Reaction mass of 2,2'-(Ethylenedioxy)diethanol and 3,6,9-Trioxaundecane-1,11-diol

Repeated dose toxicity: oral

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:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Effects of Polyethylene Glycol 400 (PEG 400) Following 13 Weeks of Gavage Treatment in Fischer-344 Rats.

Author:

Hermansky, S.J., Neptun, D.A., Loughran, K.A., Leung, H.W.

Year:

1995

Bibliographic source:

Fd. Chem. Toxicol. 2, 139-149

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Poly(oxy-1,2-ethanediyl),α-hydro-ω-hydroxy- Ethane-1,2-diol, ethoxylated

EC Number:

500-038-2

EC Name:

Poly(oxy-1,2-ethanediyl),α-hydro-ω-hydroxy- Ethane-1,2-diol, ethoxylated

Cas Number:

25322-68-3

IUPAC Name:

Poly(oxy-1,2-ethanediyl),α-hydro-ω-hydroxy- Ethane-1,2-diol, ethoxylated

Test material form:

liquid

Specific details on test material used for the study:

- Purity: > 99%

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 28 days old
- Weight at study initiation: At the time of group assignment, immediately before study initiation, only animals with body weights within ±20% of the population mean for each sex were included.
- Housing: The animals were individually housed in stainless-steel, wire mesh cages (22.5 x 15.5 x 18.0 cm) throughout the study.
- Diet: Ground was available ad libitum, except during periods of fasting prior to obtaining blood samples.
- Water: Tap water, ad libitum
- Acclimation period: Approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C (calculated from 66-77°F)
- Humidity: 40-70%
- Photoperiod: 12/12 (05:00 - 17:00)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Three graduated dose levels of the test substance were evaluated in three groups of 20 rats (10 of each sex) to produce a range of effects. Individual animal dose volumes were adjusted weekly based on the most current body weight for each animal. The high dose and control groups contained an additional 10 animals/sex that were designated for a 6-week recovery period. Control animals were treated with 5.0 mL Milli-Q filtered water kg/day.
During the treatment period, observations for mortality were made twice daily (am and pm). Observations for overt clinical signs were made once daily throughout the study. Body weight, food and water consumption data and detailed clinical observations were collected weekly for all animals. Blood samples were obtained following 13 week of treatment and 6 week of recovery (study week 13 and 19, respectively).

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

once daily, 5 d/w (65 doses)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 128 mg/kg bw/day (actual dose received)

Remarks:

Equivalent to 1.0 mL/kg/day

Doses / concentrations 2

Dose / conc.:

2 820 mg/kg bw/day (actual dose received)

Remarks:

Equivalent to 2.5 mL/kg/day

Doses / concentrations 3

Dose / conc.:

5 640 mg/kg bw/day (actual dose received)

Remarks:

Equivalent to 5.0 mL/kg/day

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

The oral route was selected based on the use pattern of the test substance as a relevant and reliable route of exposure. On the basis of the reported incidence of renal toxicity following exposure to PEGs, evaluation of this organ system was identified as a primary objective of the study.

Examinations

Observations and examinations performed and frequency:

Before study initiation, animals were examined by a clinical veterinarian. Representative animals were subjected to a comprehensive pretest health screen including full autopsy, histological examination of selected tissues, clinical pathology evaluations, serum viral antibody analyses, and examinations for faecal parasites.

CAGE SIGE OBSERVATIONS / BODY WEIGHT / FOOD CONSUMPTION / WATER CONSUMPTION / DETAILED CLINICAL OBSERVATIONS
During the treatment period, observations for mortality were made twice daily (am and pm). Observations for overt clinical signs were made once daily throughout the study. Body weight, food and water consumption data and detailed clinical observations were collected weekly for all animals.

URINALYSIS
Urine samples were obtained at week 12 and 18, 1 week prior to the collection of blood samples. Following at least 12 weeks of treatment, all animals, except those designated for the recovery period, were placed in metabolism cages. Following 5 weeks of recovery, all remaining animals were placed in metabolism cages. At the end of both the dosing and recovery phases, urine was collected for approximately 24 hours into cups containing thymol. Animals had access to both food and water while in the metabolism cages. Urine volume was measured using graduated test tubes, and urine colour and turbidity were visually assessed. Urine specific gravity was determined using a clinical refractometer. Urine osmolality was determined. The osmometer was calibrated with the appropriate standards before sample analysis. Semi-quantitative measurements were made for urine pH, protein, glucose, ketone, bilirubin, blood and urobilinogen. Urine N-acetyl-ß-D-glucosaminidase (NAG) determinations were performed on a Monarch 2000 Chemistry System. Controls for NAG were made in urine-based control material supplemented with commercially purified enzymes.

HAEMATOLOGY / CLINICAL CHEMISTRY
Following at least 13 weeks of treatment, all animals (except those designated for the recovery period) were fasted for approximately 22 hours and bled for haematology and clinical chemistry measurements before being killed. The animals assigned to the recovery group were also bled before being killed after 6 weeks of recovery. All blood was obtained from the retroorbital sinus following methoxyflurane anaesthesia. Approximately 0.5-1.0 mL blood was collected into blood collection tubes containing ethylene diaminetetraacetic acid as an anticoagulant for the haematological determinations and approximately 2.0 mL blood was collected into blood collection tubes without anticoagulant for serum chemistry analysis. The haematological analyses were performed on the day of sample collection. Commercially available quality control samples were analysed before analysis of animal samples. Blood smears for differential leucocyte counts were prepared for all animals, but only the control and high-dose groups were evaluated. Reticulocytes were measured by flow cytometry. A Monarch 2000 Chemistry System was used to analyse serum concentrations of glucose, urea nitrogen, creatinine, total protein, albumin, total and direct bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), lactate dehydrogenase (LDH), g-glutamyl transferase (GGT), sorbitol dehydrogenase (SDH), alkaline phosphatase (ALK), glutamate dehydrogenase (GLDH), calcium, phosphorus, sodium, potassium and chloride. Indirect bilirubin was calculated as the difference between total and direct bilirubin. Globulin was calculated as the difference between total protein and albumin. Serum controls were assayed with each run of samples. Controls for SDH and GLDH were made in a serum-based control material supplemented with commercially purified enzymes.

