2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol]

Repeated dose toxicity: oral

Administrative data

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.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2009

Reference 2

Reference Type:

other: Expert review

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study 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

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol]

EC Number:

245-509-0

EC Name:

2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol]

Cas Number:

23235-61-2

Molecular formula:

C12H26O5

IUPAC Name:

2-ethyl-2-{[2-ethyl-3-hydroxy-2-(hydroxymethyl)propoxy]methyl}propane-1,3-diol

Test material form:

solid: flakes

Details on test material:

- Name of test material (as cited in study report): Di-Trimethylolpropane (Di-TMP)
- Physical state: Solid white flakes.
- Analytical purity: 99.6%
- Impurities (identity and concentrations): Not specified
- Purity test date: Not specified
- Lot/batch No.: 377 41 97
- Expiration date of the lot/batch: Not specified
- Stability under test conditions: Not specified
- Storage condition of test material: To be stored at room temperature.
- Other:

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system 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

Administration / exposure

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).

Doses / concentrations

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

Examinations

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

Results and discussion

Results of examinations

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

Effect levels

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

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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).