Choline chloride

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1966

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: well documented study result, which meets basic scientific principles

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1966

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: well documented study result, which meets basic scientific principles

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

In the range-finding studies the animals (7 or 10 pregnant mice, gestation day 11 -15) were treated via daily injections with 160 mg/kg bw or 50 mg/kg bw intraperitoneally for a total of 5 times (aqueous solution).

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

NMRI

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Ivanova Kisslegg, Germany

For the investigations, NMRI mice of the company Ivanova Kisslegg in Germany, were used. Their fertility, rate of spontaneous foetal resorptions and rate of anomalies are known to us through extensive testing. The methodology corresponded to the one described in our previous reports.

Route of administration:

intraperitoneal

Vehicle:

other: the test material was administered as an aqueous solution

Details on exposure:

In the range-finding studies the animals (7 or 10 pregnant mice, gestation day 11 -15) were treated via daily injections with 160 mg/kg bw or 50 mg/kg bw intraperitoneally for a total of 5 times (aqueous solution).

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

no data

Duration of treatment / exposure:

between the 11th and 15.th day of gestation

Frequency of treatment:

once daily

Duration of test:

5 days, subsequent observation period at least up to the end of pregnancy

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

160 mg/kg bw/day (nominal)

No. of animals per sex per dose:

7 -10

Control animals:

yes, historical

Maternal examinations:

no data

Ovaries and uterine content:

no data

Fetal examinations:

mean number of offsprings, the mean foetal body weight, mean foetal length, the foetal resorption rate and the number of anomalies

Details on maternal toxic effects:

Maternal toxic effects:no data

Details on maternal toxic effects:
no details given / no adverse effects denoted

Dose descriptor:

NOEL

Effect level:

160 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOEL

Effect level:

800 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
After daily intraperitoneal injections of choline chloride on days 11 -15 of pregnacy the foetal development of mice was not influenced.
The mean number of offsprings, the mean foetal body weight, mean foetal length, the foetal resorption rate and the number of anomalies were within the range reported for the control animals.

Dose descriptor:

NOEL

Effect level:

160 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Dose descriptor:

NOEL

Effect level:

800 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1
Testing of Choline chloride for teratogenic effects in mice (injection-experiments) - 5 * 1/2 or 1/5 LD50/kg i.p. on day 11-15 of gestation
Dosis	Number of pregnant mice		mean fetus -			Foetal resportion		Number of fetus with anomalies / total number of living fetus
mg/kg	with fetus with anomalies	total	number	weight (g)	-length (cm)	absolute	%
5 x 60	3	10	9,8	1,2	2,3	5	4,8	4/98 (cleft palate)
5 x 160	1	7	9,7	1,2	2,2	8	10,5	1/68 (cleft palate)
control mice without treatment
0	50	414	9,5	1,3	2,2	343	7,99	40/3918 (cleft palate)
								6/3918 (exencephaly)
								2/3918 (mikrocephaly)
								1/3918 (mikrognathie)
								4/3918 (hypo- or -aplasia of throcic vertebra)
								1/3918 (aplasia of vertebra of ripps)
								6/3918 (rib-anomalies)

Conclusions:

The present study was well-documented and meets general scientific principles, and hence was classified as reliable with restrictions. So, the gained results can be considered as reliable and used for further assessment.
In the present range-finding study for a feeding study (Key study, “Developmental toxicity / teratogenicity - BASF, 1966 - mice - feeding experiments”), pregnant mice were injected daily on five consecutive days doses up to 160 mg/kg bw, which corresponds to 0.5 times the LD50(i.p.) of Choline chloride. The application days were chosen based on preliminary experiments showing that here foetal development was influenced most by the application of certain substances. Consequently, it was assured that by the study design every possible adverse effects of Choline chloride on the development of mice could be detected. In the present study no effect at all was detected on foetal development, i.e. on the observed parameters which are: mean number of offsprings, the mean foetal body weight, mean foetal length, the foetal resorption rate and the number of anomalies. So, Choline chloride does not need to be regarded as developmentally toxic. Additionally, the applied dose is very high, too, i.e. 0.5 times the LD50(i.p.) per day or 2.5 times the LD50(i.p.) over 5 consecutive days. So, it would be also very likely that any effects on the observed parameters can be attributed to maternal toxicity, for example the average number of foetuses due to abortion.
Although the route of application is not relevant for humans, it assesses the possible effects of the pure, mainly unmetabolized choline, as it would result from high oral dose of choline when intestinal and liver metabolism is saturated. Additionally, the effects of metabolized choline will be assessed during the main feeding study.
Therefore, in summary, the results derived in this study do not trigger any need for classification of Choline chloride as toxic to reproduction.

Executive summary:

In the range-finding studies (BASF, 1966) for the subsequent feeding experiments, NMRI mice (7 or 10 pregnant mice, gestation day 11 -15) were treated via daily injections with 160 mg/kg bw or 50 mg/kg bw intraperitoneally for a total of 5 times (aqueous solution).

After daily intraperitoneal injections of Choline chloride on days 11 -15 of pregnacy the foetal development of mice was not influenced.

The mean number of offsprings, the mean foetal body weight, mean foetal length, the foetal resorption rate and the number of anomalies were within the range reported for the control animals.

The study was classified as reliable with restrictions and meets general scientific principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1966

Report date:

1966

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

In the feeding studies the animals (16 / 12 / 11 or 7 pregnant mice, gestation day 1 -18) were treated daily via the diet with Choline chloride (1 %, 2.5 %, 5 % and 10 % in feed). The rats ingested daily approximately 5 gr of food. On gestation day 19 all animals were subjected to necropsy and the uteri and fetuses were examined.

GLP compliance:

not specified

Remarks:

study was performed prior to implementation of GLP

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

Molecular formula:

C5H14NO.Cl

IUPAC Name:

2-hydroxy-N,N,N-trimethylethanaminium chloride

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): N-Trimethyl-ß-hydroxyäthyl-ammoniumchlorid

Test animals

Species:

mouse

Strain:

NMRI

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Ivanova Kisslegg, Germany
- Housing: single in glasses
- Diet (e.g. ad libitum): every second day one piece of bread per animal per day. Thus about 5 grams of food were taken in.
- Water (e.g. ad libitum): ad libitum (out of drinking bottles)
For the investigations, NMRI mice of the company Ivanova Kisslegg in Germany, were used. Their fertility, rate of spontaneous foetal resorptions and rate of anomalies are known through extensive testing. The methodology corresponded to the one described in the laboratories previous reports.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

Feeding trials:
Food preparation:
For the preparation of feed with 1%, 2.5%, 5% and 10% of Choline chloride (= 10,000 ppm - 100,000 ppm), 5 g, 12.5 g, 25 g or 50 g of Choline chloride were finely distributed in 300 mL of a 1% aqueous traganth suspension in the Ultra-Turrex, then finely ground with 500 g of rats bread (Lab Blox from Allied Mills, Chicago) in the Star-mix and mixed with a special machine, divided in 50 approximately equal pieces and dried for 14 - 15 hours at +80 ° C. The pieces of bread weighed 9.5 to 11 g.

Experiment:
The choline containing bread was given to all experimental animals (pregnant mice from 1 - 18 day of gestation). The number of pregnant mice in the individual test groups was in the "1% - group" 16, in the "2, 5% group" 12, in the "5% group" ~ 11, and the "10% - group "7 animals.
All mice were housed singly in glases and provided with 1 piece of bread every second day and water ad libitum. Of the bread thus were taken in about 5 grams of food per day. On gestation day 19, all animals were sacrificed and the uteri and fetuses wer e examined.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

no data

Duration of treatment / exposure:

between the 1st and 18th day of gestation

Frequency of treatment:

via the feed (rat bread), the feed for 2 days was given at once every second days

Duration of test:

19 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 other: %

Remarks:

Basis: nominal conc.

Doses / concentrations 2

Dose / conc.:

2.5 other: %

Remarks:

Basis: nominal conc.

Doses / concentrations 3

Dose / conc.:

5 other: %

Remarks:

Basis: nominal conc.

Doses / concentrations 4

Dose / conc.:

10 other: %

Remarks:

Basis: nominal conc.

No. of animals per sex per dose:

1 % in rat bread - 16 animals
2.5 % in rat bread - 12 animals
5 % in rat bread - 11 animals
10 % in rat bread - 7 animals

Control animals:

yes, historical

Examinations

Maternal examinations:

On the 19th day of gestation, all animals were sacrificed and the uteri and fetuses examined in the manner described earlier.

Ovaries and uterine content:

On the 19th day of gestation, all animals were sacrificed and the uteri and fetuses examined in the manner described earlier.

Fetal examinations:

mean number of offsprings, the mean foetal body weight, mean foetal length, the foetal resportion rate and the number of anomalies

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Decrease in body weight gain with increasing dose of Choline chloride, see table 2.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

4 160 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

2

Dose descriptor:

NOEL

Effect level:

10 000 ppm (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

3

Dose descriptor:

NOEL

Effect level:

1 250 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

4

Dose descriptor:

NOEL

Effect level:

1 250 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

5

Dose descriptor:

LOAEL

Effect level:

4 160 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: other:

6

Dose descriptor:

LOAEL

Effect level:

10 800 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes.
Remark: Observed adverse effects are not related to teratogenic effects of Choline chloride but maternal toxicity.

Details on embryotoxic / teratogenic effects:
See table 3

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

10 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The administration of food with a content of 1% (=10,000 ppm) Choline chloride did not affect the development of the offsprings

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Result:
The amount of Choline chloride uptake is given per animal and day; per kg mouse/day and within the whole time of experimental treatment as well as per kg intake of Choline chloride in table 1.

Table 1
Amount of Choline chloride uptake
Choline chloride concentration in feed (%)	mean body weight during experiment	mean amount of Choline chloride uptaken
		per day		total
		per mouse	per kg mouse	per mouse	per kg mouse
1,0 %	40 g	0,05 g	1,25 g	0,90 g	22,5 g
2,5 %	30 g	0,125 g	4,16 g	2,25 g	74,8 g
5,0 %	30 g	0,250 g	10,80 g	4,50 g	194,4 g
10,0 %	25 g	0,5 g	20,00 g	9,00 g	360,0 g

The average body weight gain between day 1.-19. of gestation was in the "1%" Choline chloride group 25.2 g. The average body weight gain was reduced in a dose-dependent manner with the increased Choline chloride concentration. The reduced body weight gain in mice, as shown in table 2, is only partly due to the abortion, because in the "2.5 -group", and even more in the "5%group" dams, which did not abort, had a significantly lower body weight gain, as the mice which received the feed with 1% Choline chloride.

Table 2
mean body weight gain of pregnant mice from day 1-19 of gestation
Choline concentration in feed (%)	number of pregnant mice	mean body weiht gain (g)
1%=  10,000 ppm	total 16	+ 25,2
	without abortion 16	+ 25,2
	with abortion 0	-
2,5% =  25,000 ppm	total 12	+ 11,9
	without abortion 8	+ 16,6
	with abortion 4	+ 2,7
5 % = 50,000 ppm	total 11	+ 3,7
	without abortion 3	+ 12,6
	with abortion 8	+ 0,2
10 % = 100,000 ppm	total 7	- 5,2
	with abortion 7	- 5,2

In all 7 animals receiving the Choline chloride 10% solution (= 100,000 ppm) in the diet and in 8 of 11 mice receiving the bread with 5% (=50,000 ppm) it resulted in expulsion of all fetuses, so that in these animals after the death at the 19th day of gestation in the uterus only the implantation sites were detected. Even in the mice fed 2.5 % Choline chloride, 4 of the 12 mice aborted. Moreover, the 32 surviving fetuses of the " 5% group" were well behind in development. The administration of food with a content of 1% (=10,000 ppm) Choline chloride did not affect the development of the offsprings (Table 3).

Table 3
Testing of Choline chloride for teratogenic effects in mice (feeding-experiments) - on day 1-18 of gestation
Choline chloride concentration in feed (%)	number of pregnant mice		mean fetus -			Foetal resportion		Abortions		Number of fetus with anomalies / total number of living fetus
	with fetus with anomalies	total	number	weight (g)	length (cm)	absolute	%	absolute	%
1 % =  10,000 ppm	3	16	10,3	1,4	2,4	7	4,0		-	3/166 (2 cleft palate 1 confused ribs)
2,5 % =  25,000 ppm	0	12	5,8	1,2	2,2	4	3,6	39	34,8	0/69
5 % s»  50,000 ppm	1	11	2,9	o,9	2,0	2	1,8	77	69,4	1/32 (confused ribs)
10 % = 100,000 ppm	0	7	-	-	=	-		68	100,0	-
control mice without treatment
0 %	50	414	9,5	1,3	2,2	343	7,99	12	0,28	40/3918 (cleft palate)
										6/3918 (exencephaly)
										2/3918 (mikrocephaly)
										1/ " " (mikrognathie)
										4/ " " (hypo- or -aplasia of throcic vertebra)
										1/ " " (aplasia of vertebra of ripps)
										6/ " " (ripp-anomalies)

Evaluation of results:
Even after 5 repeated intraperitoneal injections, Choline chloride (in a dose corresponding to half LD50/kg) on gestation days 11 -15 did not affect of foetal development of NMRI mice. The feeding of Choline chloride over the entire period of gestation, in concentrations of 2.5%, 5% and 10% (25,000ppm to 100,000 ppm) on the other hand, resulted in expulstion of all fetuses and thus to complete abortion in the majority of animals. This "abortion effect"' is not an expression of a specific embryotoxic toxicity but sign of a general toxicity, because not only the mice with abortion but also the animals without abortion had a significantly reduced body weight than any untreated pregnant mice. This thesis - the lack of a specific embryotoxic effect of Choline chloride - is also supported by the fact that the gavage of food with1% (= 10,000 ppm) Choline chloride over the entire period of gestation was well tolerated by the dams without symptoms and the foetal development was not disturbed.
Choline chloride thus had under the given experimental conditions, no teratogenic effect, since the product caused only altered development in the foetuses at doses, which also had toxic effects on the dams.

Applicant's summary and conclusion

Conclusions:

The study was classified as reliable with restrictions (Klimisch 2) and meets the requirements for a developmental toxicity study. Hence, the results can be considered as reliable and be used for the assessment of possible developmentally toxic effects.
The feeding of Choline chloride over the entire period of gestation, in concentrations of 2.5%, 5% and 10% (25,000ppm to 100,000 ppm), resulted in expulsion of all fetuses and thus to complete abortion in the majority of animals, beginning with 34.8% abortions (4,160 mg/kg bw/d) to complete loss of all fetuses (20,000 mg/kg bw/d). This "abortion effect"' is not an expression of a specific embryotoxic toxicity but sign of a general toxicity, because not only the mice with abortion but also the animals without abortion had a significantly reduced body weight compared to untreated pregnant mice. This thesis - the lack of a specific embryotoxic effect of Choline chloride - is also supported by the fact that the gavage of food with 1%(= 10,000 ppm = 1,250 mg/kg bw/d) Choline chloride over the entire period of gestation was well tolerated by the dams without symptoms and the fetal development was not disturbed.
The only reason to determine the NOAEL to be 4,160 mg/kg bw/d (2.5% in feed) and to take this dose level for further risk assessment, was because the IUCLID5 software requires a numeric value and the first effects on the fetal development were seen in the next higher dose (5% in feed), although they are not related to possible developmentally toxic effects of the compound itself but to maternal toxicity.
Choline chloride thus had under the given experimental conditions no teratogenic effect, since the product caused only altered development in the fetuses at doses, which also had toxic effects on the dams.
As a consequence, Choline chloride does not need to be classified as toxic to reproduction, neither according to Regulation 1272/2008/EC nor Directive 67/548/EEC.

Executive summary:

In the feeding studies the animals (16 / 12 / 11 or 7 pregnant mice, gestation day 1 -18) were treated daily via the diet with Choline chloride (1 %, 2.5 %, 5 % and 10 %, BASF, 1966). The choline containing bread was given to all experimental animals (pregnant mice from 1 - 18 day of gestation). The number of pregnant mice in the individual test groups was in the "1% - group" 16, in the "2, 5% group" 12, in the "5% group" ~ 11, and the "10% - group "7 animals. All mice were housed singly in glases and provided with 1 piece of bread every second day and water ad libitum. Thus about 5 grams of food per day were taken in. On gestation day 19, all animals were sacrificed and subject to necropsy and the uteri and fetuses were examined and the mean number of offsprings, the mean foetal body weight, mean foetal length, the foetal resportion rate and the number of anomalies were noted.

The average body weight gain between day 1.-19. of gestation was in the "1%" Choline chloride group 25.2 g. The average body weight gain was reduced in a dose-dependent manner with the increased Choline chloride concentration. The reduced body weight gain in mice, was only partly due to the abortion, because in the "2.5 -group", and even more in the "5%group" dams, which did not abort, had a significantly lower body weight gain, as the mice which received the feed with 1% Choline chloride.

In all 7 animals receiving the Choline chloride 10% solution (= 100,000 ppm) in the diet and in 8 of 11 mice receiving the bread with 5% (= 50,000 ppm) it resulted in expulsion of all fetuses,so that in these animals after the death at the 19th day of gestation in the uterus only the implantation sites were detected. Even in the mice fed 2.5 % Choline chloride, 4 of the 12 mice aborted. Moreover, the 32 surviving fetuses of the "5% group" were well behind in development. The administration of food with a content of 1% (=10,000 ppm) Choline chloride did not affect the development of the offsprings.

The feeding of Choline chloride over the entire period of gestation, in concentrations of 2.5%, 5% and 10% (25,000 ppm to 100,000 ppm), resulted in expulsion of all fetuses and thus to complete abortion in the majority of animals. This "abortion effect" 'is not an expression of a specific embryotoxic toxicity but sign of a general toxicity, because not only the mice with abortion but also the animals without abortion had a significantly reduced body weight than any untreated pregnant mice. This thesis - the lack of a specific embryotoxic effect of Choline chloride - is also supported by the fact that the gavage of food with1%(= 10,000 ppm) Choline chloride over the entire period of gestation was well tolerated by the dams without symptoms and the foetal development was not disturbed. The only reason to determine the NOAEL to be 4,160 mg/kg bw/d (2.5% in feed) and to take this dose level for further risk assessment, was because the IUCLID5 software requires a numeric value and the first effects on the fetal development were seen in the next higher dose (5% in feed), although they are not related to possible developmentally toxic effects of the compound itself but to maternal toxicity.
Choline chloride thus had under the given experimental conditions, no teratogenic effect, since the product caused only altered development in the foetuses at doses, which also had toxic effects on the dams. The study was classified as reliable with restrictions and meets the requirements for a developmental toxicity study. Choline chloride does not need to be classified as toxic to reproduction, neither according to Regulation 1272/2008/EC nor Directive 67/548/EEC.