Choline hydrogen carbonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was performed on the read-across substance 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

Test material information

Constituent 1

Reference substance name:

Choline chloride

EC Number:

200-655-4

EC Name:

Choline chloride

Cas Number:

67-48-1

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 availabel ad libitum

Doses / concentrations

Remarks:

Doses / Concentrations:
1 %
Basis:
nominal 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

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

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

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: 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

1

Dose descriptor:

NOAEL

Effect level:

> 1 other: percent in diet

Based on:

test mat.

Remarks:

Choline chloride

Sex:

male

Basis for effect level:

other: see 'Remark'

2

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'

3

Dose descriptor:

NOAEL

Effect level:

> 1 420 mg/kg bw/day (nominal)

Based on:

other: Choline bicarbonate

Sex:

male

Basis for effect level:

other: Basis for effects: overall effects; mortality; body weight; gross pathology; organ weights Recalculated value from NOAEL > 1200 mg/kg bw Choline chloride regarding the molecular weight of each compound.

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 and the fact that it was performed on the read-across substance Choline chloride. 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 and hence, Choline bicarbonate. The study duration was 103 weeks, wherein the animals were dosed the first 72 weeks with 1 % Choline chloride 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 and so Choline hydrogen carbonate.
The read-across from Choline chloride to Choline hydrogen carbonate is justified because the absorption after oral application is very likely to remained unchanged, since the toxicologically relevant choline cation is identical in both salt, its transport mechanisms in the body are only relevant for the cation, too, and no absorption-enhancing irritating effects are expected from the anion. So, information gained from Choline chloride for this endpoint can be used as weight-of-evidence information without modification except adjusting the dose regarding the molecular weight of both choline salts.
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, 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, the read-across substance Choline chloride was administered orally 1 % 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 in food, based on all observed effects, which corresponds to NOAEL > 1200 mg/kg bw/day of Choline chloride or NOAEL > 1420 mg/kg bw/day of Choline hydrogen carbonate.

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 and hence Choline bicarbonate is practically non-toxic.