Registration Dossier

Administrative data

Description of key information

28 -day and 90- day oral studies are available for di-TMP.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 October 2007 - 13 November 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study report is well documented and is claimed to have been performed according to GLP standards, appropriate OECD Test Guideline and a Quality Assurance report is included. However, there is no indication of an appointed Study Director or formal GLP compliance statement. A separate expert review of the study is also provided.
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Remarks:
Study performed according to GLP standards, however the Laboratory is not covered by the National GLP monitor (Swedac) to conduct non-clinical studies. There is no appointed Study Director and the report does not contain a GLP compliance statement.
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: B & K Universal AB, Sollentuna, Sweden
- Age at study initiation: 5 - 6 weeks old
- Weight at study initiation: 160 - 180 g (Males), 118 - 159 g (Females).
- Fasting period before study: Not specified
- Housing: Macron 4 cages, stored in a ventilated cabinet. The animals had aspen bedding and PVC tubes for shelter in the cages.
- Diet (e.g. ad libitum): Ad libitum; standard R36 pellets from Lactamin AB, Lantmannen, Sweden.
- Water (e.g. ad libitum): Ad libitum; the water was changed twice a week.
- Acclimation period: 6 days for males, 7 days for females.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3ºC.
- Humidity (%): 40 - 60%
- Air changes (per hr): Not stated.
- Photoperiod (hrs dark / hrs light): 12h dim light/12 h dark (from dim to work friendly light when work performed in the animal room 7am to 7pm)


IN-LIFE DATES: From: 15 October 2007 To: 13 November 2007
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Prepared on a weekly basis; Di-TMP was suspended in the vehicle during stirring and heating. The high dose
required heating up to approximately 60°C to dissolve completely. At administration, all test solutions were heated to 35-40°C. The solutions were
stored at room temperature in cans for weekly use.


DIET PREPARATION
- Rate of preparation of diet (frequency): N/A
- Mixing appropriate amounts with (Type of food): N/A
- Storage temperature of food: N/A


VEHICLE
- Justification for use and choice of vehicle (if other than water): 2% Carboxymethylcellulose in deionised water was used to increase the solubility of the test substance.
- Concentration in vehicle: Formulations prepared at 0 (control), 4, 20, and 100 g/L.
- Amount of vehicle (if gavage): 1 mL / 100 g body weight
- Lot/batch no. (if required): Not specified.
- Purity: Not specified.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of cans of test solutions were analysed by Perstorp Speciality Chemicals AB (details of analysis not supplied). The deviations from the calculated concentrations were considered acceptable.
Duration of treatment / exposure:
Animals were dosed once daily for four weeks.
Frequency of treatment:
Daily. The dose was not administered to the rats on the days of urine sampling (day 23) or necropsy (day 28).
Remarks:
Doses / Concentrations:
0, 40, 200, 1000 mg/kg body weight
Basis:
actual ingested
No. of animals per sex per dose:
5 males and 5 females in each dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were decided based on an oral 4-day repeated dose-finding study
- Rationale for animal assignment (if not random): N/A (random)
- Rationale for selecting satellite groups: N/A
- Post-exposure recovery period in satellite groups: N/A
- Section schedule rationale (if not random): Randomly selected
Positive control:
Not required for this study type
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily, prior to administration.
- Cage side observations: Activity, body posture, behavioural changes, fur, breathing abnormalities, coughing, sneezing, feces, discharge from genitalia, signs of headache, signs of dehydration, other symptoms.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a week. Also performed on the day before necropsy and three days before the start of dosing.
- Observations: Nutritional status, skin, eyes, ears, paws, teeth, mucous tissues.

BODY WEIGHT: Yes
- Time schedule for examinations: Rats were weighed on days 0 (first day of administration), 7, 14, 21 and 28 (the day of necropsy).


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


OPHTHALMOSCOPIC EXAMINATION: Not conducted


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to necropsy on day 28 of the study
- Anaesthetic used for blood collection: Yes - phenobarbital (90 mg/kg bw)
- Animals fasted: No data
- How many animals: All
- Parameters checked: haemoglobin, haematocrit, mean cell haemoglobin, mean cell haemoglobin concentration, mean cell volume, red cell distribution width, red blood cell count, leukocytes, platelets and reticulocytes. The cell count was performed on basophiles, eosinophils, large unstained cells, lymphocytes, monocytes and neutrophils.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: as per haematology, above.
- Animals fasted: No data
- How many animals: All.
- Parameters checked: albumin, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, total calcium, total cholesterol, creatinine, globulin, glucose, potassium, sodium, total bilirubin, triglycerides, total protein and urea.


