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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1976
Report Date:
1976

Materials and methods

Principles of method if other than guideline:
The present experiments were initiated to provide adequate short-term investigations, with particular reference to effects on urinary oxalate excretion, using a DEG sample with a low (less than 0-01 %) MEG content.
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
The sample of DEG sed was supplied by Imperial Chemical Industrial Ltd. (Petrochemical Divisions) and gave the following analysis:
Colour (Hazen units), less than 5; specific gravity (15.5/15.5°C), 1 .120; initial boiling point, 244.4 °C; dry point, 250.3 °C; ash (w/w), less than 0.002%; acidity (as acetic), 0.0015; water (w/w), 0.035%; iron, less than 0.1 ppm; monoethylene glycol (w/w), less than 0.01%; triethylene glycol (w/w), 0.03%.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: obtained from a specified-pathogen-free colony
- Housing: five per cage
- Diet: Spillers' Laboratory Small Animal Diet, ad libitum
- Water: tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 1
- Humidity (%): 40 - 50


Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Duration of treatment / exposure:
98 days (first experiment)
225 days (second experiment)
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
0.4% in diet; equivalent to 300 and 400 mg/kg bw/day for males and females, respectively (first experiment, 98 days)
Dose / conc.:
1 500 mg/kg bw/day (nominal)
Remarks:
2.0% in diet; equivalent to 1600 and 1800 mg/kg bw/day for males and females, respectively (first experiment, 98 days)
Dose / conc.:
3 000 mg/kg bw/day (nominal)
Remarks:
4.0% in diet; equivalent to 3000 and 3700 mg/kg bw/day for males and females, respectively (first experiment, 98 days)
Dose / conc.:
64 mg/kg bw/day (nominal)
Remarks:
0.085% in diet; equivalent to 50 and 60 mg/kg bw/day for males and females, respectively (second experiment, 225 days)
Dose / conc.:
128 mg/kg bw/day (nominal)
Remarks:
0.17% in diet; equivalent to 100 and 130 mg/kg bw/day for males and females, respectively (second experiment, 225 days)
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
0.4% in diet; equivalent to 230 and 290 mg/kg bw/day for males and females, respectively (second experiment, 225 days)
Dose / conc.:
1 500 mg/kg bw/day (nominal)
Remarks:
2.0% in diet; equivalent to 1200 and 1500 mg/kg bw/day for males and females, respectively (second experiment, 225 days)
No. of animals per sex per dose:
15 rats in the first experiment (99 days)
10 rats in the second experiment (225 days)
Control animals:
yes, plain diet
Details on study design:
Groups of 15 rats of each sex were given diets containing 0, 0.4, 2.0 or 4.0 % diethylene glycol (DEG) containing less than 0.01 % monoethylene glycol for 98 days.
In a second experiment groups of 10 rats of each sex were given diets containing 0, 0.085, 0.17, 0.4 or 2.0 % of the same sample of DEG for 225 days.

Examinations

Observations and examinations performed and frequency:
FIRST EXPERIMENT
- Body weight: The animals were weighed initially, on days 1, 2, 6, 9 and 14 and then at weekly intervals throughout the study.
- Food and water consumption: the intakes of food and water were measured for each cage of rats over the 24-hr period preceding each day of weighing.
- Urinalysis: Urine was collected during wk 2, 6 and 14 of treatment and examined for microscopic constituents and content of glucose, albumin, ketones, bile salts and blood and concentration tests were carried out. These involved measuring the specific gravity and volume of urine produced in a
6-hr period of water deprivation and in the 2-hr period following a water ! load of 25 mL/kg. At wk 6 and 14 the same measurements were made on the urine produced between 16 and 20 hr after the water load.
- Haematology and chlinical chemistry: Blood samples collected at autopsy were examined for haemoglobin content, packed cell volume and counts of erythrocytes, total leucocytes, reticulocytes and differential leucocytes . In addition serum was separated and analysed for its content of urea, protein and albumin and for the
activities of glutamic-oxalacetic and glutamic-pyruvic transaminases and lactic dehydrogenase.

