Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
21 September 1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
Described in Council Regulation (EC) No. 440/2008 and subsequent revisions
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Identity: CTF
Alternative name: Cyclic Trimethylolpropane Formal
Batch no.: 150803652
EC number: 225-967-8
EC name: 5-ethyl-1,3-dioxane-5-methanol
CAS number: 5187-23-5
IUPAC name: (5-ethyl-1,3-dioxan-5-yl)methanol
Expiry date 12 August 2016
Storage conditions: Room temperature, under nitrogen

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Hsd: Sprague Dawley SD
Details on species / strain selection:
The rat is the most frequently used species and is the default species for this study under REACH. Hsd: Sprague Dawley SD rats are a well characterised strain, with which the laboratory have experience and relevant background data.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone (UD), Italy
- Age at study initiation: 27-29 days
- Weight at study initiation: 75-99 g (M), 78-93 g (F)
- Housing: Group housed by sex (5/cage)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

DETAILS OF FOOD AND WATER QUALITY:
Drinking water was supplied ad libitum to each cage via water bottles. A commercially available laboratory rodent diet (4 RF 21) was offered ad libitum throughout the study. There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet. Records of analyses of water and diet are kept on file at RTC.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: December 2015 To: April 2016

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
CTF was administered orally by gavage at a dose volume of 10 mL/kg bw. Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.
Vehicle:
water
Details on oral exposure:
The required amount of CTF was dissolved in water. The formulation was prepared daily (concentrations of 10, 27.5 and 75 mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before the study, analysis was performed to confirm that the proposed formulation procedure was acceptable. 28-hour stability at room temperature and 8-day stability at 4°C were verified for the concentration range 10-75 mg/mL. Samples of formulations prepared in Weeks 1 and 13 of the study were also analysed to check the concentration. Results of all analyses were within the acceptable range (90-110%). Chemical analysis was carried out by the Analytical Chemistry Department at RTC, according to a validated method.
Duration of treatment / exposure:
Main groups were gavaged on 90 consecutive days; recovery groups were treated for 90 consecutive days followed by a 28-day recovery period.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Low dose group
Dose / conc.:
275 mg/kg bw/day (actual dose received)
Remarks:
Mid dose group
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
High dose group
No. of animals per sex per dose:
10/sex (main groups); 5/sex (recovery groups)
Control animals:
yes, concurrent vehicle
Details on study design:
Dose levels for this study were selected based on the results of a non-GLP range-finding study.
Positive control:
Not required for this study type.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed twice daily for mortality. Once before commencement of treatment and at least daily during the study, each animal was observed and any clinical signs were recorded. Observations were performed at the same time each day, approximately 1 hour/ after dosing. All clinical signs were recorded for individual animals.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once per week from the start of treatment, each animal was given a detailed clinical examination.

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed on the day of allocation to treatment group, on the day before treatment commenced, weekly thereafter and prior to necropsy.

FOOD CONSUMPTION: The weight of food consumed by each cage of rats was recorded at weekly intervals following allocation. The group mean daily intake per rat was calculated.

FOOD EFFICIENCY: Not reported

WATER CONSUMPTION: Not measured

OPHTHALMOSCOPIC EXAMINATION: Yes
Both eyes of all animals were examined prior to treatment by ophthalmoscopy and by a slit-lamp microscope after the instillation of 0.5% Tropicamide. The eyes of all animals from high-dose and control groups were re-examined during Week 13.

HAEMATOLOGY: Yes
During Week 13 of treatment, samples of blood were withdrawn under isofluorane anaesthesia from the retro-orbital sinus of all main phase animals, under conditions of food deprivation. Prior to necropsy, blood samples were taken in fasting conditions from the abdominal vena cava of the same rats from each group, under isofluorane anaesthesia, to perform coagulation tests. During week 4 of the recovery period, blood samples were withdrawn from all surviving animals under identical conditions in order to reevaluate the haematology parameters which showed possible treatment-related changes in measurements performed during the treatment period. The following parameters were measured using an automated analyser (Siemens Advia 120): haematocrit, haemoglobin, red blood cell count, reticulocyte count, mean red blood cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, white blood cell count, differential leucocyte count, neutrophils, lymphocytes, eosinophils, basophils, monocytes, large unstained cells (LUC) and platelets. Prothrombin time was also measured (ACL 3000+).

