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Key value for chemical safety assessment

Effects on fertility

Description of key information

See "Toxicity to reproduction": The available study was performed with intraperitoneal application.

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction
Remarks:
other: one-generation study on fertility
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The endpoint addressed in this study, i.e. spermatogenesis in the male rat, does not cover completely all possible reasons for toxicity to reproduction. However, the given data indicate that the study was well-performed and meets scientific principles.
Qualifier:
no guideline available
Principles of method if other than guideline:
Male rats were injected i.p. daily over 12 or 24 days with the test item. After sacrifice, testis were weighed, fixed and examined histopathologically for the stages of spermatogenesis.
GLP compliance:
not specified
Limit test:
yes
Species:
rat
Strain:
not specified
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: animal house of the Industrial Toxicology Research Centre
- Age at study initiation: adult
- Weight at study initiation: 300 g
- Fasting period before study: no data
- Housing: in plastic cages in standard conditions of husbandry
- Use of restrainers for preventing ingestion (if dermal): not applicable
- Diet (e.g. ad libitum): standard animal feed (Lipton India) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week
Route of administration:
intraperitoneal
Vehicle:
water
Details on exposure:
intraperitoneal injection
Details on mating procedure:
not applicable
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
12 resp. 24 days
Frequency of treatment:
daily
Details on study schedule:
Animals of Group I were given ether anaesthesia and sacrificed on days 2, 5, 8, 10, and 12 after 12 days of exposure. Group II animals were similarly sacrificed on days 2, 5, 8, 10, and 12 after 24 days of exposure. Control rats were sacrificed only on day 12 after both 12- and 24-day exposure schedules.
Dose / conc.:
0 other: mg/rat/day
Remarks:
Basis: nominal injected
Dose / conc.:
25 other: mg/rat/day
Remarks:
Basis: nominal injected
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Basis: nominal injected
Dose / conc.:
83 mg/kg bw/day (nominal)
Remarks:
Basis: nominal injected
No. of animals per sex per dose:
25 males / dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: 25 mg/rat i.p. daily for 12 and 24 days. This dose was approx. 18% of the LD50 value of choline chloride in adult rats (450 mg/kg i.p.).
In earlier studies three doses of choline chloride, i.e., 8, 25, and 40 mg/rat, were used. Because the 25 mg/rat produced moderate effects, it was selected for the present studies. Further, based on the composition of the diet and average diet consumption per day/rat, 3 to 4 mg choline is ingested by a rat per day. Thus, the present dose gave a 6 to 8-fold excess availability of choline.
- Rationale for animal assignment (if not random): random
Positive control:
no data
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: no data

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): not applicable

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): not applicable
Oestrous cyclicity (parental animals):
not applicable
Sperm parameters (parental animals):
Parameters examined in males: testis weight, epididymis weight, other: Histopathological analysis
Litter observations:
not applicable
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after day 2, 5, 8, 10, 12 after exposure

HISTOPATHOLOGY / ORAGN WEIGHTS
For the qualitative histopathologic analysis, individual stages were identified and the tubules were divided into stage groups, viz., I-IV, V-VI, VII-VIII, IX-XII, and XIII-XIV (according to Leblond CP, Clermont Y. Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann NY Acad Sci. 1952;55:548-73). At least 20 tubules at each stage group (total 100 tubules) per animal were evaluated at 400 x or 1000 x magnification to analyse specific effects on testicular tissues. This included peritubular membrane status, germinal epithelial status, cytoplasmic vacuolation, cell sloughing, epithelial disorganization, cell death and cell type loss, presence of giant cells, spermatid damage, and inhibited spermiation.
The qualitative analysis, the quantitation of spermatogonia, zygotenes, and pachytenes was performed in 10 randomly selected tubules at stage XII.
Organ weights of other tissues were determined (epididymis, liver, kidney, adrenal)
Postmortem examinations (offspring):
not applicable
Statistics:
Significance for changes in data was analysed by Student t test according Snedecor GE, Cochran WH. Statistical methods. Iowa City IA: Iowa State University Press; 1967
Reproductive indices:
not applicable
Offspring viability indices:
not applicable
Clinical signs:
not specified
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The average body weight gain in control animals between days 0 and 12 following both the experimental schedules was 20.00 + 2.3 g. The weight gain in treated animals was not significantly different from control values. No data on food consumption
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The average body weight gain in control animals between days 0 and 12 following both the experimental schedules was 20.00 + 2.3 g. The weight gain in treated animals was not significantly different from control values. No data on food consumption
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
effects only transient
Other effects:
not examined
Description (incidence and severity):
Test substance intake: not applicable
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
effects only transient
Reproductive performance:
not examined
MORTALITY
No unscheduled deaths were noted.

BODY WEIGHT
The average body weight gain in control animals between days 0 and 12 following both the experimental schedules was 20.00 + 2.3 g. The weight gain in treated animals was not significantly different from control values.

REPRODUCTIVE FUNCTION: SPERM MEASURES / HISTOPATHOLOGY
12-Day choline chloride administration:
Animals administered Choline chloride for 12 days did not exhibit noticeable alteration in the organization of the germinal epithelium except on day 2, when epithelial vacuoles were seen at later stages. The nuclei of spermatogenic cells were pyknotic at these stages but not at earlier stages. The presence of cellular debris in a few tubules suggested detachment of apical cytoplasm of Sertoli cells and sloughing of spermatogenic cells. By day 5, the testis recovered from initial injuries and the epithelium showed normal architecture through day 12. The quantitation analysis showed a partial effect on both spermatogonia and primary spermatocytes (Table 2).

24-Day choline chloride adminstration
Significant changes in testicular morphology were observed in rats exposed to Choline chloride for 24 days. Prominent features observed at day 2 included disorganization of the adluminal compartment of tubules mainly beyond stage VIII. Only a few tubules at stages I-IV were damaged. At stages V-VI epithelial vacuolation was observed. The tubules at stages IX-XIII were most damaged. Blebbing of Sertoli cell apical cytoplasm and dislodging of pachytene spermatocytes were marked at these stages. The arrangement of elongating spermatid bundles was inappropriate and part of the epithelium was devoid of elongating spermatids. In the tubules at earlier stages, at day 2, a decrease or absence of round spermatids was evidence of late pachytene degeneration earlier in time. The late pachytenes were highly eosinophilic. Sloughing of cells was found in only a few tubules in Group I as compared to those in Group II, At posttreatment day 5, spermatogonia and early primary spermatocytes in almost all the tubules were normal. Several pachytenes were necrotic and the adluminal portion of such tubules showed large gaps. At stages I-IV, the population of elongated spermatids was slightly decreased. At day 8, the tubules at stages XIII-XIV also showed gaps at the expected position of the elongating spermatids. These cells were thought to have been lost due to sloughing at earlier time intervals. By day 12, most of the tubules appeared to be regenerating and contained normal spermatogenic cells except a few necrotic pachytenes at stages XI-XII. The organization of the germinal epithelium appeared normal at earlier stages. The quantitation of spermatogenesis at stage XII showed an increase in spermatogonia at posttreatment days 5, 8, 10, and 12 (Table 2). Zygotenes were not altered in comparison to control counts. The most severe adverse effect was observed in pachytenes. Maximum depletion to 64% of control counts of these cells was noted at day 2. A marked recovery in cell counts towards normal was observed at day 12.

ORGAN WEIGHTS
The testicular weights (absolute and relative) were not altered significantly following choline administration (Table 1). There were no changes in the weights of other tissues (epididymis, liver, kidney, adrenal) in the same animals. The testis from all the animals of the control group exhibited normal histoarchitecture and active spermatogenesis (Table 2).
Dose descriptor:
NOEL
Effect level:
25 other: mg/rat/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Based on mortality; body weight; organ weights; Highest dose tested.
Dose descriptor:
NOEL
Effect level:
ca. 78 - ca. 83 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Recalculated from 25 mg/rat/day with a median body weight ranging from 300-320 g. Based on mortality; body weight; organ weights Highest dose tested.
Dose descriptor:
NOAEL
Effect level:
ca. 78 - ca. 83 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'
Clinical signs:
not specified
Description (incidence and severity):
not applicable
Mortality / viability:
not specified
Description (incidence and severity):
not applicable
Body weight and weight changes:
not specified
Description (incidence and severity):
not applicable
Sexual maturation:
not specified
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
not specified
Description (incidence and severity):
not applicable
Gross pathological findings:
not specified
Description (incidence and severity):
not applicable
Histopathological findings:
not specified
Description (incidence and severity):
not applicable
not applicable
Dose descriptor:
other: not determined
Generation:
F1
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Choline chloride was administered i.p. to 25 male rats for each duration at dose levels of 0 and 25 mg/rat/rat (≙ca. 78 – 83 mg/kg bw/day) in order to assess the effects of the compound on the spermatogenesis in rats.
Remarks:
12 days of treatment showed no effects at all, i.e. did not significantly alter spermatogenesis, 24 days of treatment had only transient effects.
Reproductive effects observed:
not specified

Table 1: Testis weights of rats treated with Choline chloride

Parameter

Posttreatment day

2

5

8

10

12

12 (control)

Group I: 25 mg Choline chloride / day for 12 days

Absolute weight (g)

1.18 ± 0.03

1.30 ± 0.60

1.41 ± 0.07

1.40 ± 0.16

1.48 ± 0.03

1.36 ± 0.09

Relative weight (g)

0.66 ± 0.04

0.56 ± 0.03

0.56 ± 0.03

0.51 ± 0.01

0.51 ± 0.01

0.71 ± 0.04

Group II: 25 mg Choline chloride / day for 24 days

Absolute weight (g)

1.32 -± 0.13

1.51 ± 0.10

1.33 ± 0.05

1.41 ± 0.03

1.47 ± 0.07

1.55 ± 0.01

Relative weight (g)

0.53 ± 0.03

0.50 ± 0.01

0.48 ± 0.01

0.44 ± 0.01

0.51 ± 0.01

0.48 ± 0.01

The values are mean ± SE of 10 observations.

 

Table 2: Spermatogenic cell count at stage XII of the seminiferous epithelium following Choline chloride administration

Parameter

Posttreatment day

2

5

8

10

12

12 (control)

