Choline hydrogen carbonate

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well-documented Guideline study according GLP, but performed on formulation of Choline chloride with structural analogue (Chlorocholine chloride).

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted 1992-07-17

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

Directive 96/54/EEC

Deviations:

no

GLP compliance:

yes

Remarks:

including compliance statement

Type of study:

guinea pig maximisation test

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: David Hall Limited, Burton-on-Trent, Staffordshire, UK
- Age at study initiation: approx. 8 - 12 weeks
- Weight at study initiation: 312 - 393 g
- Housing: singly or in pairs in solid-floor polypropylene cages furnished with woodflakes
- Diet (e.g. ad libitum): Guinea Pig FD1 Diet, Special Diets Services Limited, Witham, Essex, UK; ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 22
- Humidity (%): 48 - 56
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Other: each animal was selected at random and given a number unique within the study which was written on a small area of clipped rump using a black indelible marker-pen.

Route:

intradermal and epicutaneous

Vehicle:

water

Concentration / amount:

5 % w/v solution in distilled water (Intradermal induction)
undiluted as supplied (Topical induction)
undiluted as supplied and 75 % v/v in water (Topical induction)

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

5 % w/v solution in distilled water (Intradermal induction)
undiluted as supplied (Topical induction)
undiluted as supplied and 75 % v/v in water (Topical induction)

No. of animals per dose:

10 (test group)
5 (control group)

Details on study design:

RANGE FINDING TESTS:
- Single intradermal exposure with 0.1 mL of either 1 %, 5 %, 10 % or 25 % w/v of the test item in distilled water, sighting test, for intradermal induction
- Two guinea pigs, intradermally injected with Freund's Complete Adjuvant fifteen days earlier, were topically (occlusive) exposed to 100 %, 75 %, 50 % and 25 % v/v of the test item in distilled water, for topical induction
- 100 %, 75 %, 50 % and 25 % v/v of the test item in distilled water, 14 d after induction with Freund´s Complete Adjuvant analogue to control animals in the main study, for topical challenge

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1 intradermal (row of three injections, see additional information), 1 topical 7 days later
- Exposure period: 48 h for topical induction
- Test groups:
Intradermal Induction: row of three injections, (1) Freund´s Complete Adjuvans (FCA) and H2O dest. 1:1, (2) 5% w/v solution of the test material in distilled water, (3) 5 % w/v solution of the test material in distilled water and FCA plus H2O dest. 1:1
Topical induction: undiluted test material
- Control group:
Intradermal Induction: row of three injections, (1) Freund´s Complete Adjuvans (FCA) and H2O dest. 1:1, (2) distilled water, (3) 50% distilled water and FCA plus H2O dest. 1:1
Topical induction: bare filter paper
- Site: shoulder
- Frequency of applications: Day 1 and 7
- Duration: Total 9 d
- Concentrations: 5 % w/v in water (intradermal), undiluted (topical)

B. CHALLENGE EXPOSURE
- No. of exposures:
- Day(s) of challenge: Day 21
- Exposure period: 48h
- Test groups: undiluted as supplied and 75 % v/v of test item in distilled water
- Control group: undiluted as supplied and 75 % v/v of test item in distilled water
- Site: right (100 % test item) and left (75 % v/v) shorn flank
- Concentrations: undiluted and 75 % v/v
- Evaluation (hr after challenge): 24h and 48 h

Challenge controls:

5 animals, undiluted test item and 75 % v/v of test item in distilled water

Positive control substance(s):

yes

Remarks:

historical control data from Neomycin Sulphate, 2-Mercaptobenzothiazole, 2,4-Dinitrochlorobenzene

Positive control results:

See attached background material "Driscoll_PositiveControlData" which demonstrates clearly that the laboratory has the capability to identify positive dermal sensitizers.
The incidences of sensitization of the test animals were 60% (Neomycin Sulphate), 70 - 90 % (2-Mercaptobenzothiazole) and 100 % (2,4-Dinitrochlorobenzene).

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100 % of the test item as supplied

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No skin reactions or differences in body weight gain compared to control were noted.

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100 % of the test item as supplied. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No skin reactions or differences in body weight gain compared to control were noted..