CLINICAL CHEMISTRY: Yes
During Week 13 of treatment, samples of blood were withdrawn under isofluorane anaesthesia from the retro-orbital sinus of all main phase animals, under conditions of food deprivation. During week 4 of the recovery period, blood samples were withdrawn from all surviving animals under identical conditions in order to reevaluate the clinical chemistry parameters which showed possible treatment-related changes at measurements performed during the treatment period. The following parameters were measured using an automated analyser (Siemens Advia 1200): alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, urea, creatinine, glucose, triglycerides, phosphorus, total bilirubin, total cholesterol, total protein, albumin, globulin, A/G ratio, sodium, potassium, calcium and chloride.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Once before commencement of treatment and at least once per week from the start of treatment, each animal was given a detailed clinical examination. Each animal was observed in an open arena. The test included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, unusual respiratory pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions were also recorded. Once during Weeks 12 or 13 of treatment and once during Week 4 of recovery an evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and an assessment of grip strength was also performed. The motor activity of all animals was measured once duringWeek 12 of treatment and once during Week 4 of recovery by an automated activity recording.

IMMUNOLOGY: Not specifically investigated
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were killed by exsanguination under isofluorane anaesthesia. All animals, including those found dead, were subjected to necropsy. Weights of the adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus and uterus were recorded.

HISTOPATHOLOGY:
Abnormalities
Adrenal glands
Aorta
Bone marrow (from sternum)
Brain (cerebrum, cerebellum, medulla/pons)
Caecum
Colon
Duodenum
Epididymides
Eyes
Femur with joint
Heart
Ileum
Jejunum (including Peyer’s patches)
Kidneys
Liver
Lungs (including mainstem bronchi)
Lymph nodes – cervical
Lymph nodes – mesenteric
Mammary area
Oesophagus
Ovaries
Oviducts
Pancreas
Parathyroid glands
Pituitary gland
Prostate gland
Rectum
Salivary glands
Sciatic nerve
Seminal vesicles
Skeletal muscle
Skin
Spinal column
Spinal cord
Spleen
Stomach
Testes
Thymus (where present)
Thyroid
Trachea
Urinary bladder
Uterus – cervix
Other examinations:
None
Statistics:
Standard deviations were calculated as considered appropriate. For continuous variables, the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If the data were found to be non-homogeneous, a modified t test (Cochran and Cox) was applied. The mean values, standard deviations and statistical analysis were calculated from actual values without rounding. Statistical analysis of histopathological findings was carried out by means of a nonparametric Kolmogorov-Smirnov test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
A decedent high dose male showed red staining on the muzzle on the day of death. Brown staining on head/neck was recorded in single treated females as well as in four control females on the day of necropsy. Brown staining on the eyelids was recorded on occasion in one control male. A damaged eye was seen in one high dose female. One high dose male had a single episode of convulsions during the last week of the recovery period. This was considered to be incidental.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One high dose male was found dead on Day 58 of the study. Necropsy revealed red staining on the muzzle, swollen liver, dark/red colour in the lungs and multiple dark pinpoints in the thymus. The histopathological evaluation reported haemorrhage in the lungs, as well as in the thymus and in the surrounding connective tissues up to the vascular (aorta) area in the heart. The death of this rat was attributed to misdosing.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No statisitcally significant effects were observed; however terminal bodyweight of males at 750 mg/kg bw/d is noted to be ~6.4% lower than controls.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
When compared with controls, sporadic statistically significant changes were recorded, as follows: increased platelets in males at 275 and 750 mg/kg/ bw/d (17% and 21%, respectively); increased erythrocyte count (3%), decreased MCV (2%), MCH (3%), decreased reticulocyte count (14%) and leucocyt counts (23%) in males at 275 mg/kg bw/d; decreased neutrophil count (22%) in males at 100 mg/kg bw/d; increased haemoglobin (4%), haematocrit (3%), MCV (4%), MCH (4%) and neutrophil count (41%) in females at 750 mg/kg bw/d.

A statistically significant decrease in prothrombin time was recorded for males at 750 mg/kg bw/d (8% below controls). Due to the severity and the direction of change, this finding was considered not to be of toxicological significance.

At the end of the recovery period, high dose females still showed neutrophilia (54% compared with controls). One male also showed marked neutrophilia compared to controls. Due to the low incidence and the absence of neutrophilia in males during the dosing phase, this finding was considered unrelated to treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Fluctuations of some biochemical parameters, mainly metabolic markers, were recorded for treated animals. Some of the statistically significant changes recorded during the dosing phase were still present following the recovery phase (glucose, urea and chloride in males, cholesterol and chloride in females).
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant increases in rearing were seen in all treated male groups on a single occasion during Week 2 of treatment. These values were comparable to the other values recorded during the study and, therefore, the statistical significance was probably due to a lower control value. A statistically significant decrease was seen in males receiving 275 mg/kg bw/d on a single occasion during Week 12; no toxicological significance was attributed to this finding, since it was isolated and did not show a dose-response relationship.