Group I: 25 mg Choline chloride / day for 12 days

Spermatogonia

43.23 ± 2.15

46.55±3.08

50.47±2.90

50.65 ± 3.50

51.12 ± 3.72

41.45 ± 2.05

Zygotenes

50.35±3.68

54.02±2.2

58.15±2.52

62.35 ± 3.70

65.00 ± 2.55

57.35 -+ 3.10

Pachytenes

65.55 ± 4.23

73.72±3.50

75.50 ± 3.72

75.65 ± 3.15

71.75 ± 2.60

70.62 ± 3.00

Group II: 25 mg Choline chloride / day for 24 days

Spermatogonia

50.25±3.10

54.52 ± 2.68*

52.37 ± 2.60*

55.45 ± 3.35*

53.30 ± 3.57*

42.02 ± 2.28

Zygotenes

53.23 ± 3.65

53.27±3.16

56.75±3.88

52.40 ± 2.50

53.15 ± 3.27

56.23 ± 2.68

Pachytenes

45.25 ± 4.37*

50.25±3.55*

58.60 ± 4.75

56.20 + 3.15

62.44±2.65

70.00 ± 3.20

Values are mean ± SE of 10 observations. *P < 0.05

Conclusions:
The endpoint addressed in this study, i.e. spermatogenesis in the male rat, does not cover completely all possible reasons for toxicity to reproduction. However, the given data indicate that the study was well-performed and meets scientific principles. Consequently, it was classified as Klimisch 2 and the results can be considered as reliable and, supported with data from IUCLID chapter 7.8.2, sufficient to cover this endpoint.
In this study it was examined how the intraperitoneal application of 25 mg/rat/day over 12 or 24 days influences the spermatogenesis in the rat. Although it does not cover any possible effects in the dam, only testing males gives nevertheless a good indication whether there are any effects on reproduction to be expected, as the reproductive performance is clearly dependent on a regularly functioning sperm production in the males.
The administration of 25 mg choline for 12 days did not significantly alter spermatogenesis, although treatment for 24 days increased stage XII spermatogonia count by day 5 posttreatment. Choline-induced changes were reversible and contents of epithelial germ cells reached almost normal levels. In general, Choline is present in the epididymis as glycerylphosphorylcholine, which is important for maturation of spermatozoa during their epididymal transit. Also, Choline is a constituent of cell membranes. The balance in accumulation of the methylated product (phosphatidylcholine) or the demethylated product (phosphatidylethanolamine) regulates membrane fluidity and thereby modulates the activity of membrane-bound enzymes. Hence, excess choline availability might play a role in modifying the structural and functional integrity of the Sertoli cell membrane, which has very large surface area in comparison to most other individual cells. Further, the Sertoli cell loses much of its apical membrane in carrying out its secretory functions and during processes such as sperm release. Therefore, Sertoli cells and so spermatogenesis is a very sensitive parameter for toxicity to reproduction which justifies furthermore the approach to mainly focus of the examination of possible effects of Choline only on male rats.
A second point to consider is the route of application: Choline is absorbed primarily in the jejunum and then enters the portal circulation. Some of the ingested choline is metabolized by gut bacteria to betaine and trimethylamine. Hence, the intraperitoneal route of administration was chosen to determine the worst case of possible toxic effects of pure, unmetabolized Choline chloride, and so an overestimation of the possible effects on spermatogenesis is very likely. Methylamines, resulting from intestinal metabolism, may serve as substrates for nitrosamines that have marked carcinogenic effects. Therefore, oral administration of choline, under experimental conditions, may exhibit some severe side effects due to metabolism products of choline and so a change in other biological parameters connected to reproduction may be misleadingly be attributed to choline, although being a side effect of carcinogenesis. This is avoided when choline is administered by other routes, bypassing the gut bacterial flora.
Taking into account the average body weights of the rats of 300 – 320 g, the applied amount of Choline chloride corresponds to ca. 78 – 83 mg/kg bw/day.
So, in summary, taking into account the rather high applied amount of choline, the fact that all observed effects are only transient, the sensitivity of the endpoint, and the most likely overestimation of the observed effects due to the intraperitoneal application, which is not relevant for humans, it can be clearly concluded that the administration of Choline chloride results in no adverse effects on the reproductive performance and does consequently not need to be classified as toxic to reproduction, neither according Regulation 1272/2008/EC nor Directive 67/548/EEC.
Executive summary:

In a repeated dose / reproductive toxicity study over 12 resp. 24 days with 12 days post-observation, Choline chloride was administered i.p. to 25 male rats for each duration at dose levels of 0 and 25 mg/rat/rat (ca. 78 – 83 mg/kg bw/day) in order to assess the effects of the compound on the spermatogenesis in rats.

12 days of treatment showed no effects at all, i.e. did not significantly alter spermatogenesis, the administration of the compound over 24 days had only transient effects. It depleted pachytene spermatocytes until posttreatment day 5, while slight proliferation of spermatogonia was noted from day 5 onwards. By day 12, the tubules showed almost normal cellular associations. No LOAEL could be determined up to the highest dose tested, because the transient effects are not considered adverse. The NOEL of 12 days of treatment was ca. 78 – 83 mg/kg bw/day, the NOAEL of 24 days of treatment was ca. 78 – 83 mg/kg bw/day. Choline chloride does not need to be classified as toxic to reproduction, neither according Regulation 1272/2008/EC nor Directive 67/548/EEC.

The study is acceptable to assess the possible effects of Choline chloride to reproduction and satisfies general scientific requirements.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
78 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study was classified as reliable with restrictions (Klimisch2). Although only male rats are regarded, the most sensitive endpoint (i.e. spermatogenesis) and the route of application leading to the most severe effects (intraperitoneal) were chosen. Hence, all other possible effects are supposed to occur at higher doses i.e. in females or via oral application, and the available study can serve as a surrogate for oral application testing. Since the outcome of the study was negative, first, these effects are covered within this study, too, and second, this study does not trigger any need for further testing. Hence, all the tonnage-driven data requirements under REACH are met, no data gaps were identified and hence, the database is of good quality.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

There is no reproductive toxicity study via the oral route available for Choline chloride. Hence a study with intraperitoneal applications was chosen as a surrogate, which is scientifically justified because of the following reasons: Choline is absorbed primarily in the jejunum and then enters the portal circulation. Some of the ingested choline is metabolized by gut bacteria to betaine and trimethylamine. Hence, the intraperitoneal route of administration determines the worst case of possible toxic effects of pure, unmetabolized Choline chloride, and although so an overestimation of the possible effects on spermatogenesis is very likely, it would lead to an NOAEL which already includes another safety margin.