SECOND EXPERIMENT
- Body weight: The animals were weighed initially, on days 1, 4 and 8 and then at approximately weekly intervals throughout the study.
- Food and water consumption: Food and water intake over a 24-hr period were measured at the same intervals.
- Urinalysis: Urine samples were collected in week 8, 13 and 19 from the males and in week 9, 14 and 19 from the females over 24-hr periods, during which the rats were denied access to food and water. These samples were rendered strongly acid with hydrochloric acid and analysed for oxalic acid by the method of Hodgkinson & Williams (1972). Urine analyses, renal concentration and dilution tests and urinary cell counts on samples collected during the last week of the study.
- Haematology: collection of blood for haematological examination was carried out as described for the first experiment. In the haematological examination the reticulocyte and differential leucocyte preparations were not examined.
Sacrifice and pathology:
FIRST EXPERIMENT
At autopsy a search was made for macroscopic abnormalities, and the brain, pituitary, thyroid, heart, liver, spleen, adrenal glands, kidneys, gonads, stomach, small intestine and caecum were weighed. Samples of these and of oesophagus, various lymph nodes, thymus, salivary gland, trachea, aorta, lung, skeletal muscle, pancreas, colon, rectum, urinary bladder and uterus or prostate were preserved in 10% buffered formalin. Paraffin-wax sections of these tissues were stained with haematoxylin and eosin for microscopic examination.

SECOND EXPERIMENT
Post-mortem examinations and organ-weight analysis was carried out as described in the first experiment. Histological examination was confined to the kidneys.

Results and discussion

Results of examinations

Clinical signs:
not specified
Mortality:
mortality observed, treatment-related
Description (incidence):
FIRST EXPERIMENT
Six of the male rats given the diet containing 4% DEG died, or were killed because of ill health, during the study. The deaths occurred on days 19, 39, 55 (two rats), 82 and 85 of treatment. The animals were lethargic, bad lost weight over the preceding 3-10 days, adopted a hunched position in the cage and had a rough coat. Although the body temperature serum-urea levels in three of the rats were 45, 80 and 85 mg/100 mL compared with normal values of 15-25 mg/100 mL.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
FIRST EXPERIMENT
In the female rats there were no differences between treated and control groups in the rate of body-weight gain. However, the males fed the diet containing 4% DEG gained less weight than the controls, particularly during the second half of the study, the differences in mean body weight being statistically significant from day 43. At the lower dietary levels the body weights were lower than those of the controls in the males but there were no statistically significant differences.

SECOND EXPERIMENT
The increase in body weight was less in the rats of both sexes given 2.0% DEG than in the controls. The differences in body weight were statistically significant from day 36 to 113 in the male s and from day 8 to 204 in the females.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
FIRST EXPERIMENT
The food intake was not affected by the treatment. The mean intakes at the three dietary levels being 0.3, 1.6 and 3.0 g/kg/day in the males and 0.4, 1.8 and 3.7 g/kg/day in the females.

SECOND EXPERIMENT
There were no consistent differences between the treated and control groups in food intake. The calculated intakes of DEG for the four dose levels were approximately 50, 100, 230 and 1200 mg/kg/day in the males and 60, 130, 290 and 1500 mg/kg/day in the females.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
FIRST EXPERIMENT
The mean water intake over the whole experiment was increased in both sexes at the 4% dietary level and in the males given the diet containing 2% DEG. Despite the fact that the individual observations of water intake were based on only three values, as measurements were made on a cage basis, most of the values obtained from day 6 for the males given 4% DEG showed a statistically significant increase compared with the control values. In the females, significant differences were recorded only with one or two isolated values. In males given the lowest dietary level most of the values from day 9 onwards were slightly greater than the corresponding control values and, although the overall mean was not statistically greater than the control, it was increased by 6%.

SECOND EXPERIMENT
The water intake values were variable, the overall experimental means for the males given 0.49% and females given 0.17% DEG being less than those of the corresponding controls. On the other hand, the mean value for males given the highest dose level (2.0%) was higher than that of the controls. When analysed on the basis of the means up to or after day 93, it was found that there were no significant differences in the first part of the study, but the differences in the second part were the same as those seen over the whole study. The water intakes of the female rats did not show any dose-related differences from that of the controls.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
FIRST EXPERIMENT
There were no differences in the haematological indices between the female rats given DEG and the appropriate controls. Male rats given 4% DEG for 14 wk showed a statistically significant increase in the packed cell volume, haemoglobin concentration and total erythrocytes. With the lower dietary concentration (2%), the haemoglobin concentration was J the only measurement that was affected to a statistically significant degree although there was a slight increase in the erythrocyte count and
packed cell volume. A similar change involving only the haemoglobin concentration was evident at wk 2 in the males given 4% DEG. Calculation of the absolute values showed that the erythrocytes were of a normal size and haemoglobin content. The only changes in white cells were slightly increased total counts in males after treatment for 2 wk, without any change in the distribution of the different types of white cell.

SECOND EXPERIMENT
At the end of treatment there were no differences between treated and control rats in the results of the haematological examinations, since total counts did not differ the reticulocyte and differential leucocyte preparations were not examined.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
FIRST EXPERIMENT
The only abnormal values in the serum analyses were seen in the male rats given 4% DEG for 14 wk and consisted of lowered values for glutamic-pyruvic transaminase, total protein and albumin.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
FIRST EXPERIMENT
During a 6-hr period of water deprivation, the urine volume produced by males given 4% DEG was greater than that of the controls at all three examinations but the difference was significant only at wk 6 and 14. A similar but less marked increase was evident in the females given 4% DEG. In the dilution test there was a tendency for the rats of both sexes to produce a smaller volume of more concentrated urine than the controls, although this difference reached statistically significant proportions only in the males at the first, examination (2 wk). A similar and very marked effect was found in the males given the diet containing 2% DEG for 6 wk, but this was found to be due to two animals. which produced very small volumes of highly concentrated urine and when the test was repeated 24 hr later the finding could not be reproduced.
Oxalate crystals were found in the urine from some of the rats of both sexes given 2 or 4% DEG at all examinations. At the lowest dietary level oxalate crystals were found only in the urine from female rats. There were no increases in the number of cells excreted in the urine . The only other finding in the urine was the presence of blood at wk 6 and 14 in males fed the high dietary level.

SECOND EXPERIMENT
The results of the measurements of urinary oxalic acid concentrations show that the values for the rats given 2% DEG were greater than those of the controls throughout the experiment. The differences were statistically significant in most of the comparisons. At a lower dietary concentration (0.4%) there were no differences from controls after 8-9 wk treatment or in the females at wk 14, but in males at wk 13 as well as in both sexes at wk 19 the urinary oxalate concentrations in this group were significantly higher than those of the controls. With a dietary level of 0.17% urinary oxalic acid concentrations significantly higher than those of the controls were confined to the males at wk 19 whilst with the lowest dietary level (0.085%) the oxalic acid concentrations were comparable with those of the controls throughout the experiment.
There were no significant differences between treated and control rats in the specific gravity and volume of urine produced during a 6-hr period of water deprivation or in the first 2-hr after a water load. On the other hand the volume of urine produced after the longer period without water by male rats given 0.4 or 2.0% DEG was significantly greater than that produced in the same interval by the controls. At the higher of these dietary levels there was a lower specific gravity, but this was not statistically significant. There were no statistically significant increases in urinary cell excretion or differences in urinary pH.
The analysis of the urine collected over a 6-hr period showed no differences between the groups in the incidence of rats with blood, glucose, ketones, sperm or the various concentrations of albumin in the urine. There were significant differences between controls and the group on the highest dose level in the incidence of rats with phosphate crystals. This incidence was lower in the treated males but higher in the corresponding females. Oxalate crystals were found in the urine of the treated groups and at the two highest dietary levels the incidence of this finding was significantly higher than in the controls. Oxalate crystals were found in the urine from two control rats and at the two lower dose levels there was no indication of a significantly increased incidence of this finding.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
FIRST EXPERIMENT
In the organ-weight studies at wk 2 and 6, when the body weights of all groups were similar, the only statistically significant differences from controls were heavier livers in males given 4% for 2 wk, heavier kidneys in females given 4% DEG for 6 wk and lighter small intestines and thyroids in males fiven 2% for 6 wk. When the organ weights were expressed relative to body weight, statistically significant differences from control values were apparent in the liver values, which were higher in both sexes given 4% DEG for 2 wk and in the kidney values, which were higher in females given 4% for either 2 or 6 wk. In addition, the relative kidney weights of females given 2% DEG for 2 wk were higher than those of the controls. After the longer period of exposure (14 wk) the only change in organ weight in the females was a lower kidney weight at) the lowest dietary level (0.4%), a difference that was no longer statistically significant when expressed relative to body weight. In the males given 4% DEG for 14 wk the weights of most of the organs were lower than those of the controls to a statistically significant degree, but this was associated with a markedly reduced body weight. When expressed in relation to body weight the figures for most of the organs in this group were increased compared with the controls.

