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REACH

Choline chloride

EC number: 200-655-4 | CAS number: 67-48-1

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD 422, oral, rats, with read-across substance trimethylamine (TMA): NOAEL 200 mg TMA/kg/day (NAC/AEGL Committee, 2008) (please see remarks in "Additional information")

subacute study, intraperitoneal injections, male rats, treatment for 12 or 24 days (Vachhrajani, 1993)

Link to relevant study records

Reference 1

Endpoint:

one-generation reproductive toxicity

Remarks:

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:

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, testes 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):

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

Dose descriptor:

other: not determined

Generation:

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
Parameter	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
Parameter	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.

Reference 2

Endpoint:: screening for reproductive / developmental toxicity
Remarks:: combined repeat dose and reproduction/developmental screening test
Type of information:: other: read-across
Remarks:: please refer to 'Justification for type of information'
Adequacy of study:: supporting study
Justification for type of information:: 1. HYPOTHESIS FOR THE APPROACH
In this read-across approach data of Trimethylamine (TMA) are used to fill data gaps for choline hydroxide, in accordance with Regulation No 1907/2006 (REACH), Annex XI. The read-across hypothesis for this approach is that Trimethylamine (TMA) can be formed during bacterial degradation of choline inside the intestinal tract, which is more likely at large doses of choline. Please refer to Toxicokinetic section.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source chemical:
Trimethylamine / 75-50-3 / 200-875-0

Target chemical:
Choline hydroxide / 123-41-1 / 204-625-1

3. APPROACH JUSTIFICATION
Choline is absorbed from the jejunum and ileum mainly by a saturable, energy-dependent carrier mechanism in the brush-border membrane and to a lesser extent via passive diffusion. This choline intake can be limited by its efficient metabolism by the intestinal microflora to mainly TMA, which even increases when high doses of choline are applied as e.g. found by Zeisel (1989). In this study using administration of radiolabeled choline to SD rats via orogastric intubation, it was found that low doses of choline were absorbed from the intestinal lumen before it reached the areas of gut colonized with bacteria, while at the high dose of choline, much more label reached the colon. While the authors suggested that an appreciable portion was also absorbed via diffusion across the colon, a disproportionate rise in TMA, and also oxidised TMA, excretion via urine was observed at the higher choline dose, suggesting that TMA was formed when choline reached the bacterially colonized large intestine in appreciable quantities. Thus, for the purpose of regulatory hazard assessment up to limit dose levels, information derived from an oral toxicity study conducted with TMA can be used to assess potential adverse effects which may occur after oral administration of considerably high amounts of choline compounds.
Reason / purpose for cross-reference:: read-across source
Other effects:: effects observed, treatment-related
Description (incidence and severity):: Maternal toxicity was only seen at 200 mg/kg/day, consisting of excessive salivation, abnormal breathing noise and one death at day 38. In contrast, no adverse effect on developmental or reproductive toxicity was observed up to the highest tested dose level of 200 mg/kg/day, yielding a reproductive/developmental NOAEL of 200 mg/kg/day and a NOAEL of 40 mg/kg/day for systemic toxicity
Conclusions:: Maternal toxicity was only seen at 200 mg/kg/day, consisting of excessive salivation, abnormal breathing noise and one death at day 38. In contrast, no adverse effect on developmental or reproductive toxicity was observed up to the highest tested dose level of 200 mg/kg/day, yielding a reproductive/developmental NOAEL of 200 mg/kg/day and a NOAEL of 40 mg/kg/day for systemic toxicity
Executive summary:: Gavage administration of TMA (8, 40, or 200 mg/kg/day) to Sprague-Dawley rats from 2 weeks prior to breeding through day 4 of lactation caused no developmental or reproductive toxicity in a combined repeat dose and reproductive/developmental toxicity screening test (Takashima et al. 2003, cited by NAC/AEGL Committee, 2008). Maternal toxicity was seen at only 200 mg/kg/day, consisting of excessive salivation, abnormal breathing noise, and one death on day 38 (pregnancy day 22).

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

As outlined in the toxicokinetics section in more detail, choline is absorbed from the jejunum and ileum mainly by a saturable, energy-dependent carrier mechanism in the brush-border membrane and to a lesser extent via passive diffusion. This choline intake can be limited by its efficient metabolism by the intestinal microflora to mainly trimethylamine (TMA), which even increases when high doses of choline are applied as e.g. found by Zeisel (1989). In this study using administration of radiolabeled choline to SD rats via orogastric intubation, it was found that low doses of choline were absorbed from the intestinal lumen before it reached the areas of gut colonized with bacteria, while at the high dose of choline, much more label reached the colon. While the authors suggested that an appreciable portion was also absorbed via diffusion across the colon, a disproportionate rise in TMA, and also oxidised TMA, excretion via urine was observed at the higher choline dose, suggesting that TMA was formed when choline reached the bacterially colonized large intestine in appreciable quantities. Thus, for the purpose of regulatory hazard assessment up to limit dose levels, information derived from an oral toxicity study conducted with TMA can be used to assess potential adverse effects which may occur after oral administration of considerably high amounts of choline compounds. In the above-mentioned OECD 422 study in rats exposed to 8, 40 or 20 mg/kg/day of TMA via gavage, maternal toxicity was only seen at 200 mg/kg/day, consisting of excessive salivation, abnormal breathing noise and one death at day 38. In contrast, no adverse effect on reproductive organs or any fertility parameter as well as no embryo-/fetotoxicity was observed up to the highest tested dose level of 200 mg/kg/day, yielding a reproductive/developmental NOAEL of 200 mg/kg/day and a NOAEL of 40 mg/kg/day for systemic toxicity (Takashima et al., 2003 reported in reported in NAC/AEGL Committee, 2008). As mentioned above, exposure to 200 mg/kg/day of TMA represents exposure to much higher amounts of choline due to the metabolism of choline to TMA via intestinal microbiota occurring predominantly at high choline doses exceeding capacities for absorption.

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) (BASF, 1966)

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) (BASF, 1966)

Supporting study: subacute study (oral: feed, rats, pregnant females, feeding on gestation days 2 -21 and until postnatal day 21) (Schulz et al., 2014)

Link to relevant study records

Referenceopen all close all

Reference 1

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 sacrificed and the uteri and fetuses wer e examined.
Analytical verification of doses or concentrations:: not specified
Details on mating procedure:: no data
Duration of treatment / exposure:: between the 1st and 18th day of gestation
Frequency of treatment:: via the feed (rat bread), the feed for 2 days was given at once every second days
Duration of test:: 19 days
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
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.

Reference 2

Endpoint:: developmental toxicity
Type of information:: experimental study
Adequacy of study:: supporting study
Study period:: 1966
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: other: well documented study result, which meets basic scientific principles
Reason / purpose for cross-reference:: reference to same study
Qualifier:: no guideline followed
Principles of method if other than guideline:: In the range-finding studies the animals (7 or 10 pregnant mice, gestation day 11 -15) were treated via daily injections with 160 mg/kg bw or 50 mg/kg bw intraperitoneally for a total of 5 times (aqueous solution).
GLP compliance:: not specified
Limit test:: no
Species:: mouse
Strain:: NMRI
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Ivanova Kisslegg, Germany

For the investigations, NMRI mice of the company Ivanova Kisslegg in Germany, were used. Their fertility, rate of spontaneous foetal resorptions and rate of anomalies are known to us through extensive testing. The methodology corresponded to the one described in our previous reports.
Route of administration:: intraperitoneal
Vehicle:: other: the test material was administered as an aqueous solution
Details on exposure:: In the range-finding studies the animals (7 or 10 pregnant mice, gestation day 11 -15) were treated via daily injections with 160 mg/kg bw or 50 mg/kg bw intraperitoneally for a total of 5 times (aqueous solution).
Analytical verification of doses or concentrations:: not specified
Details on mating procedure:: no data
Duration of treatment / exposure:: between the 11th and 15.th day of gestation
Frequency of treatment:: once daily
Duration of test:: 5 days, subsequent observation period at least up to the end of pregnancy
Dose / conc.:: 50 mg/kg bw/day (nominal)
Dose / conc.:: 160 mg/kg bw/day (nominal)
No. of animals per sex per dose:: 7 -10
Control animals:: yes, historical
Maternal examinations:: no data
Ovaries and uterine content:: no data
Fetal examinations:: mean number of offsprings, the mean foetal body weight, mean foetal length, the foetal resorption rate and the number of anomalies
Details on maternal toxic effects:: Maternal toxic effects:no data

Details on maternal toxic effects:
no details given / no adverse effects denoted
Dose descriptor:: NOEL
Effect level:: 160 mg/kg bw/day (nominal)
Based on:: test mat.
Basis for effect level:: other: developmental toxicity
Dose descriptor:: NOEL
Effect level:: 800 mg/kg bw/day (nominal)
Based on:: test mat.
Basis for effect level:: other: developmental toxicity
Details on embryotoxic / teratogenic effects:: Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
After daily intraperitoneal injections of choline chloride on days 11 -15 of pregnacy the foetal development of mice was not influenced.
The mean number of offsprings, the mean foetal body weight, mean foetal length, the foetal resorption rate and the number of anomalies were within the range reported for the control animals.
Dose descriptor:: NOEL
Effect level:: 160 mg/kg bw/day (nominal)
Based on:: test mat.
Basis for effect level:: other: fetotoxicity
Dose descriptor:: NOEL
Effect level:: 800 mg/kg bw/day (nominal)
Based on:: test mat.
Basis for effect level:: other: fetotoxicity
Abnormalities:: not specified
Developmental effects observed:: not specified
Conclusions:: The present study was well-documented and meets general scientific principles, and hence was classified as reliable with restrictions. So, the gained results can be considered as reliable and used for further assessment.
In the present range-finding study for a feeding study (Key study, “Developmental toxicity / teratogenicity - BASF, 1966 - mice - feeding experiments”), pregnant mice were injected daily on five consecutive days doses up to 160 mg/kg bw, which corresponds to 0.5 times the LD50(i.p.) of Choline chloride. The application days were chosen based on preliminary experiments showing that here foetal development was influenced most by the application of certain substances. Consequently, it was assured that by the study design every possible adverse effects of Choline chloride on the development of mice could be detected. In the present study no effect at all was detected on foetal development, i.e. on the observed parameters which are: mean number of offsprings, the mean foetal body weight, mean foetal length, the foetal resorption rate and the number of anomalies. So, Choline chloride does not need to be regarded as developmentally toxic. Additionally, the applied dose is very high, too, i.e. 0.5 times the LD50(i.p.) per day or 2.5 times the LD50(i.p.) over 5 consecutive days. So, it would be also very likely that any effects on the observed parameters can be attributed to maternal toxicity, for example the average number of foetuses due to abortion.
Although the route of application is not relevant for humans, it assesses the possible effects of the pure, mainly unmetabolized choline, as it would result from high oral dose of choline when intestinal and liver metabolism is saturated. Additionally, the effects of metabolized choline will be assessed during the main feeding study.
Therefore, in summary, the results derived in this study do not trigger any need for classification of Choline chloride as toxic to reproduction.
Executive summary:: In the range-finding studies (BASF, 1966) for the subsequent feeding experiments, NMRI mice (7 or 10 pregnant mice, gestation day 11 -15) were treated via daily injections with 160 mg/kg bw or 50 mg/kg bw intraperitoneally for a total of 5 times (aqueous solution).

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

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

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

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Using a rodent model, the current study implemented an intervention aimed at buffering the potential effects of prenatal stress on the developing brain cholinergic system. Specifically, control and stressed dams were fed choline-supplemented or control chow
during pregnancy and lactation, and the anxiety-related behaviours of adult offspring were assessed in the open field, elevated zero maze and social interaction tests.

GLP compliance:

not specified

Remarks:

Tox. tests performed after 2008-06-01 should be carried out in complicance with the principles of Good Laboratory Practice (GLP).However, this test was performed by university scientists. No information is given on whether or not it was according to GLP.

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Twenty four timed pregnant female Sprague Dawley rats were ordered from Charles Rivers laboratories (Portage, MI) in two cohorts (n=12 each), spaced one month apart and were 2 days pregnant upon arrival. Pregnant females were singly housed in static clear polycarbonate cages with wire bar lids and filtrated microisolator covers. All females had ad libitum access to food and water. Half of all pregnant females were fed a cholinesupplemented chow (5g/kg of choline chloride) through gestation and lactation, and half were fed a standard diet (1.1g/kg of choline chloride). Bedding (Tekfresh, Harlan Laboratories Inc., Indianapolis, IN), food (Dyets Inc., Bethlehem, Pennsylvania) and filtered water were changed weekly. One day prior to parturition, the females were transferred to larger cages (40.6×30.5×20.3) and extra bedding was provided as nesting material. At parturition, food and water continued to be replaced weekly, but the bedding and nests were left undisturbed until weaning at 21 days of age. Cage cleanliness was closely monitored during this time and additional bedding was provided when necessary. Upon weaning, offspring (n=12 overall n=96) were assigned to same-sex (male/female) groups based on stress condition [prenatally stressed (PS)/nonstressed (NS)] and diet condition (choline diet/ control diet). All offspring were housed 2 per cage in same-sex, same-condition sibling groups. Offspring had ad libitum access to food (Dyets Inc., Bethleham, Pennsylvania). All animals were maintained on a 12:12 light/dark cycle, and the room temperature was held constant at 21°C. All animals were treated in accordance with NIH guidelines and all protocols were approved by the IACUC of the University of Colorado Denver.

TEST ANIMALS
- Source: Charles Rivers laboratories (Portage, MI)
- Age at study initiation: two cohorts (n=12 each), spaced one month apart and were 2 days pregnant upon arrival
- Weight at study initiation:
- Fasting period before study:
- Housing: singly in static clear polycarbonate cages with wire bar lids and filtrated microisolator covers
- Diet (e.g. ad libitum): ad libitum access to food (Half of all pregnant females were fed a cholinesupplemented chow (5g/kg of choline chloride) through gestation and lactation, and half were fed a standard diet (1.1 g/kg of choline chloride))
- Water (e.g. ad libitum): ad libitum access to water
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Humidity (%): not specified
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12:12 light/dark cycle

IN-LIFE DATES: From: To:

Route of administration:

oral: feed

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): not specified
- Mixing appropriate amounts with (Type of food): not specified
- Storage temperature of food: not specified

VEHICLE
- Justification for use and choice of vehicle (if other than water): not applicable
- Concentration in vehicle: not applicable
- Amount of vehicle (if gavage): not applicable
- Lot/batch no. (if required): not applicable
- Purity: not applicable

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

not applicable as already pregnant dams were used

Duration of treatment / exposure:

from GD2 - end of gestation (GD21) and during subsequent lactation until weaning at 21 days of age (as such >= 40 days)

Frequency of treatment:

continuously via the diet

Duration of test:

from GD2 - end of gestation (GD21) and during subsequent lactation until weaning at 21 days of age (as such >= 40 days)

Dose / conc.:

1 100 mg/kg diet

Dose / conc.:

5 000 mg/kg diet

No. of animals per sex per dose:

12 (standard diet)
12 (choline-supplemented chow)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random): random
- Other: Half (n=12) of the pregnant female dams were randomly selected to experience unpredictable variable stress 2–3 times daily between 9am and 5pm during the last week of gestation (prenatal days 14–21). The stressors were mild in nature and included 1) restraint in cylindrical restrainers (30 min), 2) swim in water at room temperature (15 min), 3) social stress (5 rats/cage for 8–9 hours) 4) overnight fast, 5) exposure to loud radio static (80db, 60 min), and 6) transport on a noisy cart (30 min).

Maternal examinations:

not specified

Ovaries and uterine content:

not specified

Fetal examinations:

The offspring of control and prenatally stressed dams underwent anxiety-related behavioral testing in adulthood beginning at 79 days of age. All testing occurred during the light phase of the light/dark cycle and consisted of open field, elevated zero and social interaction tests, in this order. In order to prevent potential carryover effects between tests, at least one week separated open field, elevated zero, and social interaction testing, and tests were conducted in the order of least to most stressful. Observers were blind to group assignment and remained out of sight of the animals during each test.

Statistics:

Open Field—The effects of stress condition (PS/NS), and diet (choline supplemented/control) on behavior in the open field were analyzed separately for males and females by 2 factor between-subjects ANOVA.
Elevated Zero—The effects of stress condition (PS/NS), and diet (choline supplemented/control) on behavior in the elevated zero were analyzed separately for males and females by 2 factor between-subjects ANOVA. Pairwise comparisons were also conducted using t-tests tests.
Social Interaction—The effects of stress condition (PS/NS), and diet (choline supplemented/control) on social behavior were analyzed separately for males and females by mixed ANOVAs in which stress condition and diet were the between-subjects factors, and time spent sniffing the partner was the repeated measure.
Body Weight—The effects of stress condition (PS/NS), diet (choline supplemented/ control), and sex on body weight were analyzed by mixed ANOVAs in which stress condition, diet, and sex were the between-subjects factors, and time was the repeated measure.
Experimental Attrition—Three males (2 PS choline, 1 NS choline) and two females (1 NS choline, 1 NS control) fell off the elevated zero maze during testing and were therefore excluded from statistical analysis. In addition, 8 animals (2 PS choline males, 2 NS choline females, 2 PS choline females, and 2 PS control females) were not utilized for elevated zero or social interaction testing because their social experience differed from all other animals in the study. Specifically, in the week prior to testing, these animals were mistakenly housed with an unfamiliar cagemate.

Dose descriptor:

NOAEL

Remarks on result:

other: in this investigation no NOAEL was obtained.

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Prenatal stress decreases animal body weight irrespective of choline supplementation
(A significant mean increase in body weight for all animals over the course of the study. A significant main effect of sex was also detected, such that males were significantly larger than females. A significant main effect of stress condition was found, such that PS animals were slightly, yet significantly, smaller than NS animals).
Simple comparison analyses revealed the significant interaction between time and stress condition was driven by a prenatal stress-induced decrease in body weight beginning at week 9 and remained at weeks 13 and 17.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

not specified

Skeletal malformations:

not specified

Visceral malformations:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Males:
Choline treatment increased overall investigation of conspecifics, measured as sniffing duration.
A significant interaction between stress condition and time was also detected, such that NS males maintained high sniffing behavior longer than PS males.
No effects of stress condition, diet, or interactions between for time in open areas of the elevated zero, open entry number, strech-attend number per open entry, number of center visits, time spent in the center zone, or distance traveled in the center zone were found for any of the anxiety-related behaviors assessed
Females:
Only a significant effect of time was detected for sniffing durations, such that sniffing of conspecifics decreased after the first two minutes of testing [0–2 min bin vs. 2–4 min. No significant effects of stress condition, choline, or interactions between stress condition, choline, or time were found in females.
PS females spent significantly less time in the open areas than did NS females under control diet conditions. Under control diet conditions, PS females showed fewer open arm entries and increased stretch-attend number per open area entry relative to NS females.
In female offspring, choline significantly increased the number of center visits, time spent in the center zone, and distance traveled in the center zone

Dose descriptor:

NOAEL

Remarks on result:

other: in this investigation no NOAEL was obtained.

Developmental effects observed:

NS = Non Stress

PS = Prenatal Stress

Results

Open Field: Choline decreases female but not male anxiety-related behaviors in the open field

In female offspring, choline significantly increased the number of center visits [Figure 1A; F (1, 44) = 4.30, p = 0.0443], time spent in the center zone [Figure 1B; F (1, 44) = 8.33, p = 0.0060], and distance traveled in the center zone [Figure 1C; F (1, 44) = 4.62, p = 0.0372]. No main effects of stress condition or interactions between diet and stress condition were found in females. In males, no effects of stress condition, diet, or interactions between these factors were found (Figures 1A, 1B, and 1C).

Elevated Zero: Choline mitigates the anxiety-related behaviors of prenatally stressed females but not males in the elevated zero

In females, a significant effect of stress condition was found for time in the open areas of the elevated zero [F (1, 36) = 4.30, p=0.0462]. This main effect was driven primarily by differences between PS and NS females under control as opposed to choline diet conditions. Specifically, PS females spent significantly less time in the open areas than did NS females under control diet conditions [t (1, 19) = 5.16, p = 0.0349]. In contrast, differences in open area duration were mitigated under choline diet conditions, as no differences were found between PS and NS females. Similarly, ANOVA revealed trends toward effects of stress condition on both the number of open entries [F (1, 36) = 3.67, p=0.0635] and stretch-attend number per open entry [F (1, 36) = 2.88, p=0.0985]. These differences also appeared to be

driven by PS-induced alterations in anxiety-related behaviors under control but not choline diet conditions. Specifically, under control diet conditions, PS females showed fewer open arm entries [Figure 2B; t (1, 19) = 5.17, p = 0.0347] and increased stretch-attend number per open area entry [Figure 2C; t (1, 19) = 5.32, p = 0.0325] relative to NS females. In contrast, differences between PS and NS females were mitigated under choline diet conditions, as no differences were found between PS and NS females for open entry number or stretch-attend number per open entry. In males, no effects of stress condition, diet, or interactions between these factors were found for any of the anxiety-related behaviors assessed (Figures 2A, 2B, and 2C).

Social Interaction: Choline mitigates the anxiety-related behaviors of prenatally stressed males but not females during social interactions

In males, choline treatment increased overall investigation of conspecifics, measred as sniffing duration [Figure 3, right panel; F (1, 46) = 3.958, p=0.0526]. A significant interaction between stress condition and time was also detected, such that NS malesmaintained high sniffing behavior longer than PS males (Figure 3; F (3,138) = 3.176, p = 0.0262). Specifically, PS males only displayed high levels of sniffing during the first two minutes of testing, and significantly decreased thereafter [F (3, 138) = 6.40, p = 0.0006; 0–2 min bin vs. 2–4 min, p = 0.0006; 0–2 min bin vs. 4–6 min, p = 0.0003; 0–2 min bin vs. 6–8

min p = 0.0016]. In contrast, NS males maintained high levels of sniffing behavior for a full four minutes before significantly decreasing sniffing [F (3, 138) = 7.33, p = 0.0002; 2–4 min bin vs. 4–6 min, p = 0.0038; 2–4 min bin vs. 6–8 min p = 0.0040]. For females, only a significant effect of time was detected for sniffing durations [F (3, 108) = 14.111, p < 0.0001], such that sniffing of conspecifics decreased after the first two minutes of testing [0–2 min bin vs. 2–4 min bin, p = 0.0006]. No significant effects of stress condition, choline, or interactions between stress condition, choline, or time were found in

females (data not shown).

Body Weight: Prenatal stress decreases animal body weight irrespective of choline supplementation

A mixed ANOVA revealed a significant mean increase in body weight for all animals over the course of the study (F (1.51, 138.55) = 8014.05, p < 0 .001). A significant main effect of sex was also detected (F (1, 92) = 1164.67, p < 0.001), such that males were significantly larger than females. Additionally, a significant main effect of stress condition was @found, such that PS animals were slightly, yet significantly, smaller than NS animals (F (1, 92) = 18.95, p < 0 .001; PS female, x̄ = 204.92; NS female, x̄ = 219.11; PS male, x̄ = 338.20; NS male, x̄ = 355.35). These main effects were qualified by significant interactions betweentime and sex (F (1.51, 138.55) = 928.16, p < 0.001) and between time and stress condition (F (1.51, 138.55) = 6.35, p = 0.005). Simple comparison analyses revealed the significant interaction between time and stress condition was driven by a prenatal stress-induced decrease in body weight beginning at week 9 (F (1, 92) = 5.42, p = 0.0221) and remained at weeks 13 (F (1, 92) = 6.57, p = 0.120) and 17 (F (1, 92) = 10.44, p = 0.0017). The significant interaction between time and sex was driven by the difference in body weight between males and females beginning at week 9, and remaining throughout the study (data not presented graphically).

