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Repeated dose toxicity: oral

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Administrative data

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was performed on Choline chloride similar to OECD 452 with minor deviations and not all details are given. However, the available information is sufficient to consider the results as reliable.

Data source

Reference
Reference Type:
publication
Title:
Effect of methionine and choline on liver tumor promotion by phenobarbital and DDT in diethylnitrosamine-initiated rats.
Author:
Shivapurkar N, Hoover KL, Poirier LA
Year:
1986
Bibliographic source:
Carcinogenesis 7: 547-550

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
Deviations:
yes
Remarks:
only male rats used
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Choline chloride
EC Number:
200-655-4
EC Name:
Choline chloride
Cas Number:
67-48-1
Molecular formula:
C5H14NO.Cl
IUPAC Name:
2-hydroxy-N,N,N-trimethylethanaminium chloride
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): Choline chloride
- Other: Supplier: Fisher Chemical Company

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, NY
- Age at study initiation: no data
- Weight at study initiation: 50 - 60 g
- Fasting period before study: no
- Housing: three per cage in plastic shoebox cages
- Diet (e.g. ad libitum): ad libitum a natural ingredient ground chow (Wayne Laboratory Blox Allied Mills, Inc., Chicago, IL)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
chow diet was supplemented with 1.0% Choline chloride
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
five days of non-supplemented chow, i.p. injection of saline (since the choline group served as a control group in a tumor promoting study), five days of non-supplemented chow.
72 weeks of treatment with supplemented chow, followed by 31 weeks post-observation with non-supplemented chow
Frequency of treatment:
continuously, i.e. application via feed which was available ad libitum
Doses / concentrationsopen allclose all
Dose / conc.:
1 other: percent (nominal) in diet
Remarks:
Doses / Concentrations: 1%
Basis: nominal in diet
Dose / conc.:
10 000 ppm
Remarks:
calculated from the above mentioned dosage of 1 % in diet
No. of animals per sex per dose:
30 males / dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Choline treated rats served as controls in a tumor promoting study on Phenobarbital (PhB) and 1,1 bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), the levels of Choline chloride was based on previous experiments wherein these levels were shown to inhibit the drop in hepatic S-adenosylmethionine levels due to PhB and DDT
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
Positive control:
Due to original study aim (tumor promoting effects), results will be given derived from animals which were initiated with i.p. injection of 200 mg/kg bw of diethylnitrosamine dissolved in sterile normal saline followed by a diet supplemented with 0.05% 1,1 bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT)

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: No data, only body weight denoted

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were taken at weekly intervals for 16 weeks and biweekly thereafter

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: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not applicable

OTHER: Gross pathology performed
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 1)
HISTOPATHOLOGY: Yes, but no results provided
Other examinations:
Growth and survival
Statistics:
The comparisons between the incidences of tumors in different groups were analysed by Fischer's exact test. The survival data were analysed by the computer program developed by Thomas el al. (Thomas CG, Breslow N and Gart JJ (1977). Trend and homogeneity analyses of proportions and life table data. Computers Biomed. Res., 10, 373-381.). Kaplan-Meier survival curves were derived by using this procedure. Comparison among survival of different experimental groups were made by Cox's test and P value based on the chi-square test from Cox's analysis. The incidences of liver and lung tumors and of leukemias in each group were based upon the number of animals surviving at 1 year since the first instance of each of the tumors occurred between weeks 55 and 60.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
mortality: no difference compared to control animals
Mortality:
no mortality observed
Description (incidence):
mortality: no difference compared to control animals
Body weight and weight changes:
no effects observed
Description (incidence and severity):
no difference compared to control animals
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
relative liver weight: no difference compared to control animals
Gross pathological findings:
no effects observed
Description (incidence and severity):
performed, but limited data given; Liver and lung tumor formation: no difference compared to control animals
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Various lung, liver and other tumors, leukemia: no difference compared to control animals
Details on results:
CLINICAL SIGNS AND MORTALITY - no difference compared to control animals

BODY WEIGHT AND WEIGHT GAIN - no difference compared to control animals

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study) no data

FOOD EFFICIENCY no data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study) not applicable

OPHTHALMOSCOPIC EXAMINATION no data

HAEMATOLOGY no data

CLINICAL CHEMISTRY no data

URINALYSIS no data

NEUROBEHAVIOUR no data

ORGAN WEIGHTS - relative liver weight: no difference compared to control animals

GROSS PATHOLOGY - performed, but limited data given; Liver and lung tumor formation: no difference compared to control animals