SECOND EXPERIMENT
There were scattered statistically significant differences between treated and control rats in the various organ weights, but none of these were found in the rats given the highest dietary level. Similarly there were scattered statistically significant differences in relative organ weights. In this comparison, however, the relative kidney and adrenal weights of both sexes given 2.0% DEG and the relative spleen weight of males fed the same dietary level were significantly greater than those of the controls.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
FIRST EXPERIMENT
At autopsy the rats that died had enlarged kidneys with a distended pelvic area and very pale cortical tissue. In two of the animals there were obvious cortical cysts. In the animals that survived to 14 wk the only abnormalities found at autopsy 1 were in the male rats given 4% DEG and consisted of one animal with bladder calculi, containing calcium oxalate crystals, and two with kidneys having a pale cortex and distended pelvis.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
FIRST EXPERIMENT
On histological examination tubular necrosis, mainly of the proximal convoluted tubules, was evident in five rats and hydropic degeneration of the proximal tubule in the remaining animal of the six animals that died. The bladder and ureters were distended in all six animals, and obvious calculi, containing calcium oxalate crystals, were found in these bladders together with marked haematuria. Cannibalism prevented examination of the liver in two rats but in the remainder the liver was enlarged and pale. Histopathological examination revealed pale vacuolated hepatocytes in the centrilobular area with some centrilobular necrosis in one rat. In animals that survived to 14 wk the histopathological findings were confined to the kidney and liver. Lesions in the liver, hydropic degeneration and a single case of necrosis, were seen only in the females at the highest dose level. The most marked change 4 in the kidneys was tubular necrosis, encountered only in rats given 4% DEG, although at this level, one animals was affected as early as wk 2. Hydrophobic degeneration of the cells in the renal tubules was found as early as wk 2 in rats given 496 DEG and by wk 14 one male animal from the group given 2% DEG was affected. No changes were seen in the kidneys of the rats given 0.4% DEG.

SECOND EXPERIMENT
The examination of histological sections of the kidney did not reveal in any differences between the treated and control animals.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
Dietary concentrations of 0.4 and 2.0% resulted in oxalate crystalluria and mild defects in renal function as measured by concentration tests. The only finding at a dietary level of 0.176 was a 13.2 % increase in urinary oxalate excretion in male, while with the lowest level (0.085 %) no effects were observed.
Extrapolation: Since no effects were observed at the dietary level of 0.085%, application of a safety factor of 100 in the extrapolation to man indicates an acceptable daily intake for a 70 kg man of 38 mg, and on a pro rata basis a lesser or greater value for individuals of different body weight.
The significance of the 13.2% increase in oxalate excretion found in rate on the 0.17% dietary level is debatable. If this level is considered to have no significant effect on the oxalate concentration of kidney and bladder urine, the acceptable daily intake (calculated as above) would be 76 mg/day for a 70 kg individual.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
(225 days)
Effect level:
128 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
urinalysis
Key result
Dose descriptor:
NOAEL
Remarks:
(98 days)
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 600 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Applicant's summary and conclusion

Conclusions:
Since no effects were observed at the dietary level of 0.085%, application of a safety factor of 100 in the extrapolation to man indicates an acceptable daily intake for a 70-kg man of 38 mg, and on a pro rata basis a lesser or greater value for individuals of different body weight. The significance of the 13.2% increase in oxalate excretion found in rate on the 0.17% dietary level is debatable. If this level is considered to have no significant effect on the oxalate concentration of kidney and bladder urine, the acceptable daily intake (calculated as above would be 76 mg/day for a 70-kg individual.
Elevated levels of oxalic acid in urine in this study were considered to be a biomarker and do not indicate toxicity. Mild defects of renal function at 0.4% DEG (increased urine volume) were therefore considered to define the LOAEL as 230 mg/kg bw/day, and the NOAEL as 300 mg/kg bw/day.
Executive summary:

Vacuolization of the tubular epithelium (hydropic degeneration) and tubular necrosis were the histopathological findings. For the crystaluria and increased urine volumes after concentration tests, the results in the male and the female rats were inconsistent, and no clear dose-response relationships was observed for these effects. Therefore, the evaluation is based on the histopathological findings. Hydropic degeneration of the kidneys started to occur at oral dose levels of 1550 mg/kg bw/day for 14 weeks and was not seen at 300 mg/kg bw/day. The conclusion is that the NOAEL for hydropic degeneration is 300 mg/kg bw/day (0.4% 2,2’-oxydiethanol in food) in the male rats.