Neurotoxicity assessment did not reveal any toxicological differences between the treated and control groups. Increases, statistically significant in females, in landing foot splay in treated animals were seen at the end of the recovery period, when compared to controls. Due to the absence of similar finding during the dosing period, these differences were considered to be incidental and not treatment-related. A statistically significant increase in motor activity was seen in high dose males at the end of the recovery period, a decrease was seen in high dose females. Due to the different direction of the change and the absence of effects during the treatment period, these findings were considered not treatment-related.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant increases in absolute and/or relative weights were seen in animals receiving 750 mg/kg bw/d for the adrenals (+16% and +25% in males; +12% and +17% in females), liver (+22% and +30% in males; +19% and +24% in females), kidneys (+8% and +15% in males; +13% and +18% in females). Statistically significant increases in absolute and/or relative weights were seen in animals receiving 275 mg/kg bw/d for the adrenals (+14% and +21%, males) and liver (+6% and +9%, females)

Statistically significant changes observed in relative heart weight in animals receiving 750 mg/kg bw/d (+11% and +10% in males and females), when compared to controls, were minimal and considered not to be of toxicological significance. No toxicological significance was attributed to the slight statistically significant decreases in ovary weights (up to -17%) seen in females receiving 275 mg/kg bw/d, when compared to controls, since changes were slight and without a dose-relationship.

Following the recovery phase, statistically significant increases were again seen in treated males for absolute liver weight (+23%) and relative kidney weight (+18%). No relevant changes were seen in organs which showed differences during the dosing phase in treated females I ncreases in absolute and relative spleen weight seen in treated males (up to +17%) and females (+12%) when compared to controls, were considered unrelated to treatment due to the absence of similar effects during the dosing period.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Increased severity of nephropathy was noted in the high dose males, but was considered to be incidental.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
275 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
organ weights and organ / body weight ratios

Target system / organ toxicity

open allclose all
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
750 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
750 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

Bodyweights

 

M

F

0

100

275

750

0

100

275

750

Bodyweight (g)

Day 90

455.55

441.84

438.86

441.19

281.41

273.05

276.33

277.86

+28

471.20

 

 

472.45

284.54

 

 

289.34

Terminal bodyweight (g)

Day 90

433.31

415.67

409.15

405.96

268.23

257.24

260.42

258.41

+28

462.52

 

 

466.20

281.36

 

 

281.02

Haematology

 

M

F

0

100

275

750

0

100

275

750

Hb (g/dL)

Day 90

16.13

15.97

16.11

16.27

14.76

14.90

15.03

15.40**

+28

15.74

 

 

15.30

14.90

 

 

15.04

Hct (%)

Day 90

49.61

49.42

49.79

50.74

44.62

44.13

45.23

46.23*

+28

48.62

 

 

46.85

45.64

 

 

45.62

MCV (fL)

Day 90

54.51

54.12

53.15*

54.50

54.22

53.95

55.31*

56.26**

+28

53.92

 

 

53.35

55.42

 

 

55.86

Neutrophils (x103/µL)

Day 90

0.989

0.770*

0.980

1.176

0.461

0.385

0.503

0.650**

+28

0.954

 

 

2.440

0.382

 

 

0.588*

Neutrophils (%)

Day 90

10.23

8.30

12.91*

12.29

8.09

7.31

9.51

11.30**

+28

10.06

 

 

22.08

8.46

 

 

10.28

Basophils (%)

Day 90

0.86

0.89

0.87

1.08*

0.64

0.5

0.59

0.68

+28

0.78

 

 

0.85

0.52

 

 

0.58

Platelets (x103/µL)

Day 90

743.8

802.6

873.3**

902.04**

941.1

915.0

836.8

864.3

+28

773.4

 

 

842.3

790.6

 

 

780.6

PTT (s)

Day 90

23.35

23.30

23.09

21.53**

24.10

23.92

23.83

23.58

*significantly different to controls (p<0.05); **p<0.01

Clinical chemistry

 

M

F

0

100

275

750

0

100

275

750

AST (U/L)

90

81.85

72.29

76.96

69.17*

120.16

89.01

88.53

85.71

+28

73.94

 

 

84.10

77.42

 

 

90.66

Cholesterol (g/dL)

90

84.42

90.55

90.79

120.40**

90.97

105.14*

102.26*

109.83**

+28

74.20

 

 

120.53

89.22

 

 

107.42*

Glucose (mg/dL)

90

119.83

112.44

103.71*

103.86**

115.41

114.40

102.33**

107.30

+28

168.48

 

 

140.83*

132.26

 

 

122.54

Urea (mg/dL)

90

35.28

34.80

36.42

45.12**

36.14

37.41

40.33

42.31*

+28

31.78

 

 

39.95*

35.58

 

 

39.76

Cl (mM)

90

102.65

102.64

102.46

99.72**

108.32

107.86

106.60*

104.94**

+28

102.68

 

 

101.65*

104.66

 

 

102.76*

Ca (mM)

90

2.442

2.508*

2.520**

2.595**

2.466

2.473

2.500

2.538*

+28

2.508

 

 

2.528

2.474

 

 

2.432

Na (mM)

