Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented, according to accepted guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
followed 1981 guideline but reliability scoring was based on 1998 version
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Hexylene glycol
- Physical state: liquid
- Lot/batch No.: 9710P0262
- Storage condition of test material: room temperature and protected from light

Test animals

Species:
rat
Strain:
other: Sprague-Dawley Crl CD (SD) IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France
- Age at study initiation: approx. 6 weeks
- Weight at study initiation: mean bw 190 g (males) and 161 g (females)
- Housing: wire-mesh cages with 2 rats/sex/group/cage
- Diet (e.g. ad libitum): A04 C pelleted available ad libitum
- Water (e.g. ad libitum): tap available ad libitum
- Acclimation period: 13-day


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): 12 cycles
- Photoperiod (hrs dark / hrs light): 12/12


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
The test substance was prepared as a solution in the vehicle. The test substance was diluted with the required quantity of vehicle in order to achieve the concentrations of 10, 30 and 90 mg/ml and then homogenized using a magnetic stirrer. The test substance formulations were made for up to nine days on the basis of stability data and were stored at +4 "C prior to use and protected from light.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before the start of treatment, the suitability of the proposed formulation procedure was determined by the analysis of stability of solutions prepared under the same experimental conditions. These analyses were performed in common with the range-finding toxicity study (CIT/Study No. I5836 TSR) and the present study; the results of the analyses are presented in each study report but the corresponding raw data are archived with CIT/Study No. 15836 TSR. During the course of the treatment period, a check of the concentration was performed on formulations prepared for use on the study.
Duration of treatment / exposure:
13-weeks
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 150, and 450 mg/kg body weight day
Basis:
actual ingested
No. of animals per sex per dose:
20 animals/sex/control and high dose groups
10 animals/sex/mid and low dose groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: see Fabreguettes, 1999, two-week range-finding toxicity study by oral administration (gavage) in rats
- Rationale for animal assignment (if not random): Animals were selected according to body weight and clinical condition and allocated by sex to groups, according to a computerized randomization.
- Rationale for selecting satellite groups: first 10 surviving animals of eac h sexes from the control and high dose groups
- Post-exposure recovery period in satellite groups: 4 weeks

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Time schedule: 2/day during treatment, 1/day when not treated

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before treatment and in week 4, 8, and 12

BODY WEIGHT: Yes
- Time schedule for examinations: first day of treatment and once weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: once/week

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before and at the end of treatment period
- Dose groups that were examined: control and high

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of treatment period
- Anaesthetic used for blood collection: yes, isoflurane
- Animals fasted: Yes
-How many animals: 10/sex/group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of treatment period
- Animals fasted: Yes
-How many animals: 10/sex/group

URINALYSIS: Yes
- Time schedule for collection of urine: end of treatment period
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: before the first day of treatment and in week 12
- Dose groups that were examined: first five surviving animals of each sex and group
- Battery of functions tested: sensory activity / grip strength / motor activity


Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see attached tissue procedures table)
ORGAN WEIGHTS: yes (see attached tissue procedures table)
HISTOPATHOLOGY: Yes (see attached tissue procedures table)
Statistics:
See attached

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: None attibutable to treatment

BODY WEIGHT AND WEIGHT GAIN: There were no signifcant differences between treated and control groups

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Slight increases in food consumption (up to 12%), occasionally of statistical significance (at weeks 1,10,11, and 12) were observed in males at 450 mg/kg. The overall food consumption was similar between groups and the slight increase in males (maximum 12 % at week 11) did not attain statistical significance. There was no corresponding increase in bodyweight.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Slightly higher weekly mean consumption (up to 28%) was seen in males only at 150 and 450 mg/kg/day. This reached statistical signifcance at weeks 2,11, and/or 13 only

HAEMATOLOGY: The only change in haematological parameters considered of significance was a dose related statistically significant increase in mean fibrinogen level in males at 150 (+11 %) and 450 (+15 %) mg/kg and females at 450 mg/kg (+19 %). This increase was considered by the authors to be secondary to the inflammatory reaction seen in the stomach and forestomach

CLINICAL CHEMISTRY: Changes in blood chemistry, considered treatment related, were increased cholesterol in the high dose groups of both sexes (male +69 %, female +26 %) and decreased glucose levels in males and females at 150 (males -8 %, female -3 %) and 450 (males -11 %, female -8 %) mg/kg. These were considered by the authors to be related to the adaptive changes observed in the liver. Other changes which attained marginal statistical significance (1-3% over controls) were higher mean sodium levels in all treated male and female groups, also decreased chloride values in top dose males and higher chloride values in females at all dose levels. Most values were within the performing laboratory's historical control range (sodium) or without a similar trend in both sexes (chloride) and were therefore not considered of toxicological significance.