URINALYSIS: Yes
- Time schedule for collection of urine: Collected on day 23 of the study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data (food was not supplied during the time that the rats were in the metabolism cages).
- Parameters checked: pH, protein, glucose, ketone, bilirubin, erythrocyte and osmolarity.


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Three days before the start of dosing and three days before necropsy in the fourth week of administration.
- Dose groups that were examined: All
- Battery of functions tested: Locomotor activity, Behavioral changes (e.g. aggressiveness, passiveness etc.), Grabbing/grasping reflex and strength (let rat grab cage grid), Pain reflex (pinch skin between toes), Righting reflex (turn rat on its back), Hearing (click a pen behind ear)

OTHER:
Sacrifice and pathology:
At Day 28 of the study the rats were sacrificed. The necropsy, performed by the students at the toxicology programme, took place at the course laboratory at the Institute of environmental medicine, Karolinska Institutet under supervision of personnel from AstraZeneca Safety Assessment, Sodertalje, Sweden. The animals were intraperitoneally injected with sodium pentobarbital (90 mg/kg bw). When loss of consciousness was established, blood was collected and the terminal bodyweight was noted. The necropsy was directly initiated after the animal was desanguinated by cutting the carotid artery.
An external and internal examination was performed and the organs were removed. The following organs were weighed: testes, epididymis, prostate gland, ovaries, uterus, spleen, liver, kidneys, adrenal glands, thymus, heart, lungs and brain. Testes and epididymis were fixed in Bouin's solution (Picric acid, formalin and glacial acetic acid). All other organs were put in formalin. A list of all organs examined is found in appendix F. Organs from control and high dose groups of females and males were sectioned and stained at a contract laboratory, Biovet AB, Sollentuna, Sweden. As effects were found in high dose kidneys, also kidneys from the low and medium dose groups were sectioned and stained. The histopathological evaluation was performed by a pathologist at AstraZeneca Safety Assessment, Sodertalje, Sweden. The relative organ weights were calculated for all weighed organs in relation to individual brain weight.
Other examinations:
None
Statistics:
Analysis Statistical method Type of method
Body weight and food t-test Parametric
Clinical Chemistry Shirley's test Non-parametric
Organ weight Wilcoxon signed-rank test Non-parametric
Organ weight Kruskal-Wallis test Non-parametric ANOVA
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
initially, in all groups and associated with dosing
Mortality:
mortality observed, treatment-related
Description (incidence):
initially, in all groups and associated with dosing
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
elevated white blood cell counts in males at the highest dose level; values within the normal range
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
elevated serum potassium concentration; not considered to be adverse following expert review
Urinalysis findings:
no effects observed
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):
renal pathology observed in all groups but not considered to be related to treatmnet follwoing an expert review
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
During the first week of the study, symptoms such as nose bleeding and loose stool during administration were common in all dose groups. These symptoms were most likely induced by the initial stress caused by handling and new procedures. There were some observations of excessive salivation during administration especially in the high dose group, but it is unclear whether this observation is substance related or not.
At the end of the study the high dose males, but not the females, had more "dirty fur" and porphyria (causing pinkish fur) especially on their back, compared to animals in other dose groups.

BODY WEIGHT AND WEIGHT GAIN
All dose groups increased in body weight during the study. No significant differences could be seen for the females. Statistical analysis of the average weights of male rats showed significant increases (p<0.05) in the medium dose group for week 1 and in the low dose group for weeks 2, 3 and 4 . These results are not dose-related and most probably not substance-related.

FOOD CONSUMPTION
Statistical analysis of the average food consumption for the different dose groups showed no significant effects.

HAEMATOLOGY
The number of white blood cells showed a statistically significant increase in the male high dose group. No statistical effect was observed in females.
The lymphocyte count showed a statistically significant increase in the male high dose group. No statistically significant effect could be seen in the females.
In the monocyte count there was a statistical increase in the male high dose group that could not be seen in the lower dose groups or in the female groups.
There was a statistically significant increase in the basophil count in the male medium and high dose groups. In females there was no statistically significant effect.
The number of red blood cells was statistically significantly increased in females of the medium and high dose groups. No statistically significant effect was observed in males.

CLINICAL CHEMISTRY
Blood and plasma analysis
The concentration of glucose in plasma was statistically significantly increased in male medium and high dose groups, but no significant effect was observed in females.
A statistically significant decreasewas seen in plasma urea in medium and high dose females. The same effect was seen in the male high dose group.
The plasma sodium levels showed a statistically significant decrease in male medium and high dose groups. There was no statistically significant effect in females.
Plasma potassium levels were statistically significantly increased in a dose-related manner in all male dose groups. This effect was not observed in the female animals.
The concentration of total cholesterol in plasma was statistically significantly increased in all female dose groups. No difference in levels was observed in male rats.

URINALYSIS
No statistically significant effects were observed in the analyzed urinary parameters between exposed and control animals.

NEUROBEHAVIOUR
No neurological symptoms were observed in any of the animals during the study.

ORGAN WEIGHTS
Besides a statistically significant increase in final body weight in low dose males there were no statistically significant effects on body or organ weights (absolute and relative organ-to-brain).
The lower body weight of low dose males was considered not related to the test substance.

GROSS PATHOLOGY
Macroscopic findings
Observations made by the professional unaided eye were;
Stomach - Yellowish discolouration of the stomach was seen in one female in the control dose group, one male in the medium dose group and in one male in the high dose group.
Liver - Sero-sanguinous fluid in liver was discovered in one female in the low dose group. Marked lobular pattern in the liver was detected in one female in the medium dose group.
Kidneys - One cyst, which turned out to be nephroblastoma, was seen in one female in the low dose group. A yellowish-white mass in the kidneys was noticed in one female in the medium dose group.

The few findings observed were considered not related to the test substance.

HISTOPATHOLOGY: NON-NEOPLASTIC
All findings originate from the control dose group or high dose group animals, except for the findings in kidney where all dose groups are reported.

Lung
Hemorrhage in one female control animal (grade 2). 2 animals in control group male had alveolar histiocytosis (grade 1 and 2). One control female
had alveolar histiocytosis (grade d). 3 males in the high dose group showed alveolar histiocytosis (two grade 1 and one grade 2).

Stomach
In one female in control group and one male in high dose group the stomach was discoloured. No microscopic findings.

Kidneys
Tubular basophilia was detected in 2 males in the low dose group (grade 1 unilateral, grade 2 bilateral). One female in low dose group had a unilateral grade 1 tubular basophilia. One male in intermediated dose group had tubular basophilia grade 1. In the high dose group four males had tubular
basophilia (grade 2 unilateral, grade 1 unilateral, grade 1 unilateral, grade 1 unilateral). Inflammatory cell infiltrate was seen in one low dose male (grade 3) and in three low dose female (grade 2 unilateral, grade 1 unilateral, grade 3 unilateral). In the intermediate group one male and one female had a grade 1 inflammatory cell infiltrate. In the high dose male there was one grade 1 unilateral inflammatory cell infiltrate. Tubular simple dilation was
seen in two females in low dose group (grade 1 unilateral). One nephroblastoma (malignant neoplasm) was seen unilaterally in one low dose female.
One cyst was reported in one male in the high dose group.

Epididymides
One sperm granuloma (grade 2) was observed in one male in the high dose group.

Prostate gland
One control male and one male in the high dose group had inflammatory cell infiltrate (grade 1)

OTHER FINDINGS
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related adverse effects were seen at the highest dose level of 1000 mg/kg bw/d; based on the expert re-evaluation of renal fiindings
Critical effects observed:
not specified

General

Overall there were no clinical signs of toxicity from the substance. All results from the clinical observations show normal animal behaviour although the high dose males showed signs of "dirty fur" and porphyria towards the end of the study. No neurological symptoms could be observed. No treatment-related effects on body or organ weights were observed.