Also, methylamines, resulting from intestinal metabolism, may serve as substrates for nitrosamines that have marked carcinogenic effects. Therefore, oral administration of choline, under experimental conditions, may exhibit some severe side effects due to metabolism products of choline and so a change in other biological parameters connected to reproduction may misleadingly be attributed to choline, although being a side effect of carcinogenesis. This is avoided when choline is administered by other routes, bypassing the gut bacterial flora.

 

The endpoint addressed in this study, i.e. spermatogenesis in the male rat, does not cover completely all possible reasons for toxicity to reproduction. However, the given data indicate that the study was well-performed and meets scientific principles. Consequently, it was classified as Klimisch 2, the results can be considered as reliable and, supported with data from IUCLID chapter 7.8.2 as discussed below, sufficient to cover this endpoint.

In this study it was examined how the intraperitoneal application of 25 mg/rat/day over 12 or 24 days influences the spermatogenesis in the rat. Although it does not cover any possible effects in the dam, only testing males gives nevertheless a good indication whether there are any effects on reproduction to be expected, as the reproductive performance is clearly dependent on a regularly functioning sperm production in the males.

The administration of 25 mg choline for 12 days did not significantly alter spermatogenesis, although treatment for 24 days increased stage XII spermatogonia count by day 5 posttreatment. Choline-induced changes were reversible and contents of epithelial germ cells reached almost normal levels. In general, Choline is present in the epididymis as glycerylphosphorylcholine, which is important for maturation of spermatozoa during their epididymal transit. Also, Choline is a constituent of cell membranes. The balance in accumulation of the methylated product (phosphatidylcholine) or the demethylated product (phosphatidylethanolamine) regulates membrane fluidity and thereby modulates the activity of membrane-bound enzymes. Hence, excess choline availability might play a role in modifying the structural and functional integrity of the Sertoli cell membrane, which has very large surface area in comparison to most other individual cells. Further, the Sertoli cell loses much of its apical membrane in carrying out its secretory functions and during processes such as sperm release. Therefore, Sertoli cells and so spermatogenesis is a very sensitive parameter for toxicity to reproduction which justifies furthermore the approach to mainly focus of the examination of possible effects of Choline only on male rats.

 

According to REACH Annex IX, column 1, a two-generation reproductive toxicity study (e.g. OECD 416), one species, male and female, most appropriate route of administration, having regard to the likely route of human exposure, if the 28-day or 90-day study indicates adverse effects on reproductive organs or tissues, needs to be performed. In all available repeated dose toxicity studies, even in the 2 year study with an NOAEL > 1200 mg/kg bw/day, no adverse effects on any organs, including reproductive organs or tissues, were reported. Furthermore, the observed parameter in the available study on reproductive toxicity, i.e.spermatogenesis in the male rat, is a very sensitive parameter for reproductive toxicity, clearly showing no adverse effects of Choline chloride on reproduction.

According to REACH Annex X, column 1, this study is required unless already provided as part of Annex IX requirements. Furthermore, column 2 states, that the studies do not need to be conducted if i.e. the substance is of low toxicological activity (no evidence of toxicity seen in any of the tests available), it can be proven from toxicokinetic data that no systemic absorption occurs via relevant routes of exposure (e.g. plasma/blood concentrations below detection limit using a sensitive method and absence of the substance and of metabolites of the substance in urine, bile or exhaled air) and there is no or no significant human exposure. Although a study similar to OECD 416 is not available, the given data clearly show that no reproductive toxicity arises from choline chloride, i.e. the testing for the most sensitive parameter, spermatogenesis, revealed a NOAEL of 78 – 83 mg/kg bw/day, which is the highest dose tested, and the NOAEL over 2 years for any other effects on reproductive tissues or organs was > 1200 mg/kg bw/day, which is also the highest dose tested. Consequently, the available information is sufficient to show that no effects on toxicity to reproduction may arise from choline chloride, which also fully covers the purpose of a two-generation reproductive toxicity study.

Furthermore, the substance is of low toxicological activity (no evidence of toxicity seen in any of the tests available), as required in column 2 for the omission of the tests. Although the latter two criteria for the omission of the test are not met, i.e. no absorption and no exposure, it can be reasonably justified that these criteria are not applicable for choline chloride:

 

Choline is an important nutrient as a source of labile methyl groups (NRC, 1987). It plays a role in the synthesis of the phospholipid components of the cell membranes, as a methyl-group donor in methionine metabolism as well as in the synthesis of the neurotransmitter acetylcholine (Michel V, 2006). The National Academy of Sciences, USA, identified choline as an essential nutrient for humans in 1998. The quarternary amine has a physiological plasma concentration of 10-50 µM (Sweet DH, 2001).

First of all, choline is best known for its key role in neurons as the precursor of the neurotransmitter acetylcholine (Michel V, 2006).

Choline is predominantly utilized for the synthesis of essential lipid components of the cell membranes, phosphatidylcholine (PC, also lecithin), which is a lipid essential for the structural integrity and functioning of cell membranes, and sphingomyelin. Also, it is relevant for the production of potent lipid mediators such as platelet-activating factor and lysophosphatidylcholine. In the form of phosphatidylcholine, choline has structural functions in biological membranes and tissue lipid utilization. Quantitatively, PC is the most important metabolite of choline and accounts for approximately one-half of the total membrane lipid content (Michel V, 2006; NRC, 1987)

Similarly to folate, choline is a source of labile methyl groups. In the liver and kidney mitochondria, choline is oxidized to betaine (trimethylglycine), which enters then the one-carbon cycle and serves as a methyl donor in the remethylation of homocysteine to methionine, to ultimately generate the methylation agent S-adenosylmethionine. and also methylates guanidoacetic acid to creatine (Michel V, 2006; NRC, 1987; Zeisel SH, 1994).

Consequently, choline is an essential dietary component for the normal functioning of organisms. It can be also synthesized by the body itself, but an additional uptake via food is required. Under normal circumstances, it has a physiological plasma concentration of 10-50 µM, i.e.plasma/blood concentrations below the detection limit of a sensitive method cannot be achieved anyway. Also, exposure does not need to be avoided but is furthermore required via food.

The criteria of the lack of absorption and exposure are so considered only to apply to xenobiotics which are not essentially required for the normal functioning oh the human body. Consequently, they are disregarded for choline chloride and consequently, no further testing, e.g. an OECD 416 study, is required and can be omitted due to animal welfare.

 

So, in summary, taking into account the rather high applied amount of choline, the fact that all observed effects are only transient, the sensitivity of the endpoint, and the most likely overestimation of the observed effects due to the intraperitoneal application, which is not relevant for humans, it can be clearly concluded that the administration of Choline chloride results in no adverse effects on the reproductive performance and does consequently not need to be classified as toxic to reproduction, neither according Regulation 1272/2008/EC nor Directive 67/548/EEC.

Since the outcome of the study was negative, first, other related effects to reproductive toxicity are covered within this study, too, and second, this study as well as the available repeated dose toxicity studies, do not trigger any need for further testing. Hence, all the tonnage-driven data requirements under REACH are met, no datagaps were identified and hence, no additional studies are needed to be performed and can therefore be omitted due to animal welfare.


Short description of key information:
Toxicity to reproduction: subacute study (12 - 24 days), male rats, intraperitoneal application: NOEAL > 78 - 83 mg/kg bw/day (highest dose tested, based on transient effects on spermatogenesis)

Justification for selection of Effect on fertility via oral route:
Only study available.

Effects on developmental toxicity

Description of key information

Developmental toxicity:
Range finding study: subacute study (intraperitoneal), mice, pregnant females, 5 injections on gestation days 11-15: NOEL > 160 mg/kg bw/day (highest dose tested, based on developmental toxicity / fetotoxicity)
Main study: subacute study (oral: feed), mice, pregnant females, feeding on gestation days 1-18, NOAEL = 4160 mg/kg bw/day (based on developmental toxicity, which is only related to maternal toxicity)

Supporting study: subacute study (oral: feed, rats, pregnant females, feeding on gestation days 2 -21 and until postnatal day 21,

Link to relevant study records
Reference
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
Reason / purpose for cross-reference:
reference to same study
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
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.
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 sacrifices 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
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
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
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.
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:
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:
1 250 mg/kg bw/day (nominal)
Based on:
test mat.
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:
10 800 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects: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
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
Abnormalities:
not specified
Developmental effects observed:
not specified
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

I
n 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, C
holine 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.

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.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
4 160 mg/kg bw/day
Study duration:
subacute
Species:
mouse
Quality of whole database:
There are two studies available attributed to this endpoint, which are both assessed with Klimisch 2 and which are complementary and consistent to each other, because both studies do not trigger the classification of Choline chloride as a developmental toxicant. In the study more relevant for human risk assessment, doses were tested up to ca. 10 g/kg bw/d, which are magnitudes beyond every reasonably expectable intake, even by accident, in humans.
Although the test duration was shortened compared to OECD guideline 414, these studies cover the relevant time points, i.e. the timeframes most sensitive to substance applications.
So, these available, reliable studies do not trigger any need for further testing. Hence, all the tonnage-driven data requirements under REACH are met, no data gaps were identified and hence, the database is of good quality.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information
The two available mice studies were classified as reliable with restrictions (Klimisch 2) and meet the requirements for a developmental toxicity study / general scientific principles. Hence, the results can be considered as reliable and be used for the assessment of possible developmentally toxic effects.

In the range-finding study, related to the Key study, 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 a developmental toxicant. 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 fetuses due to abortion, which was seen in the key study.

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:

The feeding of Choline chloride over the entire period of gestation, i.e. days 1-18, 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, 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: The body weight of the dams is a very relevant aspect, according to Regulation 1272/2008/EC No. 3.7.2.4.4, when assessing the relevance of developmental effects due to maternal toxicity. Since the dams’ body weight gain was not only decreased but also negative in the highest dose group, it can be concluded that all effects on the foetal development were mediated by maternal toxicity. 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 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 substance 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, taking into account the outcome of both studies, neither according to Regulation 1272/2008/EC nor Directive 67/548/EEC.

The two available studies are complementary and consistent to each other, because both studies do not trigger the classification of Choline chloride as developmentally toxic. In the study more relevant for human risk assessment, doses were tested up to ca. 10 g/kg bw/d, which are magnitudes beyond every reasonably expectable intake, even by accident, in humans.

Although the test duration was shortened compared to OECD guideline 414, these studies cover the relevant time points, i.e. the timeframes most sensitive to substance applications. Hence, it is rather likely in this study to detect all possible developmental effects of the test substance.

In addition, a supporting feeding study is available, which is also classified as reliable with restrictionc (Klimisch 2). In this study the animals (12 / 12 pregnant rats, gestation day 2 -21 and until postnatal day 21) were treated daily via the diet with Choline chloride (1.1 % and 5 %, Schulz et al., 2014). The intervention aimed at - using a rodent model - buffering the potential effects of prenatal stress on the developing brain cholinergic system. This is due to the brain cholinergic dysfunction which is associated with neuropsychiatric illnesses such as depression, anxiety, and schizophrenia. Maternal stress exposure is associated with these same illnesses in adult offspring, yet the relationship between prenatal stress and brain cholinergic function is largely unexplored. Specifically, control and stressed dams were fed choline-supplemented or control chow during pregnancy and lactation, and the anxiety-related behaviors of adult offspring were assessed in the open field, elevated zero maze and social interaction tests. Pregnant rats were singly housed in static clear polycarbonate cages with wire bar lids and filtrated microisolator covers and provided with standard (choline chloride content 1.1 g/kg) or choline enriched diet (choline chloride content 5 g/kg) and water ad libitum.