URINALYSIS: Urinalysis revealed a lower mean urinary pH value (pH 6.2 compared to pH 7.0 in controls) and slightly higher specific gravity (+1.6 %) in top dose males only. These observations were considered related to the increased kidney weights in males at 150 and 450 mg/kg and the increased incidence of acidophilic globules in the cortical tubular epithelium of male rat kidneys at these dose levels. A slightly higher incidence of tubular basophilia and peritubular fibrosis were found in these same treatment groups compared to controls. This was considered by the authors to be secondary to the abnormal tubular accumulation of acidophilic globules.

ORGAN WEIGHTS: Increases in liver and kidney weights were associated with microscopic findings in these organs and are therefore considered treatment related. Liver weights were significantly increased in top dose males and females (absolute m +31%, f +14%; relative m +27%, f +8%). At the end of the recovery period top dose female absolute and relative liver weights and male absolute weights were comparable with controls while male relative liver weights showed partial reversal in recovery animals being (+11%) higher after the recovery period. Increases in females did not attain
statistical significance.

Kidney weights were significantly increased in top and mid dose males absolute kidney weights were increased 33% at the top dose while relative weights were +13% and +28% at mid and top doses respectively. Following the 4 -week recovery period absolute and relative kidney weights showed recovery the difference from controls being +16% and +15% respectively. Adrenal weights (absolute) were increased in males at all dose levels (+18%, +16% and +19% at low, mid and high dose respectively) and females at the top dose level(+17%). Relative adrenal weights were increased in top and mid dose males (+20% and +15%) respectively. At the end of the recovery period top dose female adrenal weights and relative male adrenal weights were comparable with controls while male absolute adrenal weights showed partial reversal (+11%). These increases may have been caused by cortical cell hypertrophy which occurred in 2/10 males and 3/10 females at 450 mg/kg/day. As these changes were observed in so few animals without a dose response effect, the change was not considered of toxicological significance and was possibly attributable to stress.

Changes in spleen and thymus weight were not considered of toxicological significance because they were not related to histopathological change, not dose related and not observed in both sexes.

GROSS PATHOLOGY: The only changes considered treatment related were a grey/green colouration of the kidney in 2/10 males at 150 mg/kg and 8/10 at 450 mg/kg/day. Enlarged kidneys were noted in 1/10 rats at 150 mg/kg and 5/10 rats at 450 mg/kg/day. These corresponded with microscopic kidney changes observed.

HISTOPATHOLOGY: NON-NEOPLASTIC Treatment related findings were seen in the liver, kidneys, stomach and forestomach. Hepatocellular hypertrophy in the absence of degenerative or necrotic change was observed in top dose animals of both sexes (m 10/10, f 5/10) and in males at 150 mg/kg (5/10). These effects reversed over the 4 week recovery period. This is considered an adaptive response to metabolic demand. Similar effects were observed in the range finding study. Kidney changes and increased kidney weight observed only in male rats were considered, by the authors, to be typical of male rat specific alpha 2-microglobulin nephropathy. The presence of acidophilic globules in the cortical tubular epithelium was recorded in 9/10 males for control groups (main study and recovery controls) and 10/10 males for all treatment groups. Treated recovery males showed an incidence of 5/10. The severity of the findings (based on incidence and grade) was 1.9, 2.7, 3.7 and 4.0 for controls, 50, 150 and 450 mg/kg respectively. Recovery controls were given a severity rating of 1.2 and treated animals a rating of 1.0. In males only at 150 and 450 mg/kg, increased incidences of findings in the kidneys were observed such as basophilic tubules, interstitial monocyte aggregation and dilatated medullary tubules were observed, most notably, peritubular fibrosis in males (at 150 mg/kg 6/10, and at 450 mg/kg, 9/10). These findings were partially reversed in recovery animals (450 mg/kg) with peritubular fibrosis being present in 3/10 males. These male rat specific kidney changes were also observed at both 200 and 1000 mg/kg in the 14 day range finding study. No direct measurement of alpha 2-microglobulin was made at the time these studies were carried out. Subsequently other kidney sections from the 90 day study were stained to confirm that the acidophilic globules were alpha 2-microglobulin (Massons Trichrome stain) and not indicative of hyaline droplet degeneration (PAS) All sections stained negative for PAS and positive for Massons Trichrome indicating that the droplets were alpha 2-microglobulins .Changes occured in the forestomach and, to a lesser extent, in the stomach of rats of both sexes at 150 and 450 mg/kg, which were considered to reflect local irritation. The stomach and forestomach of rats of both sex administered 1000 mg/kg/day in the 14 day range finding gavage study received microscopic examination. At this time point there were no changes in either stomach or forestomach. These changes in the forestomach included hyperplasia (males 3/10 at 150 mg/kg, males 8/10 and females 4/10 at 450 mg/kg; after recovery males 3/10, females 2/10) and hyperkeratosis (males 2/10 at 150 mg/kg, males 8/10 and females 4/10 at 450 mg/kg; after recovery males 3/10, females 2/10). Inflammatory cell infiltration and oedema of the mucosa and submucosa was also observed in high dose rats of both sexes; some changes were also observed in at 150 mg/kg. Effects in the stomach were confined to oedema and inflammatory cell infiltration of the submucosa with full recovery over 4 weeks.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
450 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Hepatocellular hypertrophy
Dose descriptor:
NOEL
Remarks:
local effects
Effect level:
50 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: local irritating effect on stomach and forestomach observed at higher doses