Discussion

Anxiety in rodents is multidimensional, and factor analysis studies demonstrate that behaviors observed in the open field, elevated plus, and social interaction tests load on distinct factors [34, 39, 40]. Thus, while each of these tests assesses anxiety-related behaviors, they appear to tap different aspects of anxiety. We report here that that prenatal stress and choline exposure influenced anxiety-related behaviors in a test- and sex-specific manner. In the open field, no effects of prenatal stress were observed for either sex. However, choline exposure increased exploratory behavior in females but not in males. For

the elevated zero, the effects of prenatal stress and choline were also female-biased. Under control diet conditions, prenatally stressed females significantly decreased their frequency and duration of visits to the open arms. In contrast, under choline-supplemented conditions, no differences were observed between PS and NS females, suggesting that choline exposure ameliorates the anxiogenic effects of prenatal stress. For the social interaction test, the effects of prenatal stress and choline were male-biased. Specifically, choline exposure increased male social behavior durations, especially in prenatally stressed males. Thus, we demonstrate here that early developmental choline exposure mitigates the deleterious effects of prenatal stress on particular dimensions of anxiety-related behaviors in males and

females.

A significant effect of prenatal stress but not choline diet on body weight was observed in the current study. This was surprising given that we previously found significant prenatal stress-induced increases in body weight in males [2]. Moreover, other studies demonstrate that postnatal dietary choline intake influences energy metabolism and lean body mass composition [41]. The differences in weight gain between these two studies may be due to differences in the fat content of the diets. In our previous study in which PS males gained weight, 17% of the calories in the chow were derived from fat (2018 Teklad Global 18% Protein Rodent Diet). In contrast, only 11% of calories were derived from fat in our current study (AIN-76A, Dyets Inc., Bethlehem, Pennsylvania). Given that prenatal stress and high

fat diet interact to increase obesity in rats [42], it is possible that differences in chow fat content between these two studies caused differential effects of prenatal stress on weight gain. Thus, our future studies will carefully control the fat content in food or manipulate chow fat content as a variable of interest.

The mechanisms by which perinatal choline mitigates the effects of prenatal stress are not yet known, but many of the neurodevelopmental consequences of prenatal stress relevant to anxiety-related behaviors are also impacted by prenatal choline exposure. For example, prenatal stress significantly reduces hippocampal neurogenesis [43], whereas prenatal choline supplementation increases hippocampal neurogenesis in offspring [44, 45].

Similarly, alterations in neurotrophic factors such as BDNF have been implicated in the pathology and treatment of psychiatric illnesses including obsessive compulsive disorder, schizophrenia and depression [46–50], and are also reduced by prenatal stress [51]. Given that prenatal choline supplementation increases several brain neurotrophic factors including BDNF, NGF and IGF2R [26, 44, 52], perinatal choline may ameliorate the deleterious effects of prenatal stress on anxiety via increased neurotrophic factors. Interestingly, BDNF may also influence anxiety and depression via changes in nAChRs, given that BDNF upregulates alpha7* nAChR receptor levels in hippocampal interneurons [53]. Therefore, further research is necessary to elucidate the potential interactions between choline supplementation, neurotrophic factors and alpha7* nAChR receptors.

In addition to possible indirect effects of perinatal choline on nAChRs, perinatal choline may also directly impact nAChR levels to influence anxiety related behaviors. For example, perinatal choline is capable of increasing nAChRs in mouse strains with low levels of hippocampal nAChRs [27, 54]. Given that we have recently found that prenatal stress alters hippocampal levels of both alpha4 beta2* and alpha7* nAChRs [19], it is plausible that prenatal choline counteracts the impact of prenatal stress by maintaining normative levels of nAChRs in the hippocampus. Studies are currently underway to compare the levels of hippocampal nAChRs in prenatally stressed animals gestated on control or choline supplemented diet.

To our knowledge, this is the first report to demonstrate that perinatal choline counteracts the effects of prenatal stress on adult anxiety-related behaviors. Interestingly, a much earlier report from Tonjes and colleagues [55] found that postnatal injections of choline chloride counteracted the effects of neonatal maternal deprivation on memory function in males in adulthood. Choline chloride treatment was most effective when administered during the same timeframe as neonatal stress on postnatal days 1–14, but also mitigated the effects of neonatal stress when administered after neonatal stress treatments on days 15–28 [55]. More recently, Corriveau & Glenn [29] employed a ‘two hit’ rodent model of schizophrenia and found that adolescent dietary choline supplementation mitigated the memory deficits in

males resulting from combined exposure to prenatal stress and the NMDA receptor antagonist MK801 in adulthood. Thus, our findings extend this budding literature by showing that perinatal choline counteracts the detrimental effects of prenatal stress on anxiety-related behaviors in both males and females.

Executive summary:

In this supporting feeding studies 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.

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

Toxicity to reproduction: other studies

Description of key information

postnatal development: rats, oral, pregnant females, feeding for 21 days starting 24 - 48 h before parturition: NOAEL = 1240 mg/kg/day (highest dose tested) (Dellschaft et al., 2015)

developmental neurotoxicity: rats, oral, lactating females, feeding for 21 days starting 24 - 48 h before parturition (Richard et al., 2017)

developmental toxicity in humans: children with FASD, oral, pre- and/or postnatal exposure, for up to 4 years (Wozniak et al., 2020 and further studies summarised therein)

developmental toxicity in humans: healthy pregnant females (summarised in Korsmo et al., 2019)

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

toxicity to reproduction: other studies

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In this read-across approach data of choline salts are used to fill data gaps for choline chloride, in accordance with Regulation No 1907/2006 (REACH), Annex XI. The basis for this read-across approach in the “Read-Across Assessment Framework” (RAAF) (ECHA 2017). The read-across hypothesis for the analogue approach is that choline salts such as chloine chloride and choline bitartrate exhibit a similar (eco)toxicological profile. This is due to the fact that the choline salts dissociate readily into the respective ions when getting into contact with water and the choline cation is what is left to be considered (US EPA, 2010). Thus, the different choline salts are used to for hazard assessment. According to the RAAF this approach is covered by scenario 1: “(Bio)transformation to common compound(s)”.
“This scenario covers the analogue approach for which the read-across hypothesis is based on (bio) transformation to common compound(s). For the REACH information requirement under consideration, the property investigated in a study conducted with one source substance is used to predict the properties that would be observed in a study with the target substance if it were to be conducted. Similar properties or absence of effect are predicted. The predicted property may be similar or based on a worst-case approach.” (ECHA 2017).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical:
Choline bitartrate 87-67-2 / 201-763-4

Target chemical:
Choline chloride / 67-48-1 / 200-655-4

3. ANALOGUE APPROACH JUSTIFICATION
Upon contact with water, choline chloride is expected to dissociate into the cationic form (choline) and the anionic form (chloride ions). Since the inorganic anion chloride is widely distributed throughout the body and not the relevant ion when assessing the toxicokinetic behaviour of choline chloride, it is scientifically justified to focus mainly on the organic cation, choline. Thus the choline cation is what is left to be considered. Due to the structural similarities, i.e. the identical organic cation, which contains a positively charged nitrogen, and small, negatively charged inorganic anion (for choline chloride: chloride), this is a reasonable and scientifically expectable conclusion, which allows to draw the generalized conclusion that choline salts in general dissociate readily in water into the corresponding positively charged quaternary hydroxyl alkylammonium ion and the negatively charged inorganic anion (OECD SIDS, 2004). This is supported by the approach of the US-EPA to use studies on choline chloride and other salts for evaluating the risk from exposure to choline hydroxide. In line with this, studies on choline or choline salts such as choline bitartrate are appropriate for the toxicological evaluation of choline chloride.
Based on the fact that in the environment and in biological fluids the same compounds are formed from the source and the target substances, the same (eco)toxicological profile of choline chloride and other choline salts is expected. Therefore, the read-across approach is justified. Thus, the available studies for the source substance choline bitartrate were used to fill data gaps for choline chloride for this endpoint.

Reason / purpose for cross-reference:

read-across source

D and C6 dams had lower final body weight, spleen weight and average pup weight than C1 dams (P<0·05). There was a linear relationship between free choline concentration in pup stomach contents with maternal dietary choline content (P<0·001, r² 0·415). Compared with C1 and C2·5, D spleens had a lower proportion of mature T cells and activated suppressor cells, and this resulted in reduced cytokine production after stimulation (P<0·05). Feeding 6·2 g choline/kg diet resulted in a higher cytokine production after stimulation with CD3/CD28 (P<0·05). Except for a higher IL-6 production after LPS stimulation with cells from the C2·5 dams (P<0·05), there were no differences between the C1 and C2·5 dams.

Conclusions:

Oral administration of choline at test concentrations up to 6.2 g choline/kg diet, corresponding to 1240 mg choline/kg bw/day, to pregnant rats for 21 d starting 24-48 h before parturition to ensure exposure at initiation of suckling was found to decrease body weights of dams and pups at the highest test concentration (-15% and -20% compared to standard diet containing 1 g choline/kg diet for dams and pups, respectively) but had no effect on the different immune cell phenotypes in the spleen and only a minimal effect on maternal immune cell function at the highest dose level. Noticeably, this study found that choline is required to maintain maternal immune function in lactating dams.

Executive summary:

The present study was designed to determine the effects of varying intakes of choline on maternal immune function during lactation.

Primiparous Sprague–Dawley rats (n=42) were randomised 24-48 h before birth and fed the following diets for 21 d: choline-devoid (0 g choline/kg diet; D, n=10); 1.0 g choline/kg diet (C1, n=11); 2.5 g choline/kg diet (C2.5, n=10); 6.2 g choline/kg diet (C6, n=11).

There was a linear relationship between free choline concentration in pup stomach contents with maternal dietary choline content. Compared with C1 and C2.5, D spleens had a lower proportion of mature T cells and activated suppressor cells, and this resulted in reduced cytokine production after stimulation. Feeding 6.2 g choline/kg diet resulted in a higher cytokine production after stimulation with CD3/CD28. Except for a higher IL-6 production after LPS stimulation with cells from the C2.5 dams, there were no differences between the C1 and C2.5 dams.

Reference 2

Endpoint:

toxicity to reproduction: other studies

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

Reason / purpose for cross-reference:

read-across source

At 10 weeks of age, there were no significant differences in body weight, spleen weight, live weight, intestinal length, and relative number of splenocytes among diet groups.
At 3 weeks, MC and HGPC pups were heavier and their splenocytes had a higher proportion of helper T cells expressing CD25 and CD28 and produced less interferon gamma (IFN-g) and tumor-necrosis factor-alpha (TNF- a) after Concanavalin A stimulation vs. Control pups (p < 0.05). At 10 weeks, MC and HGPC offspring had a lower proportion of macrophages and dendritic cells and produced less interleukin (IL)-1b but more IL-10 after lipopolysaccharide stimulation vs. Control pups (p < 0.05).

There was no significant difference in dams’ body weight at the end of pregnancy and at the end of the suckling period. Also, mean daily food intake of the dams in each diet group for the duration of the lactation period (21 days) did not differ in the Control, MC and HGPC group. At the end of the suckling period, pups from dams fed MC or HGPC diets had higher body weight, spleen weight, and liver weight compared to pups from dams fed the control diet. Spleen weight of pups from dams fed MC or HGPC were also proportionally larger relative to their body weight compared to pups from dams fed control diet.

Total choline concentration in pups’ stomach content was not significantly different among diet groups. The MC and HGPC diets had a lower proportion of phosphocholine compared to the control diet in pups’ stomach content. The HGPC diet also resulted in a lower proportion of free choline when compared to the Control diet. There was a higher proportion of PC and GPC in pups’ stomach content from dam fed the MC and the HGPC diet, respectively.

Following stimulation with a T cell mitogen ConA, splenocytes from pups from dams fed the MC and the HGPC diets produced significantly less IFN-g and TNF-a compared to pups from control-fed dams.

Feeding a MC diet also led to a lower production of IL-6 by ConA-stimulated splenocytes in 3-week-old pups compared to the control diet. At 3 weeks, there was no change in the ex vivo production of IL-2 and IL-10 by

splenocytes after ConA stimulation among diet groups. Following stimulation with LPS (bacterial challenge), splenocytes from 3-week-old pups from dams fed the MC and the HGPC diets produced significantly less IL-10 compared to pups from control-fed dams.

No change was observed in the production of IL-1b, IL-6, and TNF-a by splenocytes among diet groups after LPS stimulation in 3-week-old pups. For both ConA- and LPS-stimulated splenocytes, no differences were observed in cytokine production between the MC and the HGPC diets. There was no significant difference in the concentration of total choline adjusted for protein content in splenocytes among diet groups in 3-week-old pups. There was also no significant change in the proportion of choline coming from the different forms of choline (i.e., FC, PC, Lyso-PC, GPC, phosphocholine, and sphingomyelin) in splenocytes among diet groups.

There was no significant change among diet groups in the ex vivo production of IL-2, IL-6, IL-10, IFN-g, and TNF-a by splenocytes after ConA stimulation at 10 weeks. After LPS stimulation, offspring that received the MC and the HGPC diets at suckling produced less IL-1b but more IL-10 compared to the Control diet.

Offspring from dams fed the HGPC diet also produced less IL-6 in response to LPS. There were no differences in cytokine production between the MC and the HGPC diets in 10-week-old offspring. There was no significant

difference in the concentration of total choline adjusted for protein content in splenocytes among diet groups. There was also no significant change in the proportion of choline coming from the different forms of choline (i.e., FC, PC, Lyso-PC, GPC, phosphocholine, and sphingomyelin) in splenocytes among diet groups.

At 3 weeks of age, there was no change in the proportion of total T cells (CD3+) and helper T cells (% of CD3+ cells that also express CD4) and cytotoxic/suppressor T cells (% of CD3+ cells that also express CD8) among groups.

However, pups from dams fed the MC and the HGPC diets had a higher proportion of helper T cells expressing CD25 (IL-2 receptor) and CD28 (co-stimulatory molecule) and IgA+ cells, and a lower proportion of IgG+, OX6+ (MHC Class II), and CD45RA+CD80 + cells compared to pups from dams fed the control diet. Pups from dams fed the HGPC diet also had a lower proportion of IgM+ cells, OX6+CD80+ cells, and OX12+CD80+ cells compared to the control diet. No differences were observed in immune cells phenotypes between the MC and the HGPC diets.

At 10 weeks of age, there was no change in the proportion of total T cells and helper T cells among groups. Offspring from dams that were fed a mixture of choline forms had a lower proportion of cytotoxic T cells expressing CD28, but a higher proportion of cytotoxic T cells expressing CD152 (cytotoxic T-lymphocyte-associated protein 4) vs. the control diet. Offspring that received the MC diet also had a lower proportion of cytotoxic T cells compared to the control diet. Both MC and HGPC diets also led to a higher proportion of IgM+ cells and a lower proportion of dendritic cells (OX62+OX6+) and CD45RA+CD80+ cells vs. FC diet.

Compared to the control diet, offspring that received the MC diet had a lower proportion of macrophages (CD68+) while those that received the HGPC diet had a lower proportion of OX12+CD80+ and IgA+ cells. No differences were observed in immune cell phenotypes between the MC and the HGPC diets.

Conclusions:

It was shown, that feeding a MC diet or a HGPC diet to lactating dams enhances the growth of their offspring at the end of the suckling period.
In suckled offspring, feeding a mixture of choline forms (both a MC or a HGPC diet) led to an overall more mature lymphocyte phenotype with more helper T cells expressing activation markers and less B cells, which resulted in a beneficial effect on T cell function in that pups fed a mixture of choline forms did not have to produce as much cytokines in order to maintain a normal proliferative response. In female adult offspring, providing a mixture of choline forms during the suckling period had an overall anti-inflammatory programming effect on B cells/antigen presenting cells function with lower pro-inflammatory and higher anti-inflammatory cytokine production along with a lower proportion of macrophages and dendritic cells.

Executive summary:

This study investigates the impact of feeding lactating dams different mixtures of choline forms, similar to those in human diets, on the development and later immune function of suckled offspring.

Sprague-Dawley lactating dams (n = 6/diet) were randomized to consume one of three diets, containing 1 g/kg choline: Control (100% free choline (FC)), Mixed Choline (MC: 50% phosphatidylcholine (PC), 25% FC, 25% glycerophosphocholine (GPC)), or High GPC (HGPC: 75% GPC, 12.5% PC, 12.5% FC). At weaning, female pups (n = 2/dam) were fed the control diet until 10 weeks. At 3 weeks, MC and HGPC pups were heavier and their splenocytes had a higher proportion of helper T cells expressing CD25 and CD28 and produced less interferon gamma (IFN-g) and tumor-necrosis factor-alpha (TNF-a) after Concanavalin A stimulation vs. control pups (p < 0.05). At 10 weeks, MC and HGPC offspring had a lower proportion of macrophages and dendritic cells and produced less interleukin (IL)-1b but more IL-10 after lipopolysaccharide stimulation vs. control pups (p < 0.05). In summary, feeding mixed choline diets during lactation improved T cell phenotype/function at the end of suckling and programmed a less inflammatory response later in life.

Reference 3

Endpoint:: toxicity to reproduction: other studies
Type of information:: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:: supporting study
Justification for type of information:: REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In this read-across approach data of choline salts are used to fill data gaps for choline chloride, in accordance with Regulation No 1907/2006 (REACH), Annex XI. The basis for this read-across approach in the “Read-Across Assessment Framework” (RAAF) (ECHA 2017). The read-across hypothesis for the analogue approach is that choline salts such as chloine chloride and choline bitartrate exhibit a similar (eco)toxicological profile. This is due to the fact that the choline salts dissociate readily into the respective ions when getting into contact with water and the choline cation is what is left to be considered (US EPA, 2010). Thus, the different choline salts are used to for hazard assessment. According to the RAAF this approach is covered by scenario 1: “(Bio)transformation to common compound(s)”.
“This scenario covers the analogue approach for which the read-across hypothesis is based on (bio) transformation to common compound(s). For the REACH information requirement under consideration, the property investigated in a study conducted with one source substance is used to predict the properties that would be observed in a study with the target substance if it were to be conducted. Similar properties or absence of effect are predicted. The predicted property may be similar or based on a worst-case approach.” (ECHA 2017).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical:
Choline bitartrate 87-67-2 / 201-763-4

Target chemical:
Choline chloride / 67-48-1 / 200-655-4

3. ANALOGUE APPROACH JUSTIFICATION
Upon contact with water, choline chloride is expected to dissociate into the cationic form (choline) and the anionic form (chloride ions). Since the inorganic anion chloride is widely distributed throughout the body and not the relevant ion when assessing the toxicokinetic behaviour of choline chloride, it is scientifically justified to focus mainly on the organic cation, choline. Thus the choline cation is what is left to be considered. Due to the structural similarities, i.e. the identical organic cation, which contains a positively charged nitrogen, and small, negatively charged inorganic anion (for choline chloride: chloride), this is a reasonable and scientifically expectable conclusion, which allows to draw the generalized conclusion that choline salts in general dissociate readily in water into the corresponding positively charged quaternary hydroxyl alkylammonium ion and the negatively charged inorganic anion (OECD SIDS, 2004). This is supported by the approach of the US-EPA to use studies on choline chloride and other salts for evaluating the risk from exposure to choline hydroxide. In line with this, studies on choline or choline salts such as choline bitartrate are appropriate for the toxicological evaluation of choline chloride.
Based on the fact that in the environment and in biological fluids the same compounds are formed from the source and the target substances, the same (eco)toxicological profile of choline chloride and other choline salts is expected. Therefore, the read-across approach is justified. Thus, the available studies for the source substance choline bitartrate were used to fill data gaps for choline chloride for this endpoint.
Reason / purpose for cross-reference:: read-across source
Conclusions:: Children with FASD who received choline had higher non-verbal intelligence, higher visual-spatial skill, higher working memory ability, better verbal memory, and fewer behavioral symptoms of attention deficit hyperactivity disorder than the placebo group. No differences were seen for verbal intelligence, visual memory, or other executive functions. These data support choline as a potential neurodevelopmental intervention for FASD and highlight the need for long-term follow-up to capture treatment effects on neurodevelopmental trajectories.
Executive summary:: In this long-term follow-up study the neurodevelopmental effects of choline supplementation in children with FASD 4 years after an initial efficacy trial were evaluated.

The initial study was a randomized, double-blind, placebo-controlled trial of choline (500 mg/day) vs. placebo in 2–5 - year-olds with FASD. Participants include 31 children (16 placebo; 15 choline) seen 4 years after trial completion. The mean age at follow-up was 8.6 years. Diagnoses were 12.9% fetal alcohol syndrome (FAS), 41.9% partial FAS, and 45.1% alcohol-related neurodevelopmental disorder. The follow-up included measures of intelligence, memory, executive functioning, and behavior.

Children who received choline had higher non-verbal intelligence, higher visual-spatial skill, higher working memory ability, better verbal memory, and fewer behavioral symptoms of attention deficit hyperactivity disorder than the placebo group. No differences were seen for verbal intelligence, visual memory, or other executive functions.

These data support choline as a potential neurodevelopmental intervention for FASD and highlight the need for long-term follow-up to capture treatment effects on neurodevelopmental trajectories.

The results of this study are in line with other clinical trials, reporting that pre- or postnatal repeated administration of choline at doses ranging from 500 to 625 mg (children) or 750 to 2000 mg (pregnant females) choline per day did not cause adverse effect. Administration of choline was found to improve infants attentional/memory task (cardiac response to familiar/unfamiliar visual stimuli), visual recognition memory, or other executive functions between choline-supplemented and placebo groups. Prenatal administration of 2000 mg choline to pregnant women had a beneficial impact on the development of the offspring. These human data clearly underline absence of adverse effects of choline on pre- or postnatal development.

Reference 4

Endpoint:

toxicity to reproduction: other studies

Type of information:

other: Review

Adequacy of study:

supporting study

Study period:

no information

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

no guideline followed

Principles of method if other than guideline:

Review on adequate choline intake during pregnancy

GLP compliance:

not specified

Type of method:

in vivo

Species:

other: Human and animal data

Sex:

male/female

Details on test animals or test system and environmental conditions:

not specified

Route of administration:

other: oral or other

Vehicle:

not specified

Details on exposure:

not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

few to several weeks

Frequency of treatment:

not specified

Remarks:

550 - 900 mg/day

No. of animals per sex per dose:

not specified

Details on study design:

case control, Prospective cohort and Randomized clinical trial (RCT), animal studies

To date, none of the randomized clinical trials (RCTs) conducted in healthy pregnant women have reported any adverse effects of choline supplementation at levels ranging from 550–900 mg/day.

The Institute of Medicine (now National Academy of Medicine) recommended adequate intake levels of 425 mg choline/day for women of reproductive age, 450 mg choline/day during pregnancy and 550 mg choline/day during lactation (1998).

Rodent studies have demonstrated modulatory effects of maternal choline supplementation during pregnancy on the placental and fetal epigenome, with some reports of reduced disease risk.

Emerging data from a growing number of studies suggest that choline supply can beneficially influence functional processes of the placenta, including angiogenesis, inflammation, and macronutrient transport. Based on rodent data, prenatal choline may also be a nutritional approach to mitigate placental insufficiency.

In humans, consumption of additional choline (930 vs. 480 mg choline/day) during the third trimester of pregnancy reduced the production of placental soluble fms-like tyrosine kinase 1 (sFlt1), an anti-angiogenic protein that sequesters vascular endothelial growth factor (VEGF) in maternal circulation and contributes to endothelial dysfunction, hypertension, and proteinuria in preeclampsia.

A few prospective observational studies have explored the relationship between maternal choline status (intake or blood levels) during human pregnancy and cognitive development in children, including a positive association between concentrations of maternal plasma choline and betaine at 16 weeks of gestation with infant cognitive test scores at 18 months.

Findings from randomized clinical trials (RCTs) in support of a beneficial effect of prenatal choline on cognitive outcomes are also beginning to emerge.

In a randomized controlled feeding study, faster processing speed was observed among infants born to mothers consuming 930 versus 480 mg choline/day during their third trimester of pregnancy.

Prenatal choline may also protect against the development of congenital malformations of the central nervous system, commonly referred to as neural tube defects (NTDs). For example, both maternal choline intake and biomarkers of choline status during pregnancy have been inversely associated with offspring NTD risk.

Conclusions:

To date, none of the Randomized clinical trials conducted in healthy pregnant women have reported any adverse eects of choline supplementation at levels ranging from 550–900 mg/day.

Executive summary:

Data from both animal and human studies highlight the importance of ensuring an adequate choline intake during pregnancy. Supplementing the maternal diet with additional choline has been shown to improve offspring cognition, neurodevelopment, and placental functioning, and to protect against neural and metabolic insults.

To date, none of the Randomized clinical trials conducted in healthy pregnant women have reported any adverse eects of choline supplementation at levels ranging from 550–900 mg/day.

Justification for classification or non-classification

For the registered substance choline chloride, no GLP guideline studies concerning toxicity to reproduction are available. To support hazard assessment, read-across to choline is proposed and considered justified for the following reasons: Upon contact with water, choline chloride is expected to dissociate into the cationic form (choline) and the anionic form (chloride ions). Since the inorganic anion chloride is widely distributed throughout the body and not the relevant ion when assessing the toxicokinetic behaviour of choline chloride, it is scientifically justified to focus mainly on the organic cation, choline. Thus, the choline cation is what is left to be considered. Due to the structural similarities, i.e. the identical organic cation, which contains a positively charged nitrogen, and small, negatively charged inorganic anion (for choline chloride: chloride), this is a reasonable and scientifically expectable conclusion, which allows to draw the generalized conclusion that choline salts in general dissociate readily in water into the corresponding positively charged quaternary hydroxyl alkylammonium ion and the negatively charged inorganic anion (OECD SIDS, 2004). This is supported by the approach of the US-EPA to use studies on choline chloride and other salts for evaluating the risk from exposure to choline hydroxide. In line with this, studies on choline or choline salts such as choline bitartrate are appropriate for the toxicological evaluation of choline chloride.

Choline itself is an essential nutrient and a methyl donor with a multitude of physiological functions. It is required for normal brain growth and development; furthermore, it plays a pivotal role in maintaining structural and functional integrity of cellular membranes. It also regulates cholinergic signalling in the brain via the synthesis of acetylcholine. Via its metabolites, it participates in pathways that regulate methylation of genes related to memory and cognitive functions at different stages of development. Thus, a constant supply of choline via diet or feed is required to maintain homeostasis. Periods known for the high choline demand are the early life stage, pregnancy and lactation. In line with this, EFSA (2016) recommends a higher dietary choline intake during pregnancy and lactation compared to non-pregnant adults (480 and 520 mg/day compared to 400 mg/day, respectively) for healthy human subjects. For infants at the age of 7-11 months or children aged 1-3 years, adequate intake levels are 160 mg/day and 140 mg/day, respectively. Considering the reference body weights of 8.6 kg for infants and 11.5-12.2 kg for young children the relative adequate intakes (AI) are 18.6 mg/kg/day for infants and 8.9-9.1 mg/kg/day for young children. These recommended dietary reference values are clearly higher compared to adults (6.7 mg/kg/day, based on an adequate intake of 400 mg/day and a reference body weight of 60 kg). Similarly, the choline AI levels recommended by the US Institute of Medicine (now National Academy of Medicine) are 425 mg choline/day for women of reproductive age, 450 mg choline/day during pregnancy and 550 mg choline/day during lactation (Institute of Medicine, 1998). Indeed, studies in animals and humans reported that supplementing maternal diet with additional choline improves pregnancy outcomes (summarised in Korsmo et al., 2019). This increased requirement of choline contradicts a potentially increased sensitivity of mammalians towards choline or choline compounds in these life stages.

A series of studies addressed the impact of choline chloride on reproductive organs upon repeated exposure or potential adverse effects occurring during pregnancy, providing information on the absence of adverse effects of choline compounds on reproduction and development. An early subchronic repeated dose toxicity study in rats found no histopathological changes in ovaries or testes upon continuous administration of choline chloride up to 10 % in the diet or up to 5 % in drinking water for 3-4 months. Based on observed systemic toxicity, an overall NOAEL of approx. 1,300 – 2,900 mg/kg/day, i.e. above the limit dose level for such studies, can be derived from this study (Hodge, 1945). In line with this, in a chronic exposure of rats to 1 % of choline chlorine via diet for 72 weeks, followed by a 31 week post-exposure observation period, no indication for any test item-related effect in reproductive organs was reported (Shivapurkar, 1986). Based on findings observed in this study, a NOAEL of >1 %, i.e. >1,200 mg/kg/day can be derived, clearly supporting the findings from the earlier study. These studies are further supported by a more recent repeated dose study, in which groups of rats were treated with choline chloride at 200 mg/kg/day via oral, intranasal or intraperitoneal administration for 28 days (Mehta, 2009). Although reproductive organs were not examined in detail, no test item-related changes were observed at necropsy and an overall NOAEL of >200 mg/kg/day was identified in this study. Furthermore, daily intraperitoneal (i.p.) administration, which can reasonably be considered as worst-case administration route, of 25 mg chlorine chloride per rat for 12 days did not impact on spermatogenesis at all, and prolonged daily i.p. administration for 24 days yielded effects which were reversible and not considered adverse (Vachhrajani, 1993). These repeated dose-type studies clearly indicate the absence of adversity up to the limit dose level of 1,000 mg/kg/day, and provide no indication for any adverse effect on reproductive organs.

Potential developmental toxicity was studied in mice and rats. In mice, continuous exposure to up to 10 % of choline chloride via diet from gestational days (GD) 1-18 clearly caused maternal toxicity shown as significantly reduced body weight compared to concurrent control animals beginning at 2.5 % choline chloride, and also increased abortion rates as secondary consequence (BASF, 1966). Test concentrations up to 1 % equivalent to 1,250 mg/kg/day, i.e. again above the limit dose level for such studies, were well tolerated by the dams and did also not affect foetal development, clearly demonstrating the absence of a specific developmental toxicity in mice. In rats, daily exposure of the dams to 5 % of choline chloride via diet from GD 2 -21 and until postnatal day 21 was even found to ameliorate the effects of prenatal stress on anxiety-related behaviours of adult offspring assessed in open field, elevated zero maze and social interaction tests, while no adverse effect on development was reported (Schulz, 2014). In another study, oral administration of choline at test concentrations up to 6.2 g choline/kg diet, corresponding to 1240 mg choline/kg bw/day, to pregnant rats for 21 d starting 24-48 h before parturition to ensure exposure at initiation of suckling was found to decrease body weights of dams and pups at the highest test concentration (-15 % and -20 % compared to standard diet containing 1 g choline/kg diet for dams and pups, respectively) but had no effect on the different immune cell phenotypes in the spleen and only a minimal effect on maternal immune cell function at the highest dose level (Dellschaft et al., 2015). Noticeably, this study found that choline is required to maintain maternal immune function in lactating dams. A follow-up study investigating the impact of feeding lactating dams with mixtures of choline forms on the development and later immune function of suckled offspring revealed that feeding mixed choline diets during lactation improved T cell phenotype/function at the end of suckling and programmed a less inflammatory response later in life (Richard et al., 2017). Taken together, these data do not provide any evidence for any pre- or postnatal developmental toxicity, and also developmental neurotoxicity and immunotoxicity of choline chloride in rats and/or mice upon administration to dose levels even beyond the limit dose level of 1,000 mg/kg/day.

These conclusions are further supported by a combined repeated dose toxicity study with the reproduction/developmental screening test according to OECD TG 422 in rats conducted with trimethylamine (TMA) (Takashima et al. 2003 reported in NAC/AEGL Committee, 2008). More specifically, as outlined in the registration dossier in more detail, choline is absorbed from the jejunum and ileum mainly by a saturable, energy-dependent carrier mechanism in the brush-border membrane and to a lesser extent via passive diffusion. This choline intake can be limited by its efficient metabolism by the intestinal microflora to mainly TMA, which even increases when high doses of choline are applied as e.g. found by Zeisel (1989). In this study using administration of radiolabelled choline to SD rats via orogastric intubation, it was found that low doses of choline were absorbed from the intestinal lumen before it reached the areas of gut colonized with bacteria, while at the high dose of choline, much more label reached the colon. While the authors suggested that an appreciable portion was also absorbed via diffusion across the colon, a disproportionate rise in TMA, and also oxidised TMA, excretion via urine was observed at the higher choline dose, suggesting that TMA was formed when choline reached the bacterially colonized large intestine in appreciable quantities. Thus, for the purpose of regulatory hazard assessment up to limit dose levels, information derived from an oral toxicity study conducted with TMA can be used to assess potential adverse effects which may occur after oral administration of considerably high amounts of choline compounds. In the above-mentioned OECD 422 study in rats exposed to 8, 40 or 20 mg/kg/day of TMA via gavage, maternal toxicity was only seen at 200 mg/kg/day, consisting of excessive salivation, abnormal breathing noise and one death at day 38. In contrast, no adverse effect on reproductive organs or any fertility parameter as well as no embryo-/fetotoxicity was observed up to the highest tested dose level of 200 mg/kg/day, yielding a reproductive/developmental NOAEL of 200 mg/kg/day and a NOAEL of 40 mg/kg/day for systemic toxicity (Takashima et al., 2003 reported in reported in NAC/AEGL Committee, 2008). As mentioned above, exposure to 200 mg/kg/day of TMA represents exposure to much higher amounts of choline due to the metabolism of choline to TMA via intestinal microbiota occurring predominantly at high choline doses exceeding capacities for absorption.

These conclusions drawn from existing animal studies are clearly supported by human studies. Several human studies focussing on maternal choline intake and pregnancy/child health outcomes have been conducted. Randomized clinical trials conducted in healthy pregnant women have reported no adverse effects of choline supplementation at levels ranging from 550 to 900 mg/day (summarised in Korsmo et al., 2019). Moreover, controlled clinical trials have been conducted with the aim to use choline supplementation in young children with foetal alcohol spectrum disorder (FASD) to support their neurodevelopment for targeting the core neurocognitive and behavioural deficits associated with FASD (summarised in e.g. Wozniak et al., 2020). In these studies, pre- or postnatal repeated administration of choline at doses ranging from 500 to 625 mg (children) or 750 to 2000 mg (pregnant females) choline per day did not cause any adverse effect but was in contrast found to improve non-verbal intelligence, visual-spatial skill, working memory ability and verbal memory, and furthermore to lower behavioural symptoms of attention deficit hyperactivity disorder. No differences were seen for verbal intelligence, visual memory, or other executive functions between choline-supplemented and placebo groups. These human data clearly underline absence of adverse effects of choline on pre- or postnatal development.

Taken together, these data consistently do not provide any evidence for an adverse effect of choline on male and female fertility or pre- or postnatal developmental toxicity. Furthermore, prolonged repeated exposure to up to 72 weeks does not increase its toxicity or any effect on reproductive organs, supporting the absence of bioaccumulation potential as discussed in-depth in the registration dossier. Thus, the available dataset allows the conclusion that choline chloride is not a reproductive toxicant and classification according to CLP Regulation (Regulation (EC) No 1272/2008) is not warranted.

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

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
10 % = 100,000 ppm	with abortion 7	- 5,2

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
Choline chloride concentration in feed (%)	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)

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

EMMean (SE)	Placebo (n = 16)	Choline (n = 14)a	Statistic	Significance	Effect size
Verbal IQ	88.3 (2.8)	90.6 (3.1)	F (1, 28) = 0.29	p = 0.60	PE² = 0.01
Non-Verbal IQ	85.6 (2.1)	92.9 (2.4)	F (1, 28) = 5.17	p = 0.03*	PE² = 0.17
Fluid Reasoning	88.1 (3.7)	90.3 (4.1)	F (1, 28) = 0.15	p = 0.70	PE² = 0.01
Knowledge	85.0 (2.3)	87.5 (2.6)	F (1, 28) = 0.50	p = 0.49	PE² = 0.02
Quantitative Reasoning	93.1 (2.1)	92.7 (2.3)	F (1, 28) = 0.02	p = 0.90	PE² = 0.00
Visual-Spatial Processing	91.3 (3.0)	98.3 (3.3)	F (1, 28) = 2.38	p = 0.14	PE² = 0.08
Working Memory	84.0 (2.5)	94.4 (2.8)	F (1, 28) = 7.74	p = 0.01*	PE² = 0.23
Full-Scale IQ	86.1 (2.4)	91.1 (2.7)	F (1, 28) = 1.86	p = 0.19

Mean (SE) {n}	Placebo	Choline	Statistic	Sign.	Effect size
Memory
EI short delay components a	96.1(0.9) {16}	97.8 (0.9) {15}	F (1, 30) = 1.89	p = 0.18	PE2 = 0.06
EI short delay pairs a	63.2 (2.6) {16}	63.8 (2.7) {15}	F (1, 30) = 0.29	p = 0.87	PE2 = 0.00
EI short delay adjacent pairs a	21.3 (3.8) {16}	24.7 (4.0) {15}	F (1, 30) = 0.38	p = 0.55	PE2 = 0.13
NEPSY-II Memory for Names Delayed	6.2 (3.2) {16}	9.1 (4.3) {15}	F (1, 30) = 4.75	p = 0.04*	d = 0.77
NEPSY-II Memory for Faces Delayed	8.8 (3.7) {13}	8.7(2.3) {14}	F (1, 26) = 0.00	p = 0.96	d = 0.03
NEPSY-II Narrative Memory	7.8 (3.6) {16}	8.3 (2.4) {15}	F (1, 30) = 0.01	p = 0.93	d = 0.16
NIH Toolbox PSMT	47.4 (13.6) {16}	50.9 (14.2) {15}	F (1, 30) = 0.49	p = 0.49	d = 0.25
Executive functioning
NIH Toolbox DCCST	40.4 (7.5) {15}	44.1 (10.8) {15}	F (1, 29) = 1.29	p = 0.27	d = 0.40
NIH Toolbox Flanker test	39.8 (8.0) {16}	45.6 (9.6) {15}	F (1, 30) = 3.32	p = 0.08	d = 0.66

Registration Dossier

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Choline chloride

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Effects on fertility

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Effect on fertility: via oral route

Effect on fertility: via inhalation route

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Effect on developmental toxicity: via oral route

Effect on developmental toxicity: via inhalation route

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