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100 % of the test item as supplied

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No skin reactions or differences in body weight gain compared to control were noted.

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100 % of the test item as supplied. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No skin reactions or differences in body weight gain compared to control were noted..

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

75 % v/v of the test item in distilled water

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No skin reactions or differences in body weight gain compared to control were noted.

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 75 % v/v of the test item in distilled water. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No skin reactions or differences in body weight gain compared to control were noted..

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

75 % v/v of the test item in distilled water

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No skin reactions or differences in body weight gain compared to control were noted.

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 75 % v/v of the test item in distilled water. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No skin reactions or differences in body weight gain compared to control were noted..

Reading:

1st reading

Hours after challenge:

24

Group:

other: challenge control

Dose level:

100 % of the test item as supplied

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No skin reactions were noted.

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: challenge control. Dose level: 100 % of the test item as supplied. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No skin reactions were noted..

Reading:

2nd reading

Hours after challenge:

48

Group:

other: challenge control

Dose level:

100 % of the test item as supplied

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No skin reactions were noted.

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: challenge control. Dose level: 100 % of the test item as supplied. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No skin reactions were noted..

Reading:

1st reading

Hours after challenge:

24

Group:

other: challenge control

Dose level:

75 % v/v of the test item in distilled water

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No skin reactions were noted.

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: other: challenge control. Dose level: 75 % v/v of the test item in distilled water. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No skin reactions were noted..

Reading:

2nd reading

Hours after challenge:

48

Group:

other: challenge control

Dose level:

75 % v/v of the test item in distilled water

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No skin reactions were noted.

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: challenge control. Dose level: 75 % v/v of the test item in distilled water. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No skin reactions were noted..

 

TABLE 1
Challenge concentrations: Undiluted as supplied and 75 % v/v Vehicle: Distilled water
Animal Number	Skin Reactions (Hours After Removal of Dressing)
	24 Hours						48 Hours
	100 %			75 %			100 %			75 %
	Er	Oe	Other	Er	Oe	Other	Er	Oe	Other	Er	Oe	Other
Individual skin reactions in test animals after challenge
1	0	0	-	0	0	-	0	0	-	0	0	-
2	0	0	-	0	0	-	0	0	-	0	0	-
3	0	0	-	0	0	-	0	0	-	0	0	-
4	0	0	-	0	0	-	0	0	-	0	0	-
5	0	0	-	0	0	-	0	0	-	0	0	-
6	0	0	-	0	0	-	0	0	-	0	0	-
^7	0	0	-	0	0	-	0	0	-	0	0	-
8	0	0	-	0	0	-	0	0	-	0	0	-
^9	0	0	-	0	0	-	0	0	-	0	0	-
10	0	0	-	0	0	-	0	0	-	0	0	-
Individual skin reactions in control animals after challenge
11	0	0	-	0	0	-	0	0	-	0	0	-
12	0	0	-	0	0	-	0	0	-	0	0	-
13	0	0	-	0	0	-	0	0	-	0	0	-
14	0	0	-	0	0	-	0	0	-	0	0	-
15	0	0	-	0	0	-	0	0	-	0	0	-

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The present study is a well-documented GLP study according OECD Guideline 406 (Guinea pig maximisation test) with the restriction, that it was conducted not on Choline bicarbonate but on a formulation with Choline chloride (320 g/L) and Chlorocholine chloride (CCC, 460 g/L), whereas the latter was shown not to be a sensitizer in the study by Suresh. So in general, the study was classified as Klimisch 2 and hence, the results can be safely used to assess the sensitising potential of the read-across substance Choline chloride and hence choline bicarbonate. The test item contains to relevant parts the choline cation, i.e. 41 % of the solid ingredients are Choline chloride, which can be assumed to be the only cause for a possible sensitizing effect, and the solvent (water) is not a known sensitizer. So since neither water nor CCC, which may also serve as a read-across substance, was shown to be a dermal sensitizer, it can be concluded that every possible positive result obtained from the formulation must be attributed to Choline chloride and hence, Choline bicarbonate. Since no animal of the test group at no timepoint of observation showed any skin reaction during the challenge phase, and the dose of the test item was properly determined by preliminary sighting tests as the demanded highest non-irritating dose, it can safely be concluded that the test item did not exhibit any sensitizing properties and consequently, that choline hydrogen carbonate is non-sensitizing, too. Hence, choline carbonate does not need to be classified as skin-sensitizer, neither according Regulation 1272/2008/EC nor Directive 67/548/EEC.

Executive summary:

In a dermal sensitization study according GLP and OECD Guideline 406 with a formulation of the read-across substance Choline chloride (CC) and Chlorocholine chloride (CCC) (320 g/L CC, 460 g/L CCC) in water, 8 - 12 weeks old male albino Dunkin Hartley guinea pigs (10 animals/ test were tested in the Guinea pig maximization test (GPMT according to Magnus and Kligmann)). Induction was performed once intradermal (Day 1) and once topical (Day 7, occlusive), topical challenge was on Day 21 (occlusive). Historical data from Neomycin Sulphate, 2-Mercaptobenzothiazole and 2,4-Dinitrochlorobenzene served as positive controls and induced the anticipated effect.

No skin reactions were observed, neither in the test nor control group, and body weight gain of the test group was similar to control. Since no sensitizing effects were noted in none of the test animals in this study, the formulation of CC and CCC, and consequently also pure Choline chloride or Choline bicarbonate, is not a dermal sensitizer, and does therefore not need to be classified as sensitising to the skin, neither according Regulation 1272/2008/EC nor Directive 67/548/EEC.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

There are two well-documented guideline studies according to GLP available which would normally be classified as Klimisch 1, but since they were not conducted on Choline bicarbonate itself, they could be only classified as Klimisch 2. Nevertheless, they are still reliable enough to assess the possible skin sensitising properties of Choline bicarbonate and so no data gaps are identified for this endpoint out of the following reasons:

Both studies were performed according to OECD 406 and GLP, which is one of the reliable foreseen standard tests to cover this endpoint. The study by Driscoll, 1998, was performed on a formulation with Choline chloride (CC, 320 g/L) and chlorocholine chloride (CCC, 460 g/L), which does not only contain to relevant parts of the choline cation, i.e. 41 % of the solid ingredients are choline chloride, but is additionally performed on CCC which can serve as a read-across substance for Choline bicarbonate (CbiC). The study by Suresh, 1992, was conducted on the read-across substance for CbiC, chlormequat (CCC), only. In general, read-across, as long as it is scientifically justified, is a foreseen approach under REACH, and so the two available studies leave no data gap for the endpoint 7.4.1, Skin sensitization.

Since the chlorine moiety of CCC could lead to an altered metabolism and most likely to more reactive metabolites forming allergens by altering the bodies proteins, and hence to exhibit immunomodulating properties, the outcome of the study by Suresh on CCC only could most likely overestimate the actual sensitising effects of the relavant choline cation in CbiC. Since only two of eight animals showed a very slight erythema only at one single timepoint (72 h of challenge phase) and even no effects were noted during the rechallenge phase, it can be safely concluded that chlormequat and hence Choline bicarbonate do not exhibit sensitising properties and do therefore not need to be classified as sensitisers.

The test item in the study by Driscoll, 1998, contains to relevant parts the read-across substance Choline chloride, i.e. 41 % of the solid ingredients, and the solvent (water) is not a known sensitizer. So since neither water nor CCC, which may also serve as a read-across substance, was shown to be a dermal sensitizer, it can be concluded that every possible positive result obtained from the formulation must be attributed to Choline chloride conatining the same sensitisation-relevant organic cation than Choline bicarbonate. Since no animal of the test group at no timepoint of observation showed any skin reaction during the challenge phase, and the dose of the test item was properly determined by preliminary sighing tests as the demanded highest non-irritating dose, it can safely be concluded that the test item did not exhibit any sensitizing properties and consequently, that Choline bicarbonate is non-sensitizing, too.

One of the requirements set in OECD Guideline 406 is to choose the test doses based i.a. on skin irritating effects. The doses for the CCC-CC formulation were properly set, but might be rather low regarding CC only as it is only contained to 41 % in the solid test item. However, Choline chloride is not classified as irritating, and the challenge in the study by Driscoll, 1998, was done i.a. with the undiluted test item, and so it is not possible anyway to perform the induction in a sensitization study with the demanded concentration, i.e. which causes mild-to-moderate skin irritation. So the tested dose of Choline chloride in this study can be considered to be chosen high enough to detect a possible sensitizing effect, especially when taking into account the fact that the study by Driscoll was performed as a guinea pig maximization test.

The guinea pig maximization test (GPMT) is more sensitive than the method by Buehler, because it uses intradermal injections and Freund´s Complete Adjuvans, which is intended to stimulate unspecifically the immune system by the use of proteins as antigens. So in general, the main problem when assessing the results obtained in a GPMT, is that the skin reactions may overestimate the situation in real life and lead to false positive results. Since no skin reaction was seen in any of the test animals, it can safely be concluded that the CC-CCC formulation and hence Choline bicarbonate does not exhibit skin-sensitizing properties.

Additionally, possible mixture effects of the CC-CCC formulation can be ruled out: The formulation did not induce any skin reactions in the Magnus-Kligmann test, CCC alone was also tested negative for skin sensitizing effects in the Buehler assay except a single very slight erythema 72h during the challenge phase in two out of eight animals. Although these results are definitively not sufficient to justify a classification as skin-sensitizing of chlormequat, it can be concluded that CCC is due to its chlorine moiety the more reactive component of the formulation and hence the possibly more immuno-modulating component. In case CCC could really enhance the formation of antigens and consequently the stimulation of the adaptive immune system, the immune system would be more reactive in general and could consequently easier detect possible antigens formed by CC or CbiC, which can be assumed to be rather similar to the antigens formed by CCC and consequently be easily recognized. Since this possible stimulation would work according to the same principle as Freund´s complete adjuvans, it could to the max overestimate possible immunomodulating properties of CC or CbiC, if there should any possible mixture effects.

As a conclusion, the results derived in the two studies with occlusive dressing, especially the one from the GPMT, may only possibly overestimate the risk of skin sensitizing effects of Choline chloride and hence Choline bicarbonate in humans. First, CbiC is not intended to be injected into the human skin, and second, an occlusive dressing will not be applied on humans, which could enhance the skin penetration and reaction due to swetting, heat and softening of the skin.

Since no skin sensitizing effects were seen anyway in the available studies and the risk for humans is even lower, it can safely be concluded that Choline bicarbonate is not a skin sensitizer and hence does not need to be classified as skin sensitizer, neither according Regulation 1272/2008/EC nor Directive 67/548/EEC.

Migrated from Short description of key information:
Skin sensitisation:
- In vivo Guinea pig maximisation test on formulation of read-across substance Choline chloride (CC) and Chlorocholine chloride (CCC) (320 g/L CC, 460 g/L CCC) in water, guinea pig (Dunkin Hartley), male, OECD Guideline 406, GLP, not sensitising
- In vivo Guinea pig Bühler test Chlorocholine chloride in water, guinea pig (albino), m/f, OECD Guideline 406, GLP, not sensitising

Justification for selection of skin sensitisation endpoint:
One of two well-documented OECD Guideline 406 studies according GLP. The study by Driscoll was performed on a formulation of the read-across substance Choline chloride with structural analogue (Chlorocholine chloride), whereas the study by Suresh was only performed on the read-across substance Chlorocholine chloride only. Hence, the formulation is more similar to the registered substance Choline bicarbonate due to the lacking chlorine moiety in the only sensitisation-relevant organic cation. Additionally, the study by Driscoll was performed as guinea pig maximisation test, which is more sensitive that the method of Bühler (Study by Suresh).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Both available in vivo sensitisation studies on guinea pigs revealed negative results for a formulation with Choline chloride (320 g/L) and Chlorocholine chloride (CCC, 460 g/L) and Chlorocholine chloride, a read-across substance, only. The results were assessed as reliable and safely transferrable to choline chloride and hence, to choline bicarbonate.

Hence, choline bicarbonate does not need to be classified as skin-sensitizer, neither according Regulation 1272/2008/EC nor Directive 67/548/EEC.