In the open field test, choline supplementation significantly increased center investigation in both stressed and nonstressed female offspring, suggesting that choline-supplementation decreases female anxiety-related behavior irrespective of prenatal stress exposure. In the elevated zero maze, prenatal stress increased anxiety-related behaviors of female offspring fed a control diet (normal choline levels).

However, prenatal stress failed to increase anxiety-related behaviors in female offspring receiving supplemental choline during gestation and lactation, suggesting that dietary choline supplementation ameliorated the effects of prenatal stress on anxiety-related behaviors. For male rats, neither prenatal stress nor diet impacted anxiety-related behaviors in the open field or elevated zero maze. In contrast, perinatal choline supplementation mitigated prenatal stress-induced social behavioral deficits in males, whereas neither prenatal stress nor choline supplementation influenced female social behaviors. Taken together, these data suggest that perinatal choline supplementation ameliorates the sex-specific effects of prenatal stress.

According to REACH Annexes IX and X, column 1, a prenatal developmental (Annex IX) or developmental (Annex X) toxicity study, one species, most appropriate route of administration, having regard to the likely route of human exposure, has to be performed. Both Annexes refer to OECD 414. Although the test duration of the present key developmental toxicity study was shortened compared to OECD guideline 414, it can be reasonably expected to detect all possible developmentally toxic effects of the test substance in this study, and so the purpose of a OECD 414 study is fully covered. Furthermore, Annex IX column 2 states that a decision on the need to perform a study at this tonnage level or the next on a second species should be based on the outcome of the first test and all other relevant available data. The available data (repeated dose toxicity, toxicity to reproduction or development) clearly show that choline does not exhibit any relevant toxic effects, especially no indication on developmental toxicity is given, and the toxicokinetic behaviour of the substance does not indicate any interspecies difference to justify the testing of choline on a second, non-rodent species.

Hence, all the tonnage-driven data requirements under REACH are met, no data gaps were identified and hence, no additional studies are needed to be performed and can therefore be omitted due to animal welfare.

So, in summary, taking into account the rather high applied amount of choline, the fact there are no effects observed at already rather high doses (1250 mg/kg bw/day), the observed effects are only based on maternal toxicity, it can be clearly concluded that the administration of Choline chloride results in no adverse effects on the foetal development and does consequently not need to be classified as toxic to reproduction, neither according Regulation 1272/2008/EC nor Directive 67/548/EEC.


Justification for selection of Effect on developmental toxicity: via oral route:
There are three studies available attributed to this endpoint, which are assessed with Klimisch 2. The feeding study in mice was chosen to be the Key study and the relevant endpoint, because this study is the main study in this set of two experiments, whereas the i.p. study only served as range-finding study / supporting information. Within the feeding study, the exposure duration was prolonged, the application route is more relevant for humans, and more and higher dose levels were tested. Hence, it is rather likely in this study to detect all possible developmentally toxic effects of the test substance. In addition , a supporting feeding study in rats is reported.

Justification for classification or non-classification

In the reproductive toxicity study (Vachhrajani, 1993), only transient effects were seen on spermatogenesis in rats resulting in an NOAEL of 78 -83 mg/kg bw/day after intraperitoneal injection. Here, both a very sensitive parameter and route of application were observed, indicating that no further adverse effects on reproduction are to be expected, neither by application via oral route nor in females.

In the range finding study (BASF, 1966), no effects at all were seen after the intraperitoneal injection of 5 x 0.5 times the LD50(i.p.) (i.e. 160 mg/kg bw/day) during the most sensitive timeframe of pregnancy in mice (day 11 - 15).

Choline chloride thus had under the given experimental conditions of the feeding study (BASF, 1966) no teratogenic effect, since the substance caused only altered development in the fetuses at doses, which also had toxic effects on the dams, i.e. with an NOAEL = 4160 mg/kg bw/day.

As a consequence, Choline chloride does not need to be classified as toxic to reproduction, taking into account the outcome of all available studies, neither according to Regulation 1272/2008/EC nor Directive 67/548/EEC.

Additional information