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
HEXYLENE GLYCOL administered to Sprague-Dawley rats daily by oral route at 50, 150 and 450 mg/kg/day for a 13-week treatment period was clinically well tolerated at all dose-levels. The hepatocellular hypertrophy and the changes indicative of local irritating effect on stomach and forestomach observed at 150 and 450 mg/kg/day were not considered as a systemic adverse effect. Consequently, under our experimental conditions, the No Observed Effect Level (NOEL) is 50 mg/kg/day and the No Observed Adverse Effect Level for the systemic effect is 450 mg/kg/day.
Executive summary:

The oral toxicity of hexylene glycol has been assessed in a 90-day repeated dose study in rats (Fabreguettes, 1999). This GLP study was conducted according to OECD test guideline 408 (1981). Hexylene glycol was administered by oral gavage to groups of male and female Sprague Dawley rats daily for 13 weeks at dose levels of 0 (water vehicle), 50, 150, or 450 mg/kg body weight/day. Main study animals were killed the day after the last dose and subjected to full necropsy examinations that included gross pathology, clinical pathology, and tissue harvest for histopathology. Subsets of animals from the vehicle and high-dose groups were allowed to recover for 4 weeks following the last dose before being killed to assess the reversibility of any effects noted in the main study animals. All animals also were subjected to ophthalmology and neurological examinations.

Hexylene glycol was well tolerated at all dose levels. No effects of treatment were observed on mortality, clinical signs, body weight, food or water consumption, food efficiency, ophthalmology, or neurobehaviour. Minor and/or reversible effects associated with treatment were observed on haematology (increased fibrinogen in mid- and high-dose males and in high-dose females, considered secondary to inflammatory lesions in the stomach and forestomach), clinical chemistry (increased cholesterol in high-dose males and females and decreased glucose in mid- and high-dose animals of both sexes, considered related to increased demand in liver function noted histologically, with evidence of reversibility), urinalysis (lower pH and higher specific gravity in high-dose males, considered secondary to male rat-specific excretion of alpha-2-µ-globulin, with evidence of reversibility), and organ weights (higher liver weight in high-dose males and females and higher kidney weight in high-dose males, with evidence of reversibility). Macroscopic lesions were noted on kidneys of high-dose males at the end of the treatment period, with evidence of reversibility. Microscopic findings in the mid- and high-dose groups included hepatocellular hypertrophy in both sexes (considered a response to an increased demand in liver function), kidney changes in males (acidophilic globules in tubular epithelium, tubular basophilia, and peritubular fibrosis; all considered related to alpha-2-µ-globulin), and local irritating effects in the stomach and forestomach of both sexes (but more prominent in males). All of these microscopic effects showed evidence of reversibility. Based on these findings a NOEL of 50 mg/kg body weight/day and a NOAEL of 450 mg/kg body weight/day (the highest dose tested) were derived (Fabreguettes, 1999).

.