Clinical chemistry

A dose-dependent increase in plasma glucose was observed among male animals, but not in females. However, the glucose level in the high dose group (9.3 mmol/1) is still within the normal range for Sprague-Dawley rats (to 10.5 mmol/1) (Loeb, 1999). Elevated blood glucose could be caused by reduced tissue insulin sensitivity or alteration in insulin release (Strommer, 2002). The plasma concentration of cholesterol was increased in all female dose groups, but not in males. A dose-dependent decrease in mean plasma urea concentration was observed in both male and female rats. However, all mean values are within the reference value (8.32 mmol/1) (Loeb, 1999). Generally, an increase in serum urea is a marker for decreased glomerular filtration rate and thus impaired renal function (Klaasen, 2001). In addition, the effect in the present study was minimal (absolute difference between the control group and the highest dose group 0.8 mmol/1). A dose-dependent increase in mean plasma potassium concentration was seen in all male dose groups. Also, levels of sodium were decreased in males of medium and high dose groups. Total white blood cells and sub-populations were increased in male rats, but not changed in females. A dose-dependent increase in mean leukocyte count in blood was observed among male animals and was statistically significant in the high dose group. However, the number (10.45x109/l) is still within the normal range for rats (6-17 x109/l) (Van Zutphen, 2001). Regarding specific leukocyte types there was a dose-dependent rise in mean lymphocyte number although not exceeding normal values. Moreover, a statistically significant increase in mean monocyte count in high dose males was observed. In comparison to reference values all these values were in the range of what is considered normal. Also a dose-dependent increase in mean basophile count was seen in male rats. Altogether, the elevated number of leukocytes indicates inflammation.

Pathological findings

Macroscopic Necropsy

The macroscopic findings observed are most likely to be a matter of coincidence.

Histopathological findings

Substance- and dose-related findings were observed in the kidneys of males. The inflammatory cell infiltrate stage seen in all treated groups may be related to and precede the tubular basophilia, which is a sign of a degenerative/regenerative process. Similar effects were observed in female rats which supports that they are treatment-related. However, in females there was no clear dose-response as the effects occurred most frequently in the low dose group.

Conclusions:
Based on the independent expert review and further assessment of renal findings, a NOAEL of 1000 mg/kg bw/d is derived for this study.
Executive summary:

A 28-day repeated oral dose toxicity study was performed by students at the Institute of Environmental Medicine, Karolinska Institutet, Sweden on behalf of Perstorp Speciality Chemicals AB, to assess the toxicological effects of the test substance Di-trimethylolpropane (Di-TMP). The study was conducted comparable to OECD Guideline 407 and although the report claims that the study was performed according to GLP, there appears to be no appointed Study Director or a formal GLP compliance statement.

In this study, groups of 5 male and 5 female Sprague-Dawley rats were dosed at 40, 200, and 1000 mg/kg bodyweight/day for 28 days via oral gavage. Concurrent vehicle control groups were also administered. No overall clinical signs of toxicity or behavioural changes were observed and none of the observed macroscopic pathological findings were considered significant. A dose-dependent increase in mean plasma potassium was seen in the male test dose groups, and a decrease in sodium levels was seen in the medium and high dose levels for males. A statistically significant increase in white blood cells count was seen for the high dose level in males although the number was still within the normal range for rats. Substance- and dose-related findings were observed in the kidneys of male rats which may indicate a degenerative / regenerative process; a similar effect was seen in female rats although the effect did not appear to be dose related as the effect was seen most profoundly in the low dose level.

An expert review of this study was conducted at Huntingdon Life Sciences (Hooks & Begg, 2009). In summary, the tubular basophilia finding in the kidney was considered to be related to treatment, but in the absence of increase in severity and presence in both kidneys was not considered to be adverse in nature. In conclusion, in the absence of any adverse findings in the in-life or terminal investigations, the no-observed adverse effect level (NOAEL) in this study can be considered to be the highest dose level (1000 mg/kg bw/d).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 November 2016 to 03 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Sprague Dawley SD rat was the species and strain of choice because it is accepted by many regulatory authorities and there is ample experience and background data on this species and strain.
Sex:
male/female
Details on test animals and environmental conditions:
Body weight of animals on arrival: 83.8 to 94.7 g (males); 80.3 to 96.1 g (females)
Source of animals: Envigo RMS srl, San Pieto al Natisone, Italy
Acclimitisation period: 20 days
Housing: Upto 5 animals per cage; single sex
Caging: Clear polysulphone solid bottom cages; nesting material provided inside suitable bedding bags
Water: Drinking water; ad libitum
Feed: Laboratory rodent diet (4 RF 21, Mucedola S.r.l, Italy); ad libitum with the exception of the overnight fasting prior to blood samples being taken at the end of week 13 of dosing at the end of the 4-week recovery period.
Allocation to groups: Animals were weighed on the day of allocation (7 days prior to the start of treatment). Rats were allocated to groups by computer stratified randomisation to give equal initial group mean body weights.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
The test item was administered orally by gavage at a dose volume of 4mL/kg body weight. 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.

The required amount of Di-Trimethylolpropane, previously reduced to a powder, was dissolved/suspended in corn oil. The formulations were prepared daily for the first 9 days of treatment and weekly/every 6 days afterwards (concentrations of 25, 75 and 250mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.
The formulations prepared weekly or every six days were maintained at room temperature until the day of dosing (as per stability data: 8 days at room temperature).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the formulations, prepared on Weeks 1 and 13, were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits (85-115% for concentration and CV < 10% for homogeneity). Chemical analysis was carried out using a validated method in the range from 25 to 250mg/mL in corn oil in a separate study. In addition, the stability of formulated samples from the same range was verified after 28 hours and 8 days at room temperature in the same separate study. The software used for chemical analyses was Analyst 1.5.2 and 1.6.2.
Duration of treatment / exposure:
All animals were dosed for a minimum of 13 consecutive weeks followed by a recovery period of 4 weeks for 5 males and 5 females from Groups 1 (control group) and 4 (high dose group). Animals in the main phase were dosed up until the day before necropsy. No treatment was given during the recovery period.
Frequency of treatment:
All animals were dosed once a day, 7 days a week. No treatment was given during the recovery period.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10/sex/dose main phase
5/sex/dose recovery phase (vehicle control and 1000 mg/kg bw/day)
Control animals:
yes, concurrent vehicle
Details on study design:
Rats were allocated to groups by computer stratified randomisation to give equal initial group mean body weights. Three groups, each of 10 male and 10 female Sprague Dawley rats, received the test item by gavage at dosages of 100, 300 and 1000 mg/kg/day. A fourth similarly constituted group received the vehicle alone (corn oil) and acted as a control. Control and high dose groups included 5 additional animals per sex to be sacrificed after 4 weeks of recovery.
Positive control:
Not required
Observations and examinations performed and frequency:
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. One animal died during the study, and a complete necropsy was performed.

All clinical signs were recorded for individual animals. Once before commencement of treatment (not reported) and once daily during treatment, each animal was observed and any clinical signs recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.
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 Week 12 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 were also performed. Measurements were performed using a computer generated random order.

The motor activity (MA) of all animals was measured once duringWeek 12 of treatment and once during Week 4 of recovery by an automated activity recording. Measurements were performed using a computer generated random order.

Each animal was weighed on the day of allocation to treatment group, on the day before treatment commenced, weekly thereafter and just prior to necropsy.

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.

Both eyes of all animals were examined prior to the commencement of treatment by means of an ophthalmoscope, and by a slit-lamp microscope, after the instillation of 0.5% Tropicamide (Visumidriatic®, Visufarma, Rome, Italy). The eyes of all animals from high-dose and control groups were re-examined during Week 13 of treatment. Due to a change observed in the high-dose group, the examination was extended to the mid-dose group.

At the end of Week 13 of treatment, just prior to necropsy, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava of all surviving male and female animals from each group of the main phase, under overnight fasting conditions.
Since possible treatment-related changes were observed, blood samples for haematological investigations were taken from all animals under condition of food deprivation, at the end of Week 4 of the recovery period. Blood samples were collected and analysed in the same order.
The blood samples collected were divided into tubes as follows:
– EDTA anticoagulant for haematological investigations
– Heparin anticoagulant for biochemical tests
– Citrate anticoagulant for coagulation tests

The haematology measurements performed on blood samples are listed below:
– 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
– Platelets
These parameters were analysed by Siemens Advia 120.
Samples collected for animals nos. A2297003 (Group 1), A2297061 (Group 3) and A2297089 (Group 4) were not suitable for analysis (coagulated).

