Choline chloride

Endpoint summary

Description of key information

Skin sensitisation:
- In vivo Guinea pig maximisation test on formulation of 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
- as supporting information: In vivo Guinea pig Bühler test on chlorocholine chloride in water, guinea pig (albino), m/f, OECD Guideline 406, GLP, not sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

other: Experimental result on a formulation of Choline chloride (CC) with the structural analogue Chlorocholine chloride (CCC).

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

Justification for non-LLNA method:

The Buehler test (in vivo) was performed before the LLNA was set as preferred test method.

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 °C
- 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: 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: 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

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 only on Choline chloride (CC) but on a formulation with CC (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 Choline chloride. The test item contains two relevant parts 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. 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 chloride is non-sensitizing, too. Hence, Choline chloride 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 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, 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 is a well-documented guideline study according to GLP available, which was conducted using a formulation of Choline chloride and Chlorocholine chloride aka chlormequat (Key study, classified in this context as Klