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EC number: 204-625-1 | CAS number: 123-41-1
Toxicological information
Developmental toxicity / teratogenicity
Currently viewing:
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, conducted on the read-across substance Choline chloride
Cross-reference
- Reason / purpose for cross-reference:
- reference to same study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 1 966
- 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
- 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
Constituent 1
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 were 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 / concentrationsopen allclose all
- Dose / conc.:
- 1 other: %
- Remarks:
- Basis: nominal conc.
- Dose / conc.:
- 2.5 other: %
- Remarks:
- Basis: nominal conc.
- Dose / conc.:
- 5 other: %
- Remarks:
- Basis: nominal conc.
- 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
General toxicity (maternal animals)
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- 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.
Maternal developmental toxicity
- Number of abortions:
- effects observed, treatment-related
- Description (incidence and severity):
- 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).
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 with 1 %(= 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. - Pre- and post-implantation loss:
- effects observed, treatment-related
- Description (incidence and severity):
- 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).
- Total litter losses by resorption:
- no effects observed
- Early or late resorptions:
- no effects observed
- Dead fetuses:
- effects observed, treatment-related
- Description (incidence and severity):
- 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).
- Changes in pregnancy duration:
- effects observed, treatment-related
- Description (incidence and severity):
- 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).
- Changes in number of pregnant:
- no effects observed
- 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)
open allclose all
- Dose descriptor:
- NOAEL
- Effect level:
- 4 160 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: developmental toxicity
- Dose descriptor:
- NOAEL
- Effect level:
- 3 610 mg/kg bw/day (nominal)
- Based on:
- other: choline hydroxide
- Basis for effect level:
- other: developmental toxicity
- Dose descriptor:
- NOEL
- Effect level:
- 10 000 ppm (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: developmental toxicity
- Dose descriptor:
- NOEL
- Effect level:
- 1 250 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: developmental toxicity
- Dose descriptor:
- NOEL
- Effect level:
- 1 085 mg/kg bw/day (nominal)
- Based on:
- other: choline hydroxide
- Basis for effect level:
- other: developmental toxicity
- Dose descriptor:
- NOEL
- Effect level:
- 1 250 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Dose descriptor:
- NOEL
- Effect level:
- 1 085 mg/kg bw/day (nominal)
- Based on:
- other: choline hydroxide
- Basis for effect level:
- other: maternal toxicity
- Dose descriptor:
- LOAEL
- Effect level:
- 4 160 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: other:
- Dose descriptor:
- LOAEL
- Effect level:
- 3 610 mg/kg bw/day (nominal)
- Based on:
- other: choline hydroxide
- Basis for effect level:
- other: other:
- Dose descriptor:
- LOAEL
- Effect level:
- 10 800 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: developmental toxicity
- Dose descriptor:
- LOAEL
- Effect level:
- 9 373 mg/kg bw/day (nominal)
- Based on:
- other: choline hydroxide
- Basis for effect level:
- other: developmental toxicity
Results (fetuses)
- Reduction in number of live offspring:
- effects observed, treatment-related
- Description (incidence and severity):
- 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).
- 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)
open allclose all
- 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
- Dose descriptor:
- NOAEL
- Effect level:
- 8 678 ppm (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: Recalculated from NOAEL of choline chloride, regarding the molecular weight of both substances. Basis for effect level: 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.
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.
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).
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).
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, which was performed on the read-across substance choline chloride. Hence, the results can be considered as reliable and be used for the assessment of possible developmentally toxic effects.
The read-across from Choline chloride to Choline base is justified because the absorption after oral application is very likely to have remained unchanged, information gained from choline chloride for this endpoint can be used without modification. This is due to the fact that, if ingested orally, the contact time of the basic solution to the oesophagus is rather short for causing severe chemical burns which will be necessary to enlarge the oral uptake. Once reaching the stomach, the basic pH of the choline base solution will be immediately neutralized by the gastric acid. Only when ingesting large amounts of choline base, the neutralization capacity of the stomach acid will be used up. However, this scenario is unlikely due to expected pain in the oral cavity and pharynx caused by hydroxide. Also, in case it would have been decided to neutralize the test compound in order to avoid diminished food intake due to the undesired taste and pain, chlorous acid would be the recommended one, resulting in choline chloride anyway.
Hence, only effects of the choline cation have to be regarded and the results gained from choline chloride can be used without modifications.
The feeding of choline chloride over the entire period of gestation, in concentrations of 2.5 %, 5 % and 10 % (25 000 ppm to 100000 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 4160 mg/kg bw/d choline chloride (2.5 % in feed, corresponding to 3610 mg/kg bw/day choline hydroxide) and to take this dose level for further risk assessment, was because the IUCLID 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 and hence choline hydroxide does not need to be classified as toxic to reproduction according to Regulation 1272/2008/EC. - 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 od 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 with 1 % (= 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 4160 mg/kg bw/d (2.5 % in feed) and to take this dose level for further risk assessment, was because the IUCLID 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 according to Regulation 1272/2008/EC.
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