Cholesterol

Endpoint summary

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: oral

Remarks:

other: 4-weeks and 24-weeks

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Publication from the open literature. Study restricted to the effects of 4 weeks or 24 weeks cholesterol/choline supplemented feed on mouse liver.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Mice were fed cholesterol/choline supplemented feed for 4 or 24 weeks. In addition, after 24 weeks of cholesterol supplemented feed, mice were fed control diet for 4 weeks. Mouse livers were examined for their weight, histology, tissue cholesterol, total hexosamines in insoluble glycosaminoglycans and hydroxyproline content.

GLP compliance:

no

Limit test:

no

Species:

mouse

Strain:

other: not specified albino SPF

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 20-30 grams

Route of administration:

oral: feed

Vehicle:

other: together with 0.45% w/w choline

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Cholesterol and choline were mixed with powdered chow . To prevent auto-oxidation products of cholesterol with prolonged storage, fresh diets were prepared every 2-3 days. Experimental diets were sampled at days 2 and 3 of feeding, and glc determination of hexane extracts confirmed a single major peak of cholesterol with <1% metabolites. Control animals were fed an identical diet without the addition of cholesterol.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

4 weeks and 24 weeks

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
1% (w/w)
Basis:
other: in diet together with 0.45% choline

No. of animals per sex per dose:

10 untreated controls (before feeding experiment), 10 cholesterol 4 wks, 10 pair-fed 4 wks, 10 cholesterol 24 wks, 10 pair-fed 24 weeks, 10 cholesterol 24 wks+4 wks control diet, 10 pair-fed 24 wks+4wks control diet

Control animals:

other: pair-fed (choline-supplemented)... (see attached file)

Details on study design:

choline 0.45% w/w, approx. 0.16 mg/J or 65 mg/100 calories)

Observations and examinations performed and frequency:

OTHER:
-body weight
-liver weight
-liver histology
-liver cholesterol was determined by glc
-liver glycosaminoglycans were isolated and hexosamines were determined by glc and hydrolysed liver samples were analysed for hydroxyproline.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (livers)
HISTOPATHOLOGY: Yes (livers)

Statistics:

two-tailed Student's unpaired t-test

Clinical signs:

not specified

Mortality:

not specified

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:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

liver

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

liver

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

liver

Histopathological findings: neoplastic:

no effects observed

Details on results:

WEIGHT GAIN: normal weight gain was reported (no data)

ORGAN WEIGHTS: liver; At 4 and 24 weeks the livers from cholesterol-fed mice weighed significantly more than livers of pair-fed controls; no difference in liver weights between 4 and 24 weeks. 4 weeks of control diet after 4 weeks of cholesterol supplemented diet showed that liver weight was returned to control values.

HISTOPATHOLOGY:liver; fatty livers at 4 and 24 weeks in cholesterol supplmented mice. No difference in histological appearance of diffuse fatty infiltration without necrosis or fibrosis in livers of mice treated for 4 or 24 weeks. 4 weeks of control diet after 4 weeks of cholesterol supplemented diet showed normal histology.

OTHER FINDINGS: hepatic hydroxypproline and glycosaminoglycans (reflected by total hexosamines) were increased after 4 and 24 weeks of cholesterol feeding, without differences between 4 and 24 weeks of cholesterol feeding. 4 weeks of control diet after 4 weeks of cholesterol supplemented diet showed a return to control values.
In cholesterol-fed mice there was a 20-25-fold increase in liver cholesterol content compared to pair-fed mice, which was lowered to slightly increased values compared to pair-fed controls after 4 weeks of control diet.

Dose descriptor:

NOAEL

Remarks:

study restricted to liver effects

Effect level:

< 1 other: %

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Critical effects observed:

not specified

No changes on investigated parameters was observed between pair-fed controls and untreated controls before the feeding study.

Conclusions:

Feeding mice for 4 or 24 weeks a diet containing 1% cholesterol/0.45% choline resulted in fatty livers with increased liver weights and increased levels of liver cholesterol, glycosaminoglycans and hydroxyproline compared to choline pair-fed mice, without time-dependency. Feeding control diet for 4 weeks after the cholesterol supplemented diet showed normal histology of the livers and a return to control values of liver weight and levels of glycosaminoglycans and hydroxyproline. Liver cholesterol content was decreased but slightly higher compared to controls after the recovery period. The results indicate that the the hepatic changes were reversible after 4 weeks of cholesterol supplement.

Executive summary:

Groups of 20 male mice were fed 1% (w/w) cholesterol (equivalent to 1500 ppm)/0.45% (w/w) choline or choline-supplemented (pair-fed) diet for 4 weeks. After 4 weeks 10 mice from each group were killed and livers were examined (weight, histology, cholesterol content, glycosaminoglycans and hydroxyproline content). The remaining two groups of 10 mice were fed control diet for another 4 weeks and their livers were examined for the same parameters. Additional groups of 10 mice were fed 1% (w/w) cholesterol/0.45% (w/w) choline or choline-supplemented (pair-fed) diet for 24 weeks, and their livers were examined for the same parameters as the other groups. Feeding mice for 4 or 24 weeks a diet containing 1% cholesterol/0.45% choline resulted in fatty livers with increased liver weights and increased levels of liver cholesterol, glycosaminoglycans and hydroxyproline compared to choline pair-fed mice, without time-dependency. Feeding control diet for 4 weeks after the cholesterol supplemented diet showed normal histology of the livers and a return to control values of liver weight and levels of glycosaminoglycans and hydroxyproline. Liver glucose content was decreased but slightly higher compared to controls. The results indicate that the hepatic changes were reversible after 4 weeks of cholesterol supplement.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

Study duration:

subchronic

Species:

mouse

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Groups of 20 male mice were fed 1% (w/w) cholesterol (equivalent to 1500 ppm)/0.45% (w/w) choline or choline-supplemented (pair-fed) diet for 4 weeks. After 4 weeks 10 mice from each group were killed and livers were examined (weight, histologogy, cholesterol content, glycosaminoglycans and hydroxyproline content). The remaining two groups of 10 mice were fed control diet for another 4 weeks and their livers were examined for the same parameters. Additional groups of 10 mice were fed 1% (w/w) cholesterol/0.45% (w/w) choline or choline-supplemented (pair-fed) diet for 24 weeks, and their livers were examined for the same parameters as the other groups. Feeding mice for 4 or 24 weeks a diet containing 1% cholesterol/0.45% choline resulted in fatty livers with increased liver weights and increased levels of liver cholesterol, glycosaminoglycans and hydroxyproline compared to choline pair-fed mice, without time-dependency. Feeding control diet for 4 weeks after the cholesterol supplemented diet showed normal histology of the livers and a return to control values of liver weight and levels of glycosaminoglycans and hydroxyproline. Liver glucose content was decreased but slightly higher compared to controls. The results indicate that the hepatic changes were reversible after 4 weeks of cholesterol supplemented diet.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Reliable information from the open literature on repeated dose toxicity by the oral route (4 weeks and 24 weeks) is available.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

Based on the observed reversible effect of fatty liver in mice fed 1% (w/w) cholesterol in the diet during 4 weeks, cholesterol does not need to be classified and has no obligatory labelling requirements for repeated dose according to

-Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011),

-Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures