Choline hydrogen carbonate

Endpoint summary

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:

migrated information: read-across from supporting substance (structural analogue or surrogate)

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, and the study was performed on the read-across substance Choline chloride. 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
- 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.

Remarks:

Doses / Concentrations:
0 mg/rat/day
Basis:
other: nominal injected

Remarks:

Doses / Concentrations:
25 mg/rat/day
Basis:
other: nominal injected

Remarks:

Doses / Concentrations:
0 mg/kg bw/day
Basis:
other: nominal injected

Remarks:

Doses / Concentrations:
83 mg/kg bw/day
Basis:
other: 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:

BODY WEIGHT: Yes

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 pykontic 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:

NOEL

Effect level:

92.3 - 98.2 mg/kg bw/day (nominal)

Based on:

other: Choline bicarbonate

Sex:

male

Basis for effect level:

other: Recalculated from NOEL of Choline chloride, regarding the molecular weight of both substances. Based on mortality; body weight; organ weights Highest dose of 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'

Dose descriptor:

NOAEL

Effect level:

92.3 - 98.2 mg/kg bw/day (nominal)

Based on:

other: choline bicarbonate

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

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 on the read-across substance Choline chloride, 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.
The read-across from Choline chloride to Choline hydrogen carbonate is justified because the absorption of the toxicologically relevant choline cation after both application via the oral or intraperitoneal route is considered to be identical. Once dissociated in the aqueous environment of the vehicle or in the stomach, the bicarbonate anion will be transformed. In an aqueous solution, hydrogen carbonate is in an equilibrium with the anhydrate of the carbonic acid, carbon dioxide (CO2):
CO2 + 2 H2O ⇌ H3O+ + HCO3- with a pKs = 6.4 referring to carbon dioxide (Riedel E (1999) “Anorganische Chemie”, 4th edition, Berlin; New York, de Gruyter).
The equilibrium is far on the side of CO2, which is a regular product of cell respiration, is so ubiquitous present in the body anyway and subject of effective excretion processes.
So, information gained from Choline chloride (CC) for this endpoint can be used to cover it without regarding the bicarbonate anion or modification except adjusting the dose regarding the molecular weight of both choline salts.
In this study it was examined how the intraperitoneal application of 25 mg CC/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 the 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 chloride 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 chiefly 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 or Choline bicarbonate, 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, by passing 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, which can be recalculated for Choline bicarbonate to 92.3-98.2 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 and hence choline bicarbonate 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, the read-across substance 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 or 92.3 - 98.2 mg/kg bw/day choline bicarbonate) 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 with the read-across substance Choline chloride was ca. 78 – 83 mg/kg bw/day, the NOAEL of 24 days of treatment was ca. 78 – 83 mg/kg bw/day, both corresponding to 92.3 - 98.2 mg/kg bw/day Choline bicarbonate. Choline chloride and hence Choline bicarbonate 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 hydrogen carbonate to reproduction and satisfies general scientific requirements.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

92.3 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 and it was performed on the read-across substance Choline chloride, 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 bicarbonate. Hence a study with intraperitoneal applications of the read-across substance Choline bicarbonate was chosen as a surrogate, which is scientifically justified because of the following reasons: Choline is absorbed chiefly 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, by passing the gut bacterial flora.

The read-across from Choline chloride to Choline hydrogen carbonate is justified because the absorption of the toxicologically relevant choline cation after both application via the oral or intraperitoneal route is considered to be identical. Once dissociated in the aqueous environment of the vehicle or in the stomach, the bicarbonate anion will be transformed. In an aqueous solution, hydrogen carbonate is in an equilibrium with the anhydrate of the carbonic acid, carbon dioxide (CO2):

CO2 + 2 H2O ⇌ H3O+ + HCO3- with a pKs = 6.4 referring to carbon dioxide (Riedel E (1999) “Anorganische Chemie”, 4th edition, Berlin; New York, de Gruyter).

The equilibrium is far on the side of CO2, which is a regular product of cell respiration, is so ubiquitous present in the body anyway and subject of effective excretion processes.

So, information gained from Choline chloride (CC) for this endpoint can be used to cover it without regarding the bicarbonate anion or modification except adjusting the dose regarding the molecular weight of both choline salts.

 

The endpoint addressed in this study, i.e. spermatogenesis in the male rat, does not cover completely all possible reasons for toxicity to reproduction and was performed on the read-across substance Choline chloride (CC). 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 Choline chloride 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 the 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 chloride 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 > 1420 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 hydrogen carbonate on reproduction.

So, in summary, taking into account the rather high applied amount of choline, i.e. 78 -83 mg/kg bw/day Choline chloride, corresponding to 92.3 - 98 .2 mg/kg bw/day Choline carbonate, 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 and hence Choline bicarbonate 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 and the most sensitive endpoint was regarded, first, other possible effects on fertility 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 > 92.3 - 98.2 mg/kg bw/day (highest dose tested, based on transient effects on spermatogenesis, recalculated for Choline bicarbonate)

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 > 189.3 mg/kg bw/day (highest dose tested, based on developmental toxicity / fetotoxicity, recalculated for Choline bicarbonate)
Main study: subacute study (oral: feed), mice, pregnant females, feeding on gestation days 1-18, NOAEL = 4921 mg/kg bw/day (based on developmental toxicity, which is only related to maternal toxicity, recalculated for Choline bicarbonate)

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

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

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 g 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 peice 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

Remarks:

Doses / Concentrations:
1 %
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
2.5 %
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
5 %
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
10 %
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:

NOAEL

Effect level:

4 921 mg/kg bw/day (nominal)

Based on:

other: Choline bicarbonate

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:

1 479 mg/kg bw/day (nominal)

Based on:

other: Choline bicarbonate

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:

1 479 mg/kg bw/day (nominal)

Based on:

other: Choline bicarbonate

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:

4 921 mg/kg bw/day (nominal)

Based on:

other: Choline bicarbonate

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:

12 777 mg/kg bw/day (nominal)

Based on:

other: choline bicarbonate

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

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

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 % (25 000 ppm to 100000 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 on the read-across substance Choline chloride 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 read-across from Choline chloride to Choline hydrogen carbonate (CbiC) is justified because the absorption after oral application is very likely to remained unchanged, since the toxicologically relevant choline cation is identical in both salt, its transport mechanisms in the body are only relevant for the cation, too, and no absorption-enhancing irritating effects are expected from the anion.
The expected most relevant exposure route is the oral route. Once dissociated in the aqueous acidic environment in the stomach, the bicarbonate anion will be transformed. In an aqueous solution, hydrogen carbonate is in an equilibrium with the anhydrate of the carbonic acid, carbon dioxide (CO2):
CO2 + 2 H2O ⇌ H3O+ + HCO3- with a pKs = 6.4 referring to carbon dioxide (Riedel E (1999) “Anorganische Chemie”, 4th edition, Berlin; New York, de Gruyter).
Due to the strongly acidic environment of the stomach, the equilibrium will be furthermore shifted to the side of CO2, which is a regular product of cell respiration, is so ubiquitous present in the body anyway and subject of effective excretion processes.
So, information gained from choline chloride for this endpoint can be used to cover it without regarding the bicarbonate anion or modification except adjusting the dose regarding the molecular weight of both choline salts.

The feeding of choline chloride (CC) over the entire period of gestation, in concentrations of 2.5 %, 5 % and 10 % (25 000ppm 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 CC ≙ 4,921 mg/kg bw/day CbiC) to complete loss of all fetuses (20,000 mg/kg bw/d CC ≙ 23,661 mg/kg bw/day CbiC). 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 % Choline chloride (= 10,000 ppm ≙ 1,250 mg/kg bw/d CC ≙ 1,479 mg/kg bw/day CbiC) 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, ≙ 4,921 mg/kg bw/day CbiC) 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 and so choline bicarbonate 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 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 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 4160 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

4 921 mg/kg bw/day

Study duration:

subacute

Species:

mouse

Quality of whole database:

There are two studies on the read-cross substance Choline chloride 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 (CC) and hence choline bicarbonate as a developmental toxicant. In the study more relevant for human risk assessment, doses were tested up to ca. 10 g CC/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 studies on the read-across substance Choline chloride (CC) 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. The read-across from Choline chloride to choline hydrogen carbonate is justified because the absorption and distribution of the toxicologically relevant choline cation after i.v. or i.p. application as well as after oral application is considered to be identical.

Once dissociated in the aqueous environment in the vehicle, fluids in the abdominal cavity, blood or the aqueous acidic environment of the stomach, the bicarbonate anion will be transformed. In an aqueous solution, hydrogen carbonate is in an equilibrium with the anhydrate of the carbonic acid, carbon dioxide (CO2):

CO2 + 2 H2O ⇌ H3O+ + HCO3 - with a pKs = 6.4 referring to carbon dioxide (Riedel E (1999) “Anorganische Chemie”, 4th edition, Berlin; New York, de Gruyter).

The equilibrium is far on the side of CO2, and due to the strongly acidic environment of the stomach, the equilibrium will be furthermore shifted to the side of CO2, which is a regular product of cell respiration, is so ubiquitous present in the body anyway and subject of effective excretion processes, i.e. transitional binding on hemoglobin.

So, information gained from Choline chloride for this endpoint can be used to cover it without regarding the bicarbonate anion or modification except adjusting the dose regarding the molecular weight of both choline salts.

In the range-finding study on the read-across substance Choline chloride, related to the Key study, pregnant mice were injected daily on five consecutive days doses up to 160 mg CC/kg bw, which corresponds to 0.5 times the LD50(i.p.) of Choline chloride or 189.3 mg/kg bw/day of Choline bicarbonate (CbiC). 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 and so Choline bicarbonate 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, neither Choline chloride nor Choline bicarbonate do 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 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: 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 and so choline bicarbonate - is also supported by the fact that the gavage of food with 1%

Choline chloride

(= 10,000 ppm ≙ 1,250 mg/kg bw/d ≙ 1,479 mg/kg bw/day CbiC) 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, ≙ 4,921 mg/kg bw/day CbiC) 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 and so Choline bicarbonate 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 hydrogen carbonate as developmentally toxic. In the study more relevant for human risk assessment, doses were tested up to ca. 10 g CC/kg bw/d, corresponding to more than 12 g CbiC/kg bw, 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.

According to REACH Annex IX , column 1, a prenatal developmental toxicity study, one species, most appropriate route of administration, having regard to the likely route of human exposure, has to be performed, e.g. OECD 414. Although the test duration of the present 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 behavior 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 CC/kg bw/day, or 1479 mg CbiC/kg bw/day, respectively), the observed effects are only based on maternal toxicity, it can be clearly concluded that the administration of Choline chloride and so Choline bicarbonate 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 two studies on the read-across substance Choline chloride available attributed to this endpoint, which are both 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.

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.