90

144.12

144.28

145.24**

144.34

147.62

146.61

146.45

145.32**

+28

141.82

 

 

141.95

141.22

 

 

140.74

Protein (g/dL)

90

6.64

6.88*

6.92**

7.15**

6.77

6.67

6.77

6.87

+28

6.64

 

 

6.93*

6.40

 

 

6.42

Albumin (g/dL)

90

3.99

4.18**

4.18**

4.25**

4.11

4.07

4.11

4.17

+28

3.88

 

 

3.95

3.86

 

 

3.82

Globulin (g/dL)

90

2.65

2.70

2.74

2.90**

2.66

2.60

2.66

2.70

+28

2.76

 

 

2.98**

2.54

 

 

2.60

*significantly different to controls (p<0.05); **p<0.01

Organ weights

 

M

F

0

100

275

750

0

100

275

750

Adrenals (g)

90

0.0547

0.0566

0.0625

0.0637*

0.0583

0.0589

0.0580

0.0651*

+28

0.0496

 

 

0.0555

0.0550

 

 

0.0534

Adrenals (%)

90

0.0126

0.0137

0.0153*

0.0157**

0.0217

0.0230

0.0223

0.0253*

+28

0.0107

 

 

0.0119

0.0196

 

 

0.0191

Heart (g)

90

1.430

1.397

1.388

1.484

0.977

0.969

0.942

1.032

+28

1.514

 

 

1.463

0.950

 

 

1.014

Heart (%)

90

0.3298

0.3362

0.3389

0.3657**

0.3644

0.3771

0.3616

0.3994**

+28

0.3272

 

 

0.3142

0.3382

 

 

0.3622

Kidneys (g)

90

2.854

2.704

2.839

3.088

1.548

1.517

1.576

1.753**

+28

2.930

 

 

3.485

1.808

 

 

1.820

Kidneys (%)

90

0.6585

0.6507

0.6933

0.7599**

0.5775

0.5908

0.6054

0.6786**

+28

0.6337

 

 

0.7450**

0.6439

 

 

0.6491

Liver (g)

90

10.912

10.454

10.996

13.307**

5.877

5.772

6.243

7.003**

+28

13.988

 

 

17.275**

7.724

 

 

8.288

Liver (%)

90

2.5145

2.5128

2.6855

3.2773**

2.1925

2.2429

2.3945**

2.7110**

+28

3.0259

 

 

3.7125**

2.7525

 

 

2.9442

*significantly different to controls (p<0.05); **p<0.01

Applicant's summary and conclusion

Conclusions:
Treatment with CTF for dose levels of up to 750 mg/kg bw/d for 90 consecutive days was generally well tolerated by male and female rats. Effects of treatment were limited to slight bodyweight deficits, haematological and clinical chemistry findings and organ weight changes at the highest dose level. Minor effects seen at lower dose levels are not considered to be of clear toxicological significance.
Executive summary:

Groups of SD rats (10/sex) were gavaged with CTF (in water) at dose levels of 0 (vehicle control), 100, 275 or 750 mg/kg bw/d on 90 consecutive days. Additional control and high dose groups of 5 rats/sex were treated for 90 days, followed by a 28-day recovery period. Rats were observed daily for mortality and signs of toxicity; more detailed assessments of clinical signs were performed weekly throughout the study. Ophthalmoscopy was performed pre-test and at the end of the study period. FOB and motor activity were assessed at the end of the main study and at the end of the recovery period. Bodyweights and food consumption were measured weekly. Terminal blood samples were taken for the assessment of haematological and clinical chemistry parameters. Gross necropsy was performed on all animals; weights of the adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus and uterus were recorded. A comprehensive list of organs/tissues from all animals was investigated histopathologically. One 750 mg/kg bw/d died due to a dosing accident; there was no treatment-related mortality. With the exception of the decedent male, no signs of toxicity were observed during the study period. Haematology revealed slight effects on erythrocyte parameters and increased neutrophil counts in females at 750 mg/kg bw/d. Other haematological findings are not considered to be clearly treatment-related or not of toxicological significance. Clinical chemistry showed a number of findings at the highest dose level, including reduced AST activity, increased cholesterol, reduced glucose concentration, increased urea concentration, reduced chloride concentration and (in males) increased total protein, albumin and globulin concentrations. Findings noted at other concentrations were not considered to be of toxicological significance. Gross necropsy did not reveal any treatment-related findings. Weights of the adrenals, kidneys and liver were increased at the highest dose level, but without any histopathological correlates. Organ weight effects at lower dose levels are not considered to be of clear toxicological significance. The study report concludes that none of the effects seen in this study are of toxicological significance; however a NOAEL of 275 mg/kg bw/d is determined for this study, based on changes in haematology and clinical chemistry parameters and organ weights at the highest dose level of 750 mg/kg bw/d.