Sacrifice and pathology:

At the end of treatment, fasted animals were anaesthetized with methoxyflurane and humanely killed by severing the brachial vessels to permit exsanguination. Those animals of the control and high-dose groups designated for the recovery period were similarly treated 6 weeks later. On the day of killing, body weights were obtained to allow expression of relative organ weights. A complete autopsy was performed on all animals. The liver, kidneys, brain, heart, adrenals, spleen, testes (male) and ovaries (female) were weighed for all animals killed. An extensive range of tissues were collected and retained in 10% neutral buffered formalin. The bladder and kidneys of the high-dose and control groups were sectioned at 5 µm, stained with haematoxylin and eosin and evaluated by light microscopy.

Statistics:

The data for quantitative continuous variables were intercompared for the three treatment groups and the control group by use of Levene's test for quality of variances, analysis of variance (ANOVA), and t-tests. The t-tests were used when only one group was compared with the control group and when the F values from the ANOVA was significant. When Levene's test indicated similar variances and the ANOVA was significant, a pooled t-test was used for pairwise comparisons. When Levene's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances followed, when necessary, by a separate variance t-test for pairwise comparisons. Non-parametric data were statistically evaluated using the Kruskal-Wallis test followed by the Mann-Whitney U-test. Incidence data were compared using Fisher's exact test. For all statistical tests, the probability value of P < 0.05 (two-tailed) was used as the critical level of significance.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The only treatment-related clinical sign of toxicity observed during the study was loose faeces. Approximately 1 day after the first dose, loose faeces were observed in the majority of animals from the high-dose group of both sexes. This observation was transient and disappeared during the first week of dosing. Loose faeces were also observed near the end of the dosing phase of the study in 20 and 70% of male animals from the mid- and high-dose groups, respectively, and 61% of female animals from the high-dose group.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Six animals (one male and three females from the low-dose group and two females from the high-dose group) died during the dosing phase of the study. Autopsy or microscopic findings of trauma in the oesophagus and/or lungs of the female animals indicated that these deaths were related to the dosing procedure; these deaths were not therefore considered to be related to induced toxicity of the test substance. A cause of death for the male animal was not identified but the death of this animal was not attributed to treatment-induced toxicity, because of the lack of deaths in higher dose groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean absolute body weights of the high-dose group of both male and female animals generally were slightly decreased throughout both the dosing and recovery phases of the study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean food consumption values generally were not significantly affected in either males or females throughout the study. However, mean food consumption tended to be slightly decreased (approx. 2-8%) in the mid- and high-dose group of male rats throughout the study.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

In both male and female rats, mean water consumption values were generally increased in all dose groups throughout the dosing phase of the study. Water consumption values approximated control values during the recovery phase of the study.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urine NAG activity, osmolality and specific gravity were increased in a dose-related manner in all dose groups of male rats. The magnitude of the changes in these parameters in the low-dose group was very slight (only the specific gravity was statistically significant) and may have been due to random biologic variation. In female rats, urinary NAG activity was not significantly altered. Urinary osmolality and specific gravity tended to increase in all dose groups of female rats but only the specific gravity of the high-dose group was statistically significant. Urine pH was decreased in all dose groups of male rats and the mid- and high-dose group of female rats. The urinary concentrations of protein and bilirubin were also increased in all dose groups of male rats. The incidences of red and white blood cells in the urine were slightly increased in the high-dose group of male rats. Female rats in the mid- and high-dose groups had an increased urinary concentration of protein.
Following the recovery period (wk 18), there were no biologically significant changes in urinalysis measurements in either male or female rats.

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):

The mean weight of the male rat kidneys relative to the final body weight was increased approximately 2% in the low-dose group and approximately 4% in the mid- and high-dose groups. Absolute and relative male rat kidney weights were similar to those in the control group after the recovery period. In female rats, there was a slight increase in the weight of the kidneys relative to the body weight in the mid- and high-dose groups. Following the recovery, period, kidney weight relative to the final body weight was increased by approximately 4%. In male rats, the brain weight relative to the final body weight was increased 8% in the high-dose group, and tended to be slightly increased in the mid-dose group. The weight of the testes relative to the final body weight was increased 6% in the high-dose group and tended to be increased in the mid-dose group. Following the recovery period, there were no effects on organ weights in any dose group of male rats. The changes in the relative weight of the brain and testes in the high-dose group were attributed to the decreased final body weights and were not considered to be directly related to the test substance in those organs.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 128 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

urinalysis

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Treatment of Fischer 344 rats with PEG 400 at a constant volume of 1.0, 2.5 or 5.0 mL/kg body weight/day 5 days/week for 13 wk did not result in any mortality attributed to chemical toxicity or changes in haematology or clinical chemistry findings.