Choline chloride

Toxicity to reproduction: other studies

Administrative data

Endpoint:

toxicity to reproduction: other studies

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

no information

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Choline is required in the diet of lactating dams to maintain maternal immune function

Author:

Dellschaft NS, et al.

Year:

2015

Bibliographic source:

British Journal of Nutrition (2015), 113, 1723–1731

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Primiparous Sprague–Dawley rats (n 42) were randomised 24-48 h before birth and fed the following diets for 21 d: choline-devoid (0 g choline/kg diet; D, n=10); 1.0 g choline/kg diet (C1, n=11); 2.5 g choline/kg diet (C2.5, n=10); 6.2 g choline/kg diet (C6, n=11). The effects of varying intakes of choline on maternal immune function during lactation were determined testing several parameters (liver total fat content, choline metabolite analyses of pup stomach content, immune cell phenotype analysis and ex vivo cytokine secretion by mitogen-stimulated splenocytes).

GLP compliance:

not specified

Type of method:

in vivo

Test material

Test material information

Constituent 1

Reference substance name:

Choline

EC Number:

200-535-1

EC Name:

Choline

Cas Number:

62-49-7

Molecular formula:

C5H14NO

IUPAC Name:

2-hydroxy-N,N,N-trimethylethanaminium

Specific details on test material used for the study:

Choline was added to the diets in the form of choline bitartrate obtained from Harland Teklad

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: Primiparous Sprague–Dawley rats were obtained on day 14 of gestation
- Diet (e.g. ad libitum): Diets were fed ad libitum throughout lactation to the end of the study at 21 d postnatal. Animals were provided free access to food throughout each 24 h period

Administration / exposure

Route of administration:

oral: feed

Details on exposure:

Dams were fed standard rat chow (Lab diet 5001; PMI Nutrition International, containing 1 g choline/kg; Harland Teklad) throughout gestation, then randomised to one of four experimental diets (D, choline-deficient diet; C1, 1.0 g choline/kg diet; C2.5, 2.5 g choline/kg diet; C6, 6.2 g choline/kg diet) 24–48 h before parturition. Diets were fed ad libitum throughout lactation to the end of the study at 21 d postnatal.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The content of choline in the salt was verified by analysis before adding it to the diets.

Duration of treatment / exposure:

21 d

Frequency of treatment:

continuous

Duration of test:

After 21 days of parturition, dams and two pups per dam were weighed and killed by CO2 asphyxiation and subsequent cervical dislocation in the morning hours.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 other: g/kg diet

Remarks:

D

Doses / concentrations 2

Dose / conc.:

1 other: g/kg diet

Remarks:

C1, corresponding to 200 mg choline/kg bw/day

Doses / concentrations 3

Dose / conc.:

2.5 other: g/kg diet

Remarks:

C2.5, corresponding to 500 mg choline/kg bw/day

Doses / concentrations 4

Dose / conc.:

6.2 other: g/kg diet

Remarks:

C6, corresponding to 1240 mg choline/kg bw/day

No. of animals per sex per dose:

D (n=8, two of the original ten dams in the D group had to be killed before the end of the experiment due to significant weight loss), C1 (n=11), C2.5 (n=10), C6 (n=11)

Control animals:

yes, plain diet

Details on study design:

The effects of varying intakes of choline on maternal immune function during lactation were determined in primiparous Sprague–Dawley rats treated for 21 days testing the following parameters: Anthropometric characteristics, Liver total fat content, Choline metabolite analyses of pup stomach content, immune cell phenotype analysis and ex vivo cytokine secretion by mitogen-stimulated splenocytes.

Statistics:

Statistical analyses were conducted using SAS statistical software (version 9.3; SAS Institute, Inc.). Data were analysed for normal distribution in each dietary group with the use of a Kolmogorov–Smirnov test. Parametric data were subsequently analysed for differences by ANOVA and Tukey post hoc testing was used. Regression analysis was used to test the relationship between dietary choline intake and the concentration of free and total choline in the stomach content of the offspring. Non-parametric data were log-transformed before analysis by ANOVA as above. In some instances, logtransformation did not lead to normal distribution of the data, and groups were compared similarly using Mann–Whitney U-tests. For all statistical tests, significance was accepted with a CI of 95% (P<0.05). All results are presented as means with their standard errors and the actual number of values available for each measure is indicated with the results.

Results and discussion

Observed effects

D and C6 dams had lower final body weight, spleen weight and average pup weight than C1 dams (P<0.05). There was a linear relationship between free choline concentration in pup stomach contents with maternal dietary choline content (P<0·001, r² 0.415). Compared with C1 and C2.5, D spleens had a lower proportion of mature T cells and activated suppressor cells, and this resulted in reduced cytokine production after stimulation (P<0.05). Feeding 6.2 g choline/kg diet resulted in a higher cytokine production after stimulation with CD3/CD28 (P<0.05). Except for a higher IL-6 production after LPS stimulation with cells from the C2.5 dams (P<0.05), there were no differences between the C1 and C2.5 dams.

Applicant's summary and conclusion

Conclusions:

Oral administration of choline at test concentrations up to 6.2 g choline/kg diet, corresponding to 1240 mg choline/kg bw/day, to pregnant rats for 21 d starting 24-48 h before parturition to ensure exposure at initiation of suckling was found to decrease body weights of dams and pups at the highest test concentration (-15% and -20% compared to standard diet containing 1 g choline/kg diet for dams and pups, respectively) but had no effect on the different immune cell phenotypes in the spleen and only a minimal effect on maternal immune cell function at the highest dose level. Noticeably, this study found that choline is required to maintain maternal immune function in lactating dams.

Executive summary:

The present study was designed to determine the effects of varying intakes of choline on maternal immune function during lactation.

Primiparous Sprague–Dawley rats (n 42) were randomised 24-48 h before birth and fed the following diets for 21 d: choline-devoid (0 g choline/kg diet; D, n=10); 1.0 g choline/kg diet (C1, n=11); 2.5 g choline/kg diet (C2·5, n=10); 6.2 g choline/kg diet (C6, n=11).

There was a linear relationship between free choline concentration in pup stomach contents with maternal dietary choline content. Compared with C1 and C2.5, D spleens had a lower proportion of mature T cells and activated suppressor cells, and this resulted in reduced cytokine production after stimulation. Feeding 6.2 g choline/kg diet resulted in a higher cytokine production after stimulation with CD3/CD28. Except for a higher IL-6 production after LPS stimulation with cells from the C2.5 dams, there were no differences between the C1 and C2.5 dams.

Oral administration of choline at test concentrations up to 6.2 g choline/kg diet, corresponding to 1240 mg choline/kg bw/day, to pregnant rats for 21 d starting 24-48 h before parturition to ensure exposure at initiation of suckling was found to decrease body weights of dams and pups at the highest test concentration (-15% and -20% compared to standard diet containing 1 g choline/kg diet for dams and pups, respectively) but had no effect on the different immune cell phenotypes in the spleen and only a minimal effect on maternal immune cell function at the highest dose level. Noticeably, this study found that choline is required to maintain maternal immune function in lactating dams.