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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Principles of method if other than guideline:
In addition, a satellite recovery group was included for the high level dose animals and controls and the study also included a functional observation battery assessment.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Commercial grade substance. Purity >98%.

Test animals

Species:
rat
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, UK
- Strain Crl:CD BR
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 135-197 (males), 127-171 (females)
- Fasting period before study: none specified
- Housing: groups of five (single sex) in suspended stainless steel cages with grid floors (36.5x55x25 cm)
- Diet (ad libitum): pelleted SDS rat and mouse maintenance diet #1
- Water (ad libitum): tap
- Acclimation period: 12 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-3C
- Humidity (%): 55 +/-15
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 21/6/00 To: 21/9/00 (1 day less for males)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: prepared by serial dilution on a weekly basis. Dosing solutions stored at 4C.

VEHICLE
- Concentration in vehicle: 5, 22.5 and 100mg/ml
- Amount of vehicle (if gavage): 10ml/kg.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples prepared for use in weeks 1, 6 and 12 were analytically verified. Analysis by GC/FID. Analysis confirmed solutions were within the range 2.7% above to 2.2% below nominal, which is regarded as acceptable (+/-10%). The dilutions were also confirmed as stable when stored at 4C for 15 days.
Duration of treatment / exposure:
Test duration: 90 days plus 4 week recovery satellite groups for control and high dose animals.
Frequency of treatment:
Dosing regime: 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
225 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:
Male: 15 animals at 0 mg/kg bw/day
Male: 10 animals at 50 mg/kg bw/day
Male: 10 animals at 225 mg/kg bw/day
Male: 15 animals at 1000 mg/kg bw/day
Female: 15 animals at 0 mg/kg bw/day
Female: 10 animals at 50 mg/kg bw/day
Female: 10 animals at 225 mg/kg bw/day
Female: 15 animals at 1000 mg/kg bw/day
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: based on results from a sub-acute study (liver effects).
- Rationale for selecting satellite groups: To check if effects seen in a sub-acute study were reversible adaptations.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes, measured on a 'per cage of 5 animals' weekly basis

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 : Yes, visual daily appraisal only. No formal measurements

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before study and at week 12. Pupils dilated with 0.5% tropicamide solution prior to examination.
- Dose groups that were examined: all.