The coagulation measurements performed on blood samples are listed below:
– Prothrombin time
This parameter was analysed by Instrumentation Laboratory ACL 3000 PLUS.
Samples collected for animals nos. A2297035 (Group 2), A2297071 (Group 4) were not suitable for analysis (coagulated).

The clinical chemistry measurements performed on blood samples are listed below:
– 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
– Chloride
These parameters were analysed by Siemens Advia 1200.

Sacrifice and pathology:
Animals that had completed the scheduled test period were killed by exsanguination under isofluorane anaesthesia. All animals, including those found dead, were subjected to necropsy, supervised by a pathologist, as detailed below.

The clinical history of the animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.

From all animals completing the scheduled test period, the adrenal glands, brain (cerebrum, cerebellum, medulla/pons), epididymides, heart, kidneys, liver, ovaries (including oviducts), spleen, testes, thymus (where present), thyroid gland (including parathyroid glands) and uterus - cervix were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.

Samples of all the tissues listed in section 4.5.6 were fixed and preserved in 10% neutral buffered formalin (except eyes, testes and epididymides which were fixed in Modified Davidson’s fluid and preserved in 70% ethyl alcohol).

The tissues required for histopathological examination are listed in section 4.5.6. After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometre thickness and stained with haematoxylin and eosin.
The examination was conducted as detailed below:
i Tissues specified in section 4.5.6 from all animals in the control and high dose groups dying during the treatment period or killed at the end of the 13 weeks of treatment.
ii All abnormalities in all main phase groups.
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 inhomogeneous aModified t test (Cochran and Cox) was applied. The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off. Statistical analysis of histopathological finding was carried out by means of a nonparametric Kolmogorov-Smirnov test.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant clinical signs were observed during the study. The clinical signs were limited to hairloss in different regions of the body surface (head and/or neck and/or interscapular region) for individual female animals pertaining to Groups 1, 3 and 4 observed in the first occasion from Week 7 forward. In addition, scab on the neck was seen in one male rat of Group 1 on Weeks 12 and 13 (from Day 72 to Day 89). In a single male animal of Group 2 (no. A2297044), starting from Week 8 up to the end of the treatment period, a palpable mass was noted in the dorsal surface of the neck.
No changes of toxicological significance were found at the weekly clinical examination during treatment and recovery periods, which included an evaluation of neurotoxicity.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female of the control group (no. A2297029) was found dead on Day 59 (Week 9). No clinical signs were observed in the animals prior to death.
At post mortem examination, the animal showed dark red oily and fluid content in the thoracic cavity. At histopathology examination, the most relevant findings observed were mild granulomatous inflammation and haemorrhage in the lungs. In addition, numerous clear optic empty vacuoles (fatty change like) were present in the cytoplasm of alveolocytes in the lung, glomerular epithelium in the kidneys and cardiomyocytes in the heart, where the vacuoles were observed also within the vessel lumina. The nature of these vacuoles could be corn oil (vehicle) deposits and the cause of death was considered related to a misdosing.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant body weight differences were noted between treated animals and controls during the treatment and recovery periods.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Slight but statistically significant increase (not higher than 13%) of food consumption was sporadically observed during the study in Groups 3 and 4 of both sexes. No toxicological relevance was attributed to the sporadically slight increase due to the direction and value of the change.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Ophthalmoscopic examination performed in Week 13 in control and high-dose groups animals revealed a bilateral cataract detected in one male of Group 4 (no. A2297074). The examination was extended to Group 3 animals, and no ocular lesions were observed in the mid-dose group. The finding in Group 4 animal was considered spontaneous and not treatment-related.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
During the dosing phase, slight leucopenia was recorded in some animals of both sexes dosed at 300 and 1000 mg/kg/day. Mean group values were approximately 19% and 24% below controls, respectively, and changes were mainly due to reduced lymphocytes. The statistically significant difference of mean corpuscular haemoglobin concentration, recorded between controls and females receiving 1000 mg/kg/day, was of minimal severity (2%), therefore it was considered to be of no toxicological relevance. Other erythrocyte parameters were unaffected by treatmnet During the recovery phase, leucopenia was no longer recorded in treated animals, which showed slight leucocytosis. The other statistically significant differences between control and treated males (mean corpuscular volume, mean corpuscular haemoglobin concentration, platelets) were not recorded during the dosing phase, therefore they were considered unrelated to treatment.

No changes were recorded for coagulation.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Compared with mean control data, during the dosing phase, treated animals showed statistically significant fluctuations of some biochemical parameters, such as: increase of bilirubin in males dosed at 300 mg/kg/day (59%), increase of glucose in males treated at 100 mg/kg/day (19%), decrease of the same parameters in those receiving 1000 mg/kg/day (31%), increase of urea in males dosed at 1000 mg/kg/day (17%), decrease of protein in males receiving 100 mg/kg/day (4%), decrease of alkaline phosphatase in females dosed at 300 and 1000 mg/kg/day (26% and 23%, respectively) and decrease of phosphorus in those receiving 300 mg/kg/day. In general, changes were of slight severity, some of them showed no dose-relation (bilirubin, glucose and protein in males, phosphorus in females) and the decrease of alkaline phosphatase usually does not reflect any pathological condition. For these reasons, the above findings were considered to be of no toxicological relevance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No differences between treated animals and controls, which could be considered of toxicological relevance, were observed at functional tests (sensory reactivity, landing footsplay, grip strength) performed at the end of treatment and recovery periods. Motor activity measurements performed at the end of the treatment and recovery periods did not show any toxicologically significant differences between treated animals and controls.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Terminal body weight of treated animals of the three dosed groups (Groups 2, 3 and 4) was comparable to the control group. No treatment-related absolute and relative organ weight changes were seen. The minimal statistically significant decrease in relative liver mean weight (-8%) seen in the low dose females at final sacrifice was considered incidental as unrelated to the dose and as all individual values were within the physiological range of variability.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes were noted, following gross pathology examination at both final and recovery sacrifice. All observed changes were considered spontaneous and incidental, having a comparable incidence in control and treated groups and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were noted, following histopathology evaluation. All reported changes were considered spontaneous and incidental, having a comparable incidence in control and treated groups, and/or are characteristically seen in untreated Sprague Dawley rats of the same age.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no

Summary of findings in main group rats

 

M

F

Dose level (mg/kg bw/d)

0

100

300

1000

0

100

300

1000

N

20

20

20

20

20

20

20

20

Mortality

-

-

-

-

1

-

-

-

Terminal bodyweight (g)

470.6

473.0

479.0

477.6

274.9

282.5

291.9

290.9

Cataract

-

-

-

1

-

-

-

-

WBC (x103/µL)

7.074

8.346

5.665*

5.219*

4.493

4.719

3.748

3.490

Lymphocytes (x103/µL)

5.639

6.881

4.379*

3.974*

3.934

4.153

3.201

2.961

RBC (x106/µL)

8.270

8.259

8.031

8.320

7.210

.243

7.153

7.262

Hb (g/dL)

15.15

15.19

14.63

15.18

13.79

13.87

13.61

13.84

Hct (%)

44.99

45.20

43.14

44.87

40.69

40.37

39.58

39.99

MCV (fL)

54.40

54.74

53.72

53.97

56.43

55.75

55.40

55.08

MCH (pg)

18.30

18.38

18.22

18.25

19.13

19.14

19.02

19.06

MCHC (g/dL)

33.67

33.59

33.92

33.80

33.91

34.36

34.38

34.60*

Bilirubin

0.032

0.049

0.051*

0.027

0.053

0.061

0.062

0.031

Glucose (mg/dL)

138.16

164.79*

127.66

94.86**

97.13

116.25

117.10

118.11

Urea (mg/dL)

30.72

30.11

33.17

36.09*

36.81

39.61

36.36

40.33

Total protein (g/dL)

6.58

6.30*

6.44

6.68

6.18

6.10

6.36

6.14

ALP (U/L)

178.49

174.69

176.74

157.26

119.65

112.91

88.39**

91.75**

P (mg/dL)

5.635

5.819

5.401

5.572

5.520

5.119

4.563*

5.237

Terminal bodyweight (g)

461.21

453.87

453.67

445.62

257.68

265.72

267.15

269.02

Liver weight (g)

12.252

11.819

11.572

12.151

6.672

6.329

6.949

6.724

Liver weight (%)

2.66

2.61

2.55

2.73

2.59

2.78*

2.60

2.50

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

Conclusions:
In the absence of any effects of treatment, a NOAEL of 1000 mg/kg bw/d can be determined for this study.
Executive summary:

