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toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
Pregnant rats were fed for 15 days predelivery until 15 days postpartum a choline (Ch)-deficient diet (CD diet) or a CD diet supplemented with 0.8 % Ch-CI (CS) or 1 % N-methylaminoethanol (MMAE). Brain of rats was examined histologically and cytologically. Levels of choline and acetylcholine (ACh) and total phospholipids were measured in the brain of pups.
GLP compliance:
Type of method:
in vivo

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
no data

Test animals

Details on test animals or test system and environmental conditions:
Female rats of the Sprague-Dawley strain at 3rd day of pregnancy were obtained from Zivic- Miller Laboratories (Allison Park, PA.). The animals were placed in plastic cages, one per cage, and housed in an animal room with controlled temperature and humidity.

Administration / exposure

Route of administration:
oral: feed
unchanged (no vehicle)
Details on exposure:
Starting at the 6th day of pregnancy, the rats were randomly divided into four separate groups and were fed one of the following diets: (a) CD diet; (b) CD diet supplemented with 1.0% MMEA (MMEA diet); (c) CD diet supplemented with 1.0% DMAE (DMAE diet); and (d) CD diet supplemented with an adequate (0.8%) level of ChCI. In other experiments, as specified in the text, an additional group of pregnant rats was fed laboratory chow (Purina, Ralston Purina Co., St. Louis, MO).
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
15 days predelivery until 15 days postpartum
Frequency of treatment:
in diet (assessed by measurement of food consumption per day)
Duration of test:
not applicable
Doses / concentrations
Dose / conc.:
1 other: %
Basis: nominal in diet
No. of animals per sex per dose:
no data
Control animals:
yes, plain diet
Details on study design:
Food consumption was measured daily, and body weight twice a week.
After parturition, the number of pups in each litter, their body weights and behaviour were recorded. Pups used for neurochemical and morphologic studies were killed within 24 h after birth.
Dams were fed the diets for an additional 15 days after parturition at which time they were killed. For determination of survival rates, pups were left to be nursed by the respective dams for up to 15 days.
An analysis of variance was applied to the experimental data. If the overall difference among the groups was found to be significant (P < 0.05), differences between the means were then checked with Student t-test and regarded to be significant if P < 0.05. CD treated animals served as controls for the other groups.

Results and discussion

Effect levels

Dose descriptor:
Basis for effect level:
other: please refer to 'Remarks'
Remarks on result:
not determinable
no NOAEL identified

Observed effects

Levels of Ch and acetylcholine (ACh) were elevated in the brain of pups born of dams fed the MMAE and DMAE diets. The phosphatidyl-Ch and phosphatidylaminoethanol (PAE) contents in the brain of the MME- and DMEexposed pups were markedly reduced.

Any other information on results incl. tables

The daily food intake and body weight gain of the pregnant rats fed the various diets, and the rate of survival of newborn pups are shown in Table 1. The amount of food ingested by the CD, CS and DMAE groups of dams was the same, while the intake of the MMAE group was only about 40% of that of the others. The daily gain in body weight of the dams reflected this intake of the diets. Gestation proceeded normally in all the pregnant rats. Litters of approximately equal size (average of 12) were delivered by the dams, but the average body weight of the pups born of the MMAE group of dams was significantly lower than that of the other litters. It is worth noting that the gain in body weight of dams fed the CD, CS and DMAE diets, as well as the body weight of their respective pups, were comparable to those of dams, and their pups, fed laboratory chow (LC). It is apparent, therefore, that the synthetic diets are fairly adequate even though they lack Ch, and contain only marginal amounts of methionine. All the pups delivered by the dams fed the LC, CD and CS diets survived for more than 15 days. In contrast, only 18 out of 253, and none out of 120, survived for more than 36 h in the groups of pups delivered by mothers fed the DMAE and MMAE diets, respectively.


No significant histological or cytological abnormalities were seen in the brains of pups born of dams fed the various diets. Heavy deposits of glycogen were seen in the livers of pups born of dams fed the CS diet. In the liver of pups born of dams fed the MMAE and DMAE diet, glycogen and fatty infiltrations of moderate degrees were present.

Brain and liver weight

As seen in Table 2, apart from the MMAE pups, the weights of liver and brain in all the other dietary categories of pups were quite similar within one representative experimental group of animals.

Brain and liver DMAE, Ch and ACh

As shown in Fig. 1 (attached document), day-old MMAE- and DMAE exposed pups had measurable amounts of DMAE in their brains (11.7±1.8 and 72.7±12.7nmol/g, respectively), while DMAE was not detectable in the brains of pups born of dams fed the CD and CS diets. The brain level of Ch was elevated 43% in the MMAE-exposed and 53% in the DMAE-exposed pups when compared with that in pups born of dams fed the CD diet. Levels of ACh in the brain were also increased by 27% in the MMAE-exposed pups, and by 36% in the DMAE-exposed pups. Concentrations of Ch and ACh in the brains of the day-old pups born of dams fed the CS diet, while elevated, did not differ significantly from those of the pups in the CD group of dams (t = 1.4839, df = 10, P > 0.2) but Ch levels were significantly lower than those of pups in the MMEA and DMAE exposed groups of pups.

Figure 2 shows the concentrations of DMAE, Ch and ACh in the livers of the pups. From a comparison of Figs. 1 and 2, it is apparent that the four diets led to liver profiles of DMAE, Ch, and ACh qualitatively similar to those in the brain. However, there was one quantitative difference. In the pups of the CS group of dams, the liver content of Ch was significantly higher than that in the pups of the CD group, whereas the brain Ch content was not.

The concentrations of DMAE, Ch and ACh, which were measured separately in the cortex and striatum of the dams, are shown in Table 3. As was the case with the pups, DMAE was present in the brain tissues of both the MMAE and DMAE groups of dams but not in the CD or CS groups. The concentration of DMAE in the brain areas of the DMAE-treated dams was, however, approx 30-fold higher than that of the MMAE-treated dams. Unlike the whole brain Ch levels in the pups, the Ch content in both brain areas of the CS and MMAE groups of dams was not different from that of the CD group. The Ch concentration in the DMAE-treated dams brain areas could not accurately be determined because of methodologic complications in measuring Ch content in the presence of tissue concentrations of DMAE 10-fold greater than those of Ch (for discussion see Zahniser et al., 19776). Also, in contrast with the results of the pup brain ACh determinations, the cortical and striatal levels of ACh in the CS, MMEA and DMAE groups of dams were similar when compared with those of the CD group.

Brain phospholipids

Table 4 shows that there was no striking difference between the various groups of pups in either the concentration or the content of brain total phospholipids. The concentration of total phospholipid in brains of the CS group of pups was significantly lower (P<0.05) than that in the CD pups, but this difference amounted to only 11%. Significant differences did, however, exist in the relative content of the individual phospholipids. The contents of sphingomyelin and phosphatidic acids in pups of dams fed the CD diet were lower than those in pups born of mothers fed the other diets. In pups of mothers fed the MMEA or DMAE diets, the contents of phosphatidyl-Ch and phosphatidylaminoethanol (PAE) were markedly lower than those in pups delivered by dams fed either the CD or CS diets. Large amounts of PMMEA and PDMAE were present in the brain phospholipids of the MME-exposed pups and PDMAE in the DMAE-exposed pups. Other phospholipids examined were equally distributed in the various groups of pups.

Applicant's summary and conclusion

The inclusion of 1% MMAE or DMAE in the CD diet of dams limits the survival of pups after birth, most likely by interfering with the development or maturation of one or more vital systems of the fetuses.
Executive summary:

" Pregnant rats were fed for 15 days predelivery until 15 days postpartum a choline (Ch)-deficient diet (CD diet) or a CD diet supplemented with 0.8% Ch-CI (CS), 1% N-methylaminoethanol (MMAE) or 1 % N,N-dimethylaminoethanol (DMAE). Gestation and parturition of the pregnant rats proceeded normally. However, all the pups born of dams fed the MMAE diet, and most of those born of dams fed the DMAE diet, died within 36 h of birth. No histological or cytological alterations were detected in the brain of the pups. Levels of Ch and acetylcholine (ACh) were elevated in the brain of pups born of dams fed the MMAE and DMAE diets, but not the CS diet. The content of total phospholipids in the brain of the pups was not altered by the diet fed to the dams. However, the phosphatidyl-Ch and phosphatidylaminoethanol (PAE) contents in the brain of the MMAE- and DMAE exposed pups were markedly reduced. At the same time, significant amounts of DMAE, phosphatidyl-N-monomethylaminoethanol (PMMAE) and of phosphatidyl-N,N-dimethylaminoethanol( PDMAE) were present in the same brain areas."

"It has been concluded that, in the presence of a dietary deficiency of Ch, MMAE increases the demand for methyl groups and is thus more toxic than DMAE which is already two-thirds methylated. Our observations (Table 1) support this conclusion in as much as supplementation of the CD diet with 1% MMEA, but not with 1% DMAE, appears to compromise the growth of the dams as well, and indicates that availability of methyl groups may be the critical factor."