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Toxicological information

Toxicity to reproduction: other studies

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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

Constituent 1
Chemical structure
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 / concentrationsopen allclose all
Dose / conc.:
0 other: g/kg diet
Remarks:
D
Dose / conc.:
1 other: g/kg diet
Remarks:
C1, corresponding to 200 mg choline/kg bw/day
Dose / conc.:
2.5 other: g/kg diet
Remarks:
C2.5, corresponding to 500 mg choline/kg bw/day
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.