A GLP compliant 13-week study was conducted in Sprague Dawley rats according to OECD test guideline 408 with di-trimethylolpropane. Male and female rats were dosed at 100, 300 or 1000 mg/kg bw/d by oral gavage for 13 weeks. A control group was also included which comprised treatment with the vehicle control only (corn oil). Additional groups (vehicle control and test article group dosed with 1000 mg/kg bw/d) were dosed for the same period of time and then allowed a 4 -week recovery prior to termination of the study. There was no treatment-related mortality and no clinical signs which were of toxicological relevance. There were no toxicologically significant changes to body weight, food consumption, clinical chemistry or coagulation parameters. There were no relevent changes detected post mortem for terminal body weight, organ weights, gross macroscopic examinations and microscopic examinations. There was evidence of leucocytosis, which was reversible during the recovery phase. The overall conclusion of the study is that the NOAEL (no adverse effect level) is considered to be 1000 mg/kg bw/d.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
A sub-chronic GLP and OECD guideline compliant study is available for this endpoint. A sub-acute study is also available which was conducted according to OECD test guideline, but is not GLP compliant as no Study Director was assigned and there is statement of GLP compliance.

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

28-day study with di-TMP

A 28-day repeated oral dose toxicity study was performed to assess the toxicological effects of the test substance Di-trimethylolpropane (Di-TMP). The study was conducted comparable to OECD Guideline 407 and although the report claims that the study was performed according to GLP, there appears to be no appointed Study Director or a formal GLP compliance statement.

In this study, groups of 5 male and 5 female Sprague-Dawley rats were dosed at 40, 200, and 1000 mg/kg bw/d for 28 days via oral gavage. Concurrent vehicle control groups were also administered. No overall clinical signs of toxicity or behavioural changes were observed and none of the observed macroscopic pathological findings were considered significant. A dose-dependent increase in mean plasma potassium was seen in the male test dose groups, and a decrease in sodium levels was seen in the medium and high dose levels for males. A statistically significant increase in white blood cells count was seen for the high dose level in males although the number was still within the normal range for rats. The original report concludes that substance- and dose-related findings were observed in the kidneys of male rats which may indicate a degenerative / regenerative process; a similar effect was seen in female rats although the effect did not appear to be dose related as the effect was seen most profoundly in the low dose level. Results seemed to indicate signs of disturbed kidney function. As the effects were seen in all dose groups, no NOAEL could be derived from the study; a LOEL of 40 mg/kg bodyweight/day for renal effects in male rats was determined. The results of this study indicate a sex-difference in the sensitivity to Di-TMP as the majority of the treatment-related effects were seen in male rats. An expert review of this study was conducted at Huntingdon Life Sciences (Hooks & Begg, 2009). In summary, the tubular basophilia finding in the kidney was considered to be related to treatment, but in the absence of increase in severity and presence in both kidneys was not considered to be adverse in nature. In conclusion, in the absence of any adverse findings in the in-life or terminal investigations, the no-observed adverse effect level (NOAEL) in this study can be considered to be the highest dose level (1000 mg/kg bw/d).

13-week study with di-trimethylolpropane

A GLP compliant 13-week study was conducted in Sprague Dawley rats according to OECD test guideline 408 with di-trimethylolpropane. Male and female rats were dosed at 100, 300 or 1000 mg/kg bw/d by oral gavage for 13 weeks. A control group was also included which comprised treatment with the vehicle control only (corn oil). Additional groups (vehicle control and test article group dosed with 1000 mg/kg bw/day) were dosed for the same period of time and then allowed a 4 -week recovery prior to termination of the study. There were no clinical signs which were of toxicological relevence and no toxicologically significant changes to body weight, food consumption, clinical chemistry or coagulation parameters. There were no relevent changes detected post mortem for terminal body weight, organ weights, gross macroscopic examinations and microscopic examinations. There was evidence of leucocytosis, which was reversible during the recovery phase. The overall conclusion of the study is that the NOEL (no adverse effect level) is considered to be 1000 mg/kg bw/d.

Justification for classification or non-classification

The available data indicate Di-Trimethylolpropane to be of low toxicity following repeated oral exposure: classification for STOT-RE is not required according to Regulation (EC) No 1272/2008.