HISTOPATHOLOGY: NON-NEOPLASTIC no data

HISTOPATHOLOGY: NEOPLASTIC (if applicable) - various lung, liver and other tumors, leukemia: no difference compared to control animals

HISTORICAL CONTROL DATA (if applicable) controls see table 1

OTHER FINDINGS See table 1

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
> 10 000 ppm
Based on:
test mat.
Remarks:
Choline chloride
Sex:
male
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: originally reported as 1 % in diet
Dose descriptor:
NOAEL
Effect level:
> 1 200 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
Choline chloride
Sex:
male
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Results – Comparison of rats fed 1% choline chloride in diet to control group (plain diet)

Endpoint

Treatment Group (1% Choline chloride in diet)

Control Group (plain diet)

Positive Control Group (i.p. 200 mg/kg bw of diethylnitrosamine, 0.05% DDT in diet)

Survival at week

52

28

28

28

78

28

28

21

102

24

23

3

Body weight at week / g

10

258

253

262

50

406

408

394

Relative liver weight (%)

3.4

3.60

13.42

Number of animals bearing liver tumors

Neoplastic nodules

2

2

1

Hepatocellular carcinomas

0

1

28

Cholangiomas + cholangiocarcinomas

0

0

6

Lung metastases

0

0

13

Number of animals with tumors / extrahepatic lesions

Lung

1

2

4

Leukemia

2

8

4

Others

4

7

8

Applicant's summary and conclusion

Conclusions:
The present study was classified as reliable with restrictions due to the limited information provided. However, since the available information is sufficient to consider the study as reliable, the results obtained can be used to assess the repeated dose toxicity of Choline chloride. The study duration was 103 weeks, wherein the animals were dosed the first 72 weeks with 1% Choline chloride (10,000 ppm) in diet. Consequently, considering the total life span of a rat of approx. 1.5 - 2 years, the study duration was chosen long enough to detect all possible effects arising from Choline chloride.
No adverse effects were detected compared to control. In fact, although not statistically significant, Choline chloride treated animals developed less tumors than control animals. Also, no effects were seen regarding body weight gain, and the relative liver weight was also slightly, but not significantly decreased compared to control. This could be due to the fact that Choline chloride, which is also used as a feed additive, is an effective methyl donor, which does not require extensive metabolic pathways, which could possibly lead to additional liver damage due to hazardous degradation products. Hence, it is likely that CC does not only exhibit no adverse effects but also liver-protecting effects. Most likely effects for an increased liver weight can be (non)-neoplastic lesions, fatty liver or scar formation / cirrhosis due to necrosis already on only single cellular level, and also an increased requirement of metabolic enzymes. These effects are diminished by an additional gavage of Choline chloride. Furthermore, the positive control (i.p. 200 mg/kg bw of diethylnitrosamine, 0.05% DDT in diet) led to a decreased body weight and increased relative liver weight and tumor formation compared to control, which is an additional reason why the study, and so the results, can be considered as valid.
So, taking further into account the average food consumption of 120 g/kg bw/day of a male rat as given in ECHAs guidance document R.8, and the fact that no adverse effects were denoted compared to control at an average Choline chloride (CC) consumption of 1% in diet, the NOAEL was determined to be > 1% CC in diet (10,000 ppm), which corresponds to >1200 mg/kg bw/day (nearly life time duration).
In conclusion, it can be stated that Choline chloride does not induce any adverse effects and can be considered as non-toxic when administered chronically to rats, and no classification, neither as carcinogenic or STOT-RE, is required.
Executive summary:

In a chronic toxicity study equivalent to OECD Guideline 452, Choline chloride was administered orally 1% (10,000 ppm) in feed to male Fischer 344 rats, 30 animals per group, over 72 weeks with 31 weeks post-observation period.

There were no compound-related adverse effects denoted compared to control regarding the observed endpoints, i.e. body weight and body weight gain, relative liver weight, Tumor formation in liver and lung, leukemia and other tumors. So the NOAEL is >1% choline chloride (10,000 ppm) in food, based on all observed effects, which corresponds to NOAEL > 1200 mg/kg bw/day.

This chronic toxicity study in rats is acceptable with restrictions, satisfies the guideline requirements for a chronic oral toxicity study (OECD 452) in rats, and allow to draw the conclusion that Choline chloride is practically non-toxic.