examined from all control and high dose group animals (stomach examined from all animals): adrenal gland (2), aorta abdominalis, bone marrow (os femoris), brain (3 levels: cerebrum, cerebellum, medulla/pons), epididymis (2), eye with optic nerve, gross lesions, heart (3 levels: left and right ventricle, septum), large intestine (colon, rectum), small intestine (duodenum, jejunum, ileum incl. Peyer’s patches), kidney and urethra (2), liver, lungs (with mainstem bronchi and bronchioles), lymph node (cervical and mesenteric), mammary gland, nerve (sciatic), oesophagus, ovary (2), pancreas, pituitary, prostate, salivary glands (mandibular, sublingual and parotid gland), skin (left flank), spinal cord (3 levels: cervical, midthoracic, lumbar), spleen, stomach, testicle (2), thymus, thyroid (2) including parathyroids, tissue masses or tumours (including regional lymphnodes), trachea (including larynx), urinary bladder, uterus (including cervix and oviducts), vagina
Other examinations:
None
Statistics:
Student’s t-test: all numerical function tests
Multiple t-test based on Dunnet, C.W.: body weights, food consumption, haematology, clinical chemistry, relative and absolute organ weights
Fisher’s exact test: histology

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see Table 1
Mortality:
mortality observed, treatment-related
Description (incidence):
see Table 1
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see Table 1
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:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
see Table 2
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see Table 3
Urinalysis findings:
not examined
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:
effects observed, treatment-related
Description (incidence and severity):
see Table 4
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality, salivation at 150 and 500 mg/kg post-dosing

BODY WEIGHT AND WEIGHT GAIN
Reduced in males females at 500 mg/kg

FOOD CONSUMPTION
No effects

FOOD EFFICIENCY
Not determined

WATER CONSUMPTION
No effects

OPHTHALMOSCOPIC EXAMINATION
No effects

HAEMATOLOGY
50 mg/kg bw.
No effects

150 mg/kg bw.
Slightly reduced thromboplastin time in males (-8%)

500 mg/kg bw.
Haemoglobin and haematocrit were reduced in males by -21 or -15% respectively and also MCV, MCH and MCHC by -10%, -17% or -8%. Platelets were increased in males (+ 50%) and females (+32%). The same applied to reticulocytes (males +88%, females +43%). Clotting parameters were reduced in males (thromboplastin time -8% and activated thromboplastin time -18%).
Neutrophiles rel./abs. were increased in females by +49%/+94%. All changed parameters attained statistical significance at p ≤ 0.01.


CLINICAL CHEMISTRY
50 mg/kg bw.
No effects

150 mg/kg bw.
No effects

500 mg/kg bw.
The following parameters were reduced in males and female animals: albumin (-6%/-6%), bilirubin (-20%/-17%) and protein (-16%/-14%). Calcium was reduced in males by -3%

URINALYSIS
Not determined

NEUROBEHAVIOUR
No effects

ORGAN WEIGHTS
No effects

GROSS PATHOLOGY
No effects

HISTOPATHOLOGY: NON-NEOPLASTIC
50 mg/kg bw.
No effects

150 mg/kg bw.
Fundus region of the stomach: subepithelial mixed cell infiltrations
Forestomach: epithelial hyperplasia

500 mg/kg bw.
Fundus region of the stomach: subepithelial mixed cell infiltrations, damaged mucosa, mucosal erosions with neutrophilic granulocytes
Forestomach: epithelial hyperplasia

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: local effects
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic effects

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

 

Table 1  Results of clinical clinical signs and body weight development

Parameter (changed 13 weeks after start of treatment)

Control

0 mg/kg bw

Low dose

50 mg/kg bw

Medium dose

150 mg/kg bw

High dose

500 mg/kg bw

Dose-response

+/-

 

m

f

m

f

m

f

m

f

m

f

Clinical signs

Salivation

 

-

 

-

 

-

 

-

 

+

 

+

 

+

 

+

 

+

 

+

Body weights

Week 13 (means, g)

Gains, week 0-13
(means, g)

 

459


216

 

269


102

 

461


221

 

272


112

 

472


228

 

270


109

 

399↓ns


176↓ns

 

250↓ns


91↓ns

 

-


-

 

-


-

** = p ≤ 0.01

 

 

 

Table 2  Results of haematology (mean values, n = 10)

Parameter (changed 13 week after start of treatment)

Control

0 mg/kg bw

Low dose

50 mg/kg bw

Medium dose

150 mg/kg bw

High dose

500 mg/kg bw

Dose-response

+/-

 

m

f

m

f

m

f

m

f

m

f

Haemoglobin

10.24

9.65

10.32

10.01

10.12

9.71

8.06**

9.49

 

 

Reticulocytes

17.60

18.50

14.00

19.30

20.33

21.40

33.10**

26.40

 

 

Platelets

1096.0

1083.6

1088.2

1076.2

1186.5

1138.0

1647.6**

1434.4**

 

 

Neutrophils (rel.)
Neutrophils (abs.)

16.89
2.230

10.72
0.832

9.67
1.061

10.89
0.906

14.11
1.382

13.59
1.092

18.46
2.515

15.93↑
1.615**





Haematocrit

46.6

43.3

47.0

44.3

46.5

43.1

39.5**

43.1

 

 

Thromboplastin time

10.04

8.87

9.71

8.75

9.25**

8.80

9.24**

8.59

 

 

Activated partial thromboplastin time

16.71

13.55

15.40

14.30

14.94

14.39

13.61**

14.17

 

 

MCV

51.83

52.72

51.09

53.23

52.38

53.24

46.46**

52.62

 

 

MCH

18.429

18.963

18.156

19.398

18.383

19.282

15.241**

18.720

 

 

MCHC

355.34

359.78

355.53

364.41

350.75

361.96

327.23**

355.29

 

 

** = p ≤ 0.01

 

 

 

Table 3  Results of clinical chemistry (mean values, n = 10)

Parameter (changed 13 week after start of treatment)

Control

0 mg/kg bw

Low dose

50 mg/kg bw

Medium dose

150 mg/kg bw

High dose

500 mg/kg bw

Dose-response

+/-

 

m

f

m

f

m

f

m

f

m

f

Albumin

31.58

33.97

31.82

34.92

31.13

33.82

29.76**

32.07**

+

+

Bilirubin

2.71

3.09

2.82

3.20

2.71

3.08

2.17**

2.55

+

+

Protein

62.4

66.5

63.2

67.9

59.9

66.0

52.4**

57.2**

+

+

Calcium

2.708

2.682

2.713

2.710

2.651

2.661

2.628**

2.660

+

+

** = p ≤ 0.01

 

 

 

 

Table 4  Results of histopathology (no. of animals affected/10 animals)

 

Parameter (changed 13 weeks after start of treatment)

Control

0 mg/kg bw

Low dose

50 mg/kg bw

Medium dose

150 mg/kg bw

High dose

500 mg/kg bw

Dose-response

+/-

 

m

f

m

f

m

f

m

f

m

f

Stomach
(fundus region)

 

 

 

 

 

 

 

 

 

 

Mixed cell infiltrat., subepithelial


0


0


0


0


2


3


9***


10***





Lymphoid hyperplasia
submucosa


0


0


0


0


0


0


9***


10***





Neutroph. granulocyt.
surface mucosa


0


0


0


0


0


0


3


3





Erosion with neutrophilic granulocytes, mucosa


0


0


0


0


0


0


9***


9***





Stomach
(forestomach)

 

 

 

 

 

 

 

 

 

 

Epithelial hyperplasia

0

0

0

1

7**

3

9***

8***

 

 

** = p ≤ 0.01
*** = p ≤ 0.001

 

 

Applicant's summary and conclusion

Conclusions:
As EUF (TPI 1618) is a formaldehyde releasing compound, the mucosal damage of the stomach is considered as local effect due to the irritant properties of formaldehyde. Other effects as seen in haematology parameters such as decrease in haemoglobin/haematocrit with concomitant stimulation of the erythropoietic activity (increase of reticulocytes) and the increase of neutrophil granulocytes and platelets may also be due to the inflammatory changes in the stomach. A decrease of clotting times as seen in the present study is not regarded as biological significant change. The same applies to the changes in clinical chemistry, where the significant decrease of some parameters is not considered as relevant systemic change. All changes observed are considered to be due to the local irritating properties of EUF (TPI 1618) as a formaldehyde releasing compound. Therefore no systemic changes were observed. The NO(A)ELsystemic was above 500 mg/kg bw./day. The NOELlocal was at 50 mg/kg bw./day due to the changes in the stomach.
Executive summary:

Study performed according to OECD guideline 408. 90-Day oral (gavage) toxicity study in male and female Sprague-Dawley rats. Dose levels were 0, 50, 150 and 500 mg/kg bw.

All changes observed were considered to be due to the local irritating properties of EUF (TPI 1618) as a formaldehyde releasing compound.

The NO(A)ELsystemicwas above 500 mg/kg bw./day. The NOELlocalwas at 50 mg/kg bw./day due to the changes in the stomach.