Choline hydrogen carbonate

Endpoint summary

Description of key information

Repeated dose Toxicity:
- Chronic (72 weeks + 31 weeks post-observation) study oral (feed), rat (Fischer 344) male (similar to OECD guideline 452): NOAEL > 1200 mg/kg bw/day (Choline chloride, highest / only dose tested), corresponding to 1420 mg/kg bw/day Choline bicarbonate
- Subchronic (3 – 4 months) study oral (feed and drinking water), rat m/f (similar to OECD Guideline 408): NOAEL = 1300 – 2900 mg/kg bw/day Choline chloride, or 1540 - 3430 mg/kg bw/day Choline bicarbonate, LOAEL = 3400 – 5000 mg/kg bw/day Choline chloride, or 4020 - 5915 mg/kg bw/day Choline bicarbonate
- Subacute (28 day) study oral (gavage), intraperitoneal and intranasal, mouse (Balb/c) m/f (GLP, OECD Guideline 407 / no guideline available): NOEL > 200 mg/kg bw/day (Choline chloride, highest / only dose tested), corresponding to 236.6 mg/kg bw/day Choline bicarbonate

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: oral

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was performed on the read-across substance Choline chloride similar to OECD 452 with minor deviations and not all details are given. However, the available information is sufficient to consider the results as reliable.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Deviations:

yes

Remarks:

only male rats used

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, NY
- Age at study initiation: no data
- Weight at study initiation: 50 - 60 g
- Fasting period before study: no
- Housing: three per cage in plastic shoebox cages
- Diet (e.g. ad libitum): ad libitum a natural ingredient ground chow (Wayne Laboratory Blox Allied Mills, Inc., Chicago, IL)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
chow diet was supplemented with 1.0 % Choline chloride

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

five days of non-supplemented chow, i.p. injection of saline (since the choline group served as a control group in a tumor promoting study), five days of non-supplemented chow.
72 weeks of treatment with supplemented chow, followed by 31 weeks post-observation with non-supplemented chow

Frequency of treatment:

continuously, i.e. application via feed which was availabel ad libitum

Remarks:

Doses / Concentrations:
1 %
Basis:
nominal in diet

No. of animals per sex per dose:

30 males / dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Choline treated rats served as controls in a tumor promoting study on Phenobarbital (PhB) and 1,1 bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), the levels of Choline chloride was based on previous experiments wherein these levels were shown to inhibit the drop in hepatic S-adenosylmethionine levels due to PhB and DDT
- Rationale for animal assignment (if not random): random

Positive control:

Due to original study aim (tumor promoting effects), results will be given derived from animals which were initiated with i.p. injection of 200 mg/kg bw of diethylnitrosamine dissolved in sterile normal saline followed by a diet supplemented with 0.05 % 1,1 bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT)

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: No data, only body weight denoted

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were taken at weekly intervals for 16 weeks and biweekly thereafter

OTHER: Gross pathology performed

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 1)
HISTOPATHOLOGY: Yes, but no results provided

Other examinations:

Growth and survival

Statistics:

The comparisons between the incidences of tumors in different groups were analysed by Fischer's exact test. The survival data were analysed by the computer program developed by Thomas el al. (Thomas CG, Breslow N and Gart JJ (1977) Trend and homogeneity analyses of proportions and life table data. Computers Biomed. Res., 10, 373-381.). Kaplan-Meier survival curves were derived by using this procedure. Comparison among survival of different experimental groups were made by Cox's test and P value based on the chi-square test from Cox's analysis. The incidences of liver and lung tumors and of leukemias in each group were based upon the number of animals surviving at 1 year since the first instance of each of the tumors occurred between weeks 55 and 60.

Clinical signs:

no effects observed

Description (incidence and severity):

mortality: no difference compared to control animals

Mortality:

no mortality observed

Description (incidence):

mortality: no difference compared to control animals

Body weight and weight changes:

no effects observed

Description (incidence and severity):

no difference compared to control animals

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

relative liver weight: no difference compared to control animals

Gross pathological findings:

no effects observed

Description (incidence and severity):

performed, but limited data given; Liver and lung tumor formation: no difference compared to control animals

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Various lung, liver and other tumors, leukemia: no difference compared to control animals

Details on results:

CLINICAL SIGNS AND MORTALITY - no difference compared to control animals

BODY WEIGHT AND WEIGHT GAIN - no difference compared to control animals

ORGAN WEIGHTS - relative liver weight: no difference compared to control animals

GROSS PATHOLOGY - performed, but limited data given; Liver and lung tumor formation: no difference compared to control animals

HISTOPATHOLOGY: NEOPLASTIC (if applicable) - various lung, liver and other tumors, leukemia: no difference compared to control animals

HISTORICAL CONTROL DATA (if applicable) controls see table 1

OTHER FINDINGS See table 1

Dose descriptor:

NOAEL

Effect level:

> 1 other: percent in diet

Based on:

test mat.

Remarks:

Choline chloride

Sex:

male

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Effect level:

> 1 200 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

Choline chloride

Sex:

male

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Effect level:

> 1 420 mg/kg bw/day (nominal)

Based on:

other: Choline bicarbonate

Sex:

male

Basis for effect level:

other: Basis for effects: overall effects; mortality; body weight; gross pathology; organ weights Recalculated value from NOAEL > 1200 mg/kg bw Choline chloride regarding the molecular weight of each compound.

Critical effects observed:

not specified

Table 1: Results – Comparison of rats fed 1% choline chloride in diet to control group (plain diet)

Endpoint		Treatment Group (1 % Choline chloride in diet)	Control Group (plain diet)	Positive Control Group (i.p. 200 mg/kg bw of diethylnitrosamine, 0.05 % DDT in diet)
Survival at week	52	28	28	28
	78	28	28	21
	102	24	23	3
Body weight at week / g	10	258	253	262
	50	406	408	394
Relative liver weight (%)		3.4	3.60	13.42
Number of animals bearing liver tumors	Neoplastic nodules	2	2	1
	Hepatocellular carcinomas	0	1	28
	Cholangiomas + cholangiocarcinomas	0	0	6
	Lung metastases	0	0	13
Number of animals with tumors / extrahepatic lesions	Lung	1	2	4
	Leukemia	2	8	4
	Others	4	7	8

Conclusions:

The present study was classified as reliable with restrictions due to the limited information provided and the fact that it was performed on the read-across substance Choline chloride. However, since the available information is sufficient to consider the study as reliable, the results obtained can be used to assess the repeated dose toxicity of Choline chloride and hence, Choline bicarbonate. The study duration was 103 weeks, wherein the animals were dosed the first 72 weeks with 1 % Choline chloride in diet. Consequently, considering the total life span of a rat of approx. 1.5 - 2 years, the study duration was chosen long enough to detect all possible effects arising from Choline chloride and so Choline hydrogen carbonate.
The read-across from Choline chloride to Choline hydrogen carbonate is justified because the absorption after oral application is very likely to remained unchanged, since the toxicologically relevant choline cation is identical in both salt, its transport mechanisms in the body are only relevant for the cation, too, and no absorption-enhancing irritating effects are expected from the anion. So, information gained from Choline chloride for this endpoint can be used as weight-of-evidence information without modification except adjusting the dose regarding the molecular weight of both choline salts.
No adverse effects were detected compared to control. In fact, although not statistically significant, Choline chloride treated animals developed less tumors than control animals. Also, no effects were seen regarding body weight gain, and the relative liver weight was also slightly, but not significantly decreased compared to control. This could be due to the fact that Choline chloride, which is also used as a feed additive, is an effective methyl donor, which does not require extensive metabolic pathways, which could possibly lead to additional liver damage due to hazardous degradation products. Hence, it is likely that CC does not only exhibit no adverse effects but also liver-protecting effects. Most likely effects for an increased liver weight can be (non)-neoplastic lesions, fatty liver or scar formation / cirrhosis due to necrosis already on only single cellular level, and also an increased requirement of metabolic enzymes. These effects are diminished by an additional gavage of Choline chloride. Furthermore, the positive control (i.p. 200 mg/kg bw of diethylnitrosamine, 0.05 % DDT in diet) led to a decreased body weight and increased relative liver weight and tumor formation compared to control, which is an additional reason why the study, and so the results, can be considered as valid.
So, taking further into account the average food consumption of 120 g/kg bw/day of a male rat as given in ECHAs guidance document R.8, and the fact that no adverse effects were denoted compared to control at an average Choline chloride (CC) consumption of 1 % in diet, the NOAEL was determined to be > 1 % CC in diet, which corresponds to > 1200 mg/kg bw/day (nearly life time duration).
In conclusion, it can be stated that Choline chloride does not induce any adverse effects and can be considered as non-toxic when administered chronically to rats, and no classification, neither as carcinogenic or STOT-RE, is required.

Executive summary:

In a chronic toxicity study equivalent to OECD Guideline 452, the read-across substance Choline chloride was administered orally 1 % in feed to male Fischer 344 rats, 30 animals per group, over 72 weeks with 31 weeks post-observation period.

There were no compound-related adverse effects denoted compared to control regarding the observed endpoints, i.e. body weight and body weight gain, relative liver weight, Tumor formation in liver and lung, leukemia and other tumors. So the NOAEL is > 1 % Choline chloride in food, based on all observed effects, which corresponds to NOAEL > 1200 mg/kg bw/day of Choline chloride or NOAEL > 1420 mg/kg bw/day of Choline hydrogen carbonate.

This chronic toxicity study in rats is acceptable with restrictions, satisfies the guideline requirements for a chronic oral toxicity study (OECD 452) in rats, and allow to draw the conclusion that Choline chloride and hence Choline bicarbonate is practically non-toxic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 420 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Three equivalently reliable studies on the read-across substance Choline chloride, assessed with Klimisch 2, are available to cover the endpoint "Repeated Dose Toxicity: oral". Three different study durations are covered, one subacute, one subchronic and one chronic. The former covers the standard information requirements as demanded by REACH Annex IX column 1 No. 8.6.1 (28 days), the latter two ones, which are due to the test duration the more important ones, cover REACH Annex IX column 1 No. 8.6.2 (90 days resp. column 2, chronic). Hence, the available information meets fully the tonnage-driven data requirements of REACH.
Additionally, all available studies revealed equivalent, plausible and consistent results over all three durations, i.e. give all no rise to concern of compound-related toxic effects when applying choline chloride, and so Choline bicarbonate, repeatedly and trigger no classification as STOT-RE. So, the whole database is of high quality.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There are three studies available evaluating the possible hazards of the read-across substance Choline chloride, and hence Choline bicarbonate (CbiC), by repeated oral application. The read-across from Choline chloride to Choline hydrogen carbonate is justified because the absorption after oral application is very likely to remain unchanged, since the toxicologically relevant choline cation is identical in both salt, its transport mechanisms in the body are only relevant for the cation, too, and no absorption-enhancing irritating effects are expected from the anion.

The expected most relevant exposure route is the oral route. Once dissociated in the aqueous acidic environment in the stomach, the bicarbonate anion will be transformed. In an aqueous solution, hydrogen carbonate is in an equilibrium with the anhydrate of the carbonic acid, carbon dioxide (CO2):

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

Due to the strongly acidic environment of the stomach, the equilibrium will be furthermore shifted to the side of CO2, which is a regular product of cell respiration, is so ubiquitous present in the body anyway and subject of effective excretion processes.

So, information gained from choline chloride for this endpoint can be used as weight-of-evidence information without modification except adjusting the dose regarding the molecular weight of both choline salts.

The relevant studies are:

a) Chronic study (Shivapurkar et al., 1986):

No adverse effects were detected when applying 1200 mg/kg bw/day Choline chloride (CC), corresponding to 1420 mg/kg bw/day CbiC, compared to control. In fact, although not statistically significant, choline treated animals developed less tumors than control animals. Also, no effects were seen regarding body weight gain, and the relative liver weight was also slightly, but not significantly decreased compared to control. This could be due to the fact that choline, which is also used as a feed additive, is an effective methyl donor, which does not require extensive metabolic pathways, which could possibly lead to additional liver damage due to hazardous degradation products. Hence, it is likely that CC does not only exhibit no adverse effects but also liver-protecting effects.

In conclusion, it can be stated that Choline chloride, and so Choline bicarbonate, does not induce any adverse effects and can be considered as non-toxic when administered chronically to rats, and no classification, neither as carcinogenic or STOT-RE, is required.

b) Subchronic study (Hodge, 1945):

With an increasing dose of the read-across substance Choline chloride applied either in the feed or drinking water over 3-4 months to rats, the body weight gain decreases at doses ≥ 2.7 % in feed (≙1869.2 - 2554.6 mg CC/kg bw/day, or 2211.3 - 3022.2 mg CbiC/kg bw/day, respectively) or ≥ 1 % in drinking water (≙1337.6 - 2153.8 mg CC/kg bw/day, or 1582.4 - 2548.0 mg CbiC/kg bw/day, respectively), as do the organ weights. Nevertheless the relative organ weights remain unchanged. Also, the average food intake and water consumption decreases with an increasing dose of Choline chloride. Hence, it is most likely that the observed effects, i.e. body weights gain, are due to the decreased feed and water consumption because of a possibly undesired taste of the feed or water. So, the required nutrients for growth are not or not fully available to permit body weight gain comparable to control. As a conclusion, the observed effects are not due to intrinsic toxic effects of Choline chloride, and so Choline bicarbonate, but only a consequence of feed and water refusal due to the taste. This conclusion is furthermore supported by the fact that only the organ weights, but not the relative organ weights were diminished compared to control and also all histopathological examinations were negative or not CC-dose-related. So it is only possible to determine mainly NOELs but not NOAELs since no adverse effects can be detected which are directly attributable to toxic effects of Choline chloride, and so Choline bicarbonate.

Additionally, the dose may increase when taking into account the nominal CC percentage in feed or water. Nevertheless, when correcting the dose to the actual feed or water intake to units of mg/kg bw/day, all effects are in the same range of approx. 1300 – 2900 mg/kg bw/day choline chloride or 1540 - 3430 mg/kg bw/day choline bicarbonate, respectively. Since, as stated above, these effects are not due to intrinsic toxic effects of CC or CbiC, this value should be considered to be the NOAEL within this study. One may argue that all animals in the groups receiving 4 % and 5 % CC in water died. However, also these effects can be explained by limited water consumption which is in the range of only 10 % of the control groups, and also not due to intrinsic toxic properties of CC or CbiC, because limited water consumption is in general accompanied with more severe effects than limited feed consumption.

The only observed exemption is made by the 10 % dose group with substance application via feed. This is the only dose group in which also the relative organ weights deviate from control and deaths occurred during the feed study. Also, the absolute CC intake is markedly increased, compared to the other dose groups, to approx. 3400 – 5000 mg CC/kg bw/day or 4020 - 5915 mg CbiC/kg bw/day, respectively. Hence, these effects may be considered as directly related to the Choline chloride intake and therefore as the LOAEL in th subchronic study.

c) Subacute study (Mehta et al., 2009):

The results are not only available for oral administration, but also for i.p. and i.n. routes, which broadens the insight in possible effects by choline.

In general, all observed parameters (body weights gain, organ weights, haematological parameters or clinical bio-chemistry parameters) did not deviate from the control groups, when Choline chloride was applied at a dose of 200 mg/kg bw/d, which corresponds to 236.6 mg/kg bw/day Choline bicarbonate. The only observed derogation from control was an elevated creatinine level. Since this is most probably due to the application route, which is not relevant for humans, this effect can be neglected: A direct absorption via diffusion into the kidney tissue and therefore direct effects of the unmetabolized choline on the rather sensitive organ is the most likely reason for a diminuished creatinine clearance. When applied via other routes, a massive metabolism can be assumed, for example via intestinal bacteria or first pass effect, e.g. by cytochrome P450 3A4 (oral application), or cytochrome P450 1A (intranasal and consequently lung tissue).

So, the NOEL was determined as > 200 mg/kg bw/d Choline chloride or 236.6 mg/kg bw/ day choline bicarbonate, respectively. Since this was the highest dose tested and not a single relevant effect was observed, it can probably be much higher, which is supported by a NOAEL > 1420 mg/kg bw/day (chronic study, Shivapurkar et al., 1986), and the results on Choline chloride and so Choline bicarbonate do not indicate any need for classification.

 

No studies are available for the repeated application via inhalation or dermal route. However, they were not conducted due to animal welfare as they were not considered necessary for human risk assessment:

The oral route is considered to be the most likely one for humans, because the test substance has a very low vapor pressure (9.13 * E-10 Pa at 25 °C, EPIWIN, modified grain method) and is expected to have a high melting point, so the potential for the generation of inhalable forms is low. Furthermore, the substance is distributed as an aqueous solution, so no dust with inhalable particles will be formed and therefore no acute inhalation test was performed.

Additionally, the study performed by Mehta et al., 2009 (IUCLID chapter 7.5.1), covers three different applications routes of the read-across substance Choline chloride over 28 days including intranasal application, which is the most similar application route to inhalation, as the absorption will be over the trachea and lung, too, and so the compound is objected to the same absorption rates and tissue-specific metabolism, e.g. rather via cytochrome P450 1A than CYP 3A4. In this study, no substance-related toxic effects were denoted up to the highest dose tested, i.e. the NOEL by i.n. application is >200 mg/kg bw/day Choline chloride, corresponding to 236.6 mg/kg bw/day Choline bicarbonate.

Although skin contact may possibly occur when using Choline bicarbonate, the physico-chemical and toxicological properties do not suggest potential for a significant rate of absorption through the skin. Skin absorption is influenced by several factors, i.a.:

- Molecular weight: Less than 100 favours dermal uptake. Above 500 the molecule may be too large. With a molecular weight of 165.2 g/mole, absorption is possible, but not highly favoured.

- LogPow: For substances with Log Pow values <0, poor lipophilicity will limit penetration into the stratum corneum and hence dermal absorption. Values < –1 suggest that a substance is not likely to be sufficiently lipophilic to cross the stratum corneum, therefore dermal absorption is likely to be low. Since Choline bicarbonate, due to its ionic structure and rather small molecular weight, has a negative logPow, dermal absorption may practically not occur.

- Water solubility: If water solubility is above 10,000 mg/L and the LogPow value below 0, the substance may be too hydrophilic to cross the lipid rich environment of the stratum corneum. Dermal uptake for these substances will be low. As stated above, LogPow is expected to be below 0, and additionally, water solubility is very high, i.e. > 750 g/L. Also here, dermal absorption may practically not occur due to the high hydrophilicity of the compound.

- Skin irritation / corrosion: If the substance is a skin irritant or corrosive, damage to the skin surface may enhance penetration. Choline bicarbonate was not classified as irritant to the skin, therefore, no additional penetration enhancement must be considered. Hence, based on the physico-chemical properties of Choline bicarbonate, a dermal absorption is much hindered and so unlikely. Consequently, no additional testing is required.

 

In summary, there are three repeated dose studies with oral application available on the read-across substance Choline chloride, all assessed with Klimisch 2. Hence, the results obtained can be used to assess the repeated dose toxicity of Choline hydrogen carbonate. All available studies revealed equivalent, plausible and consistent results over all three durations including a nearly lifetime exposure with an NOAEL > 1200 mg/kg bw Choline chloride, corresponding to 1420 mg/kg bw/day Choline bicarbonate, i.e. all studies give no rise to concern of compound-related toxic effects when applying Choline chloride and so Choline bicarbonate repeatedly and trigger no classification as STOT-RE.

There are no repeated dose toxicity studies available with dermal application or via inhalation. However, these studies are not considered necessary due to exposure or other considerations regarding the unlikeliness of absorption as stated above.

Consequently, there are no data gaps in repeated dose toxicity. Although no human data is available on the possible hazards of the substance, there is no reason to believe that the observed lack of adverse effects in all studies would not be relevant for Humans, as the available animal models are all accepted as an scientifically justified surrogate for human testing. All possible deviations regarding absorption, metabolism or other factors are considered to be negligible when assessing the possible risk for humans with the available animal data.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Three equally reliable studies on the read-across substance Choline chloride are available for this endpoint. Hence, the study with the longest test duration was chosen as this is the likeliest study to detect all possible substance-related effects with repeated application, which is the actual purpose of the endpoint.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
According to REACH Annex IX column 1, the most appropriate route of administration, having regard to the likely route of human exposure, has to be chosen for repeated dose toxicity testing. There are three different studies available in which theread-across substance Choline chloride was applied orally: This route is considered to be the most likely one for humans, because the test substance has a very low vapor pressure and is expected to have a high melting point, so the potential for the generation of inhalable forms is low. Furthermore, the substance is distributed as an aqueous solution, so no dust with inhalable particles will be formed and therefore no acute inhalation test was performed.
Additionally, the study performed by Mehta et al., 2009 (IUCLID chapter 7.5.1), covers three different applications routes over 28 days including intranasal application, which is the most similar application route to inhalation, as the absorption will be over the trachea and lung, too, and so the compound is objected to the same absorption rates and tissue-specific metabolism, e.g. rather via cytochrome P450 1A than CYP 3A4. In this study, no substance-related toxic effects were denoted up to the highest dose tested, i.e. the NOEL by i.n. application is >200 mg/kg bw/day Choline chloride or >236.6 mg/kg bw/day Choline bicarbonate. According to REACH Annex IX column 2 No. 8.6.2, the sub-chronic toxicity study (90 days) does not need to be conducted if the substance is unreactive, insoluble and not inhalable and there is no evidence of absorption and no evidence of toxicity in a 28-day "limit test", particularly if such a pattern is coupled with limited human exposure. This condition contributes additionally to the conclusion of the applicant, that no repeated dose testing via inhalation route needs to be performed and can consequently be waived due to animal welfare.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
In general, an inhalation study does not need to be provided due to exposure and other considerations as laid down above.
Additionally, the study by Mehta et al., 2009 (IUCLID chapter 7.5.1), covering also the intranasal application route, did not state any local effects after the application of 200 mg/kg bw / day Choline chloride over 28 days, which can be transferred to Choline bicarbonate. Also here, no need for additional testing was apparent.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
According to REACH Annex IX column 1, the most appropriate route of administration, having regard to the likely route of human exposure, has to be chosen for repeated dose toxicity testing. There are three different studies available in which the read-across substance Choline chloride was applied orally. This route is considered to be the most likely one for humans.
Also, according to REACH Annex IX column 2 testing by the dermal route is appropriate if (1) skin contact in production and/or use is likely; and (2) the physico-chemical properties suggest a significant rate of absorption through the skin; and (3) one of the following conditions mentioned therein is met.
Although inhalation of Choline bicarbonate is unlikely and skin contact may possibly occur when using Choline bicarbonate, the latter conditions do not apply. The physico-chemical and toxicological properties do not suggest potential for a significant rate of absorption through the skin. Skin absorption is influenced by several factors, i.a.:
- Molecular weight: With a molecular weight of 165.2 g/mole, absorption is possible, but not highly favoured
- LogPow: Since Choline bicarbonate, due to its ionic structure, has a negative logPow, dermal absorption may practically not occur because poor lipophilicity will limit penetration into the stratum corneum.
- Water solubility: Water solubility is very high, i.e. >750 g/L. Also here, dermal absorption may practically not occur due to the high hydrophilicity of the compound.
- Skin irritation / corrosion: Choline bicarbonate was not classified as irritant to the skin, so skin penetration is not enhanced, no additional penetration enhancement must be considered.
Hence, based on the physico-chemical properties of Choline bicarbonate, a dermal absorption is very hindered and so unlikely. Consequently, no additional testing is required and the testing for acute toxicity via dermal route for Choline bicarbonate can be waived.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
In general, a study via dermal the route does not need to be provided due to exposure and other considerations regarding the unlikeliness of absorption as laid down above. Furthermore, the substance was neither classified as skin or eye irritant and also, both available sensitization studies on guinea pigs with the read-across substance Choline chloride did not show any local effects after repeated application during the different phases of the test. So, also here, no need for additional testing was apparent.

Justification for classification or non-classification

In a chronic toxicity study, the NOAEL was determined to be > 1 % Choline chloride (CC) in diet, which corresponds to > 1200 mg/kg bw/day CC or > 1420 mg/kg bw/day Choline bicarbonate (CbiC) (over nearly life time duration). No substance-related effects on specific organs were reported. This is supported by the results of a subchronic study (NOAEL ca. 1300 – 2900 mg CC/kg bw/day or 1540 - 3430 mg CbiC/kg bw/day, LOAEL ca. 3400 – 5000mg CC/kg bw/day or 4020 - 5915 mg CbiC/kg bw/day) and a subacute study (NOEL >200 mg CC/kg bw/day or > 236.6 mg CbiC/kg bw/day), without any detectable specific organ toxicity after repeated exposure. Additionally, all mentioned doses for the no effect levels are above the guidance values for classification as STOT-RE Cat. 2, as given in section 3.9.2.9.7 of Regulation 1272/2008/EC.

Hence, Choline hydrogen carbonate does not need to be classified as STOT-RE.