HAEMATOLOGY: Yes / No / No data
- Time schedule for collection of blood: end of study (day 92/93) and end of recovery period for satellite animals
- Anaesthetic used for blood collection: Yes (isofluorane), from retroorbital sinus.
- Animals fasted: Yes, overnight prior to sampling
- How many animals: all main study males and all recovery group animals.
- Parameters checked: Hct, Hb, RBC, MCHC, MCV, MCH, WBC, Platelet counts, differential WBC count, cell morphology., prothrombin time, activated partial prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule, Animals fasted, animal number: as for haematology.
- How many animals:
- Parameters checked: urea, creatinine, NA, K, Ca, P, Cl, cholesterol, triglycerides, glucose, bilirubin, ALP. ALT, AST, GGT, total protein, albumin, albumin/globulin ratio.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Full battery before study, at weeks 12 and end of recovery period. Handling response recorded daily prior to treatment.
- Dose groups that were examined: all.
- Battery of functions tested: handling response, field activity (full battery only), motor activity (full battery only)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus (where present), uterus with cervix.
HISTOPATHOLOGY: Yes: Control and high dose : adrenals, alimentary tract (oesophagus, stomach, duodenum, jejenum, ileum, caecum, colon, rectum), aorta, brain, epididymides, eyes, head (nasal cavity, paranasal sinuses, nasopharynx), heart, kidneys (males only), liver, lungs (including bronchi), lymph nodes, (mandibular & mesenteric), ovaries, pancreas, pituitary, prostate, salivary glands, sciatic nerves, seminal vesicles, spinal cord, spleen, sternum (bone marrow), testes, thymus, thyroids, trachea, urinary bladder, uterus, vagina, plus any macroscopic abnormalities:
HISTOPATHOLOGY: Recovery animals, low and mid dose animals: liver and macroscopic abnormalities only.
Statistics:
If data consisted predominantly of one particular value (relative frequency >75%) the proportion of values different from the mode was analysed (Fisher/Mantel test), otherwise:
- Bartlett test: for heterogeneity of variance between treatments. (Where significant, log transformation was tried to look for more stable variance structure).
- One way ANOVA followed by Student's t-test and Williams' test where no heterogeneity found above to look for dose response significance; Kruskal Wallis test if significant heterogeneity found.
- For organ weight data analysis: ANOVA using terminal body weight as a covariate to allow for overall body weight effects.
- Histopathology: Fisher's exact test.
Significance levels in all cases set at p<0.05.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Treatment at 1000mg/kg/day was associated with a transient post dose salivation in both sexes in increasing numbers of animals throughout the study. It was also seen in two mid dose males in the last week of treatment only. The observation was not regarded as adverse. No clinical signs were seen in the low dose group.
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):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Slightly lower levels of monocytes and large unsustained cells were seen in both sexes in all dose groups compared to the control animals. One control animal showed anomalously high levels for these parameters. Lower lymphocyte levels were also noted among treated female groups compared to those of control animals. While these findings were of statistical significance, there were no associated histological findings and these changes were not considered to be of toxicological significance. RBC and clotting parameters were considered unaffected by treatment despite occasional small but statistically significant differences from control values. The blood smear results revealed a higher incidence of males showing colour variation at 1000mg/kg/day compared to controls but this was considered to be of no toxicological consequence. Assessment of haematological parameters measured on Day 29 of the recovery period revealed that the differences from control leucocyte levels only remained for females but not (at least in part) for males. There were no longer any differences in the blood smear results. These results were considered to reflect normal variation of haematological parameters.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Investigations performed on Day 92/93 of treatment revealed slightly higher cholesterol levels for all treated male groups and females receiving 225 and 1000 mg/kg/day, when compared to controls, although it was noted that values among female groups were not dosage-related. Triglyceride levels were noted as variable but generally lower for both sexes at all dosages except females receiving 225 mg/kg/day, compared to controls. Differences from control were generally statistically significant at 1000 mg/kg/day (p<0.05 for male cholesterol; p<0.01l for triglycerides) and these changes may have been related to the minimal degree of hepatic change noted histologically at 225 and 1000mg/kg/day. All other intergroup variations seen on Day 92/93 for the remaining parameters, including those attaining statistical significance, were generally slight and inconsistent between the sexes, and were therefore considered not to be of toxicological importance. Assessment of biochemical parameters measured on Day 29 of the recovery period showed smaller differences from controls in cholesterol and triglyceride levels for both sexes, suggesting that any possible effects on these parameters were adaptations that had reversed.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
The only observed difference was in the hindlimb grip strength of females in the high dose group. There was no other difference in behaviour nor were there similar effects in males. There was no difference in the grip strength performance of the females during the recovery phase. In the absence of corroborating evidence, this single difference is not considered to be attributable to treatment.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Assessment of organ weights from animals killed after the I 3 week treatment period revealed increased group mean liver weight for males and females receiving I 000 mg/kg/day, compared to controls while group mean kidney weights were elevated among males treated with 1000 mg/kg/day. In support of the organ weight changes being treatment related were low levels of statistical significance and histological findings in sections from both of these tissues. No differences in liver and kidney weight were seen between the high dose animals and controls at the end of the recovery period suggesting that the findings were non-adverse and reversible adaptations to treatment. Higher adrenals and uterus plus cervix weights were noted among the treated groups but were considered not to be related to treatment in the absence of histological findings or statistical significance. No other effects of treatment were identified.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Liver - Minimal centrilobular hepatocyte hypertrophy was reported in seven male and six female rats receiving 1000 mg/kg/day, and two males receiving 225 mg/kg/day. This finding was considered to be associated with the increased group mean liver weights of both sexes ~ receiving 1000 mg/kg/day.
Kidneys - There were an increased degree and incidence of cortical tubular eosinophilic inclusions in male rats receiving 225 or 1000 mg/kg/day and a marginal increased incidence of this change in males receiving 50 mg/kg/day, when compared with the background incidence among control group males. Although this was not statistically significant, it was considered likely to be related to treatment. This change was not seen in any female rats. The effect may be related similar to hydrocarbon nephrosis which is a male rat specific effect that is not relevant to humans. The effect was also seen in 60% of control animals and the incidence and severity only increased slightly albeit significantly with treatment.
The histopathology findings are shown in detail below. All other findings were considered to be incidental and not treatment related,
Histopathological findings: neoplastic:
not examined

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: See remark below

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Any other information on results incl. tables

Histopathology findings

Incidence of liver findings

 

Males

Females

Dose level (mg/kg/day)

0

50

225

1000

0

50

225

1000

Centrilobular hepatocyte hypertrophy

Total

0

0

2

7

0

0

0

6

Minimal

0

0

2

7

0

0

0

6

10 animals examined in all dose groups

Incidence of kidney findings

 

Males

Dose level (mg/kg/day)

0

50

225

1000

Cortical tubular eosinophilic inclusions

Total

6

8

9

10

Minimal

6

8

6

7

Slight

0

0

3

3

10 animals examined in all dose groups. No incidence in females

Statistically significant haematological findings. 

 

Males – mean (SD)

 

 

Control animals

High dose animals

Leucocytes (x1E-09)

0.22 (0.059), n=14

0.16 (0.036), n=13

 

Females – mean (SD), n=15

 

 

Control animals

High dose animals

Hct (L/L)

0.434 (0.0297)

0.431 (0.0163)

MCH (g/dL)

33.7 (0.79)

34.2 (0.49)

WBC (x1E-09)

9.45 (0.79)

6.69 (1.598)

Lymphocytes (x1E-09)

7.24 (2.293)

5.53 (1.374)

Monocytes (x1E-09)

0.25 (0.189)

0.16 (0.061)

Leucocytes (x1E-09)

0.24 (0.374)

0.10 (0.037)

Applicant's summary and conclusion

Conclusions:
No classification for repeat dose effects required
Executive summary:

In a guideline and GLP 90 day repeat dose oral gavage toxicity study using the substance ethoxypropoxy propanol, a no adverse effect level of 1000mg/kg/day was established. The study also included a functional observation battery and a satellite 4 week recovery group. Observed effects attributed to treatment included salivation post dosing. At the end of the study, slight differences were seen in clinical chemistry, in liver and kidney weight and histopathology and in one of the FOB parameters. All of these were seen in the high dose animals and were attributed to treatment. Slight haematological statistically significant changes were seen but were not attributed to treatment. Of the treatment related changes seen, all were fully reversed in the recovery group animals and were therefore regarded as being adaptive and reversible changes that were not adverse.