Registration Dossier

Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Accepted, well-documented publication which meets basic scientific principles

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1978

Materials and methods

Test guideline
Qualifier:
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 (MME). 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:
no
Type of method:
in vivo

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
no data

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals 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
Vehicle:
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% MME (MME diet); (c) CD diet supplemented with 1.0% DME (DME 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
Remarks:
Doses / Concentrations:
1%
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 behavior
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.
Statistics:
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:
NOAEL
Basis for effect level:
other: 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 DME and MME diets, resp.
Remarks on result:
not determinable
Remarks:
no NOAEL identified

Observed effects

Levels of Ch and acetylcholine (ACh) were elevated in the brain of pups born of dams fed the MME and DME 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 gainof 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 DME groups of dams was the same, while the intake of the MME 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 MME 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 DME 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 I5 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 DME and MME diets, respectively.

Histopathology

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 MME and DME diet, glycogen and fatty infiltrations of moderate degrees were present.

Brain and liver weight

As seen in Table 2, apart from the MME 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 DME, Ch and ACh

As shown in Fig. 1 (attached document), day-old MME- and DMEexposed pups had measurable amounts of DME in their brains (11.7±1.8 and 72.7±12.7nmol/g, respectively), while DME 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 MME-exposed and 53% in the DME-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 MME-exposed pups, and by 36% in the DME-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 MME and DME exposed groups of pups.

Figure 2 shows the concentrations of DME, 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 DME, 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 DME, 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, DME was present in the brain tissues of both the MME and DME groups of dams but not in the CD or CS groups. The concentration of DME in the brain areas of the DME-treated dams was, however, approx 30-fold higher than that of the MME-treated dams. Unlike the whole brain Ch levels in the pups, the Ch content in both brain areas of the CS and MME groups of dams was not different from that of the CD group. The Ch concentration in the DME-treated dams brain areas could not accurately be determined because of methodologic complications in measuring Ch content in the presence of tissue concentrations of DME 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, MME and DME 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 MME or DME 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 PMME and PDME were present in the brain phospholipids of the MME-exposed pups and PDME in the DME-exposed pups. Other phospholipids examined were equally distributed in the various groups of pups.

Applicant's summary and conclusion

Conclusions:
The inclusion of 1% MME or DME 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 (MME) or I % N,N-dimethylaminoethanol (DME). Gestation and parturition of the pregnant rats proceeded normally. However, all the pups born of dams fed the MME diet, and most of those born of dams fed the DME 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 MME and DME 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 MME- and DMEexposed pups were markedly reduced. At the same time, significant amounts of DME, phosphatidyl-Nmonomethylaminoethanol (PMME) and of phosphatidyl-N,N-dimethylaminoethanol( PDME) were present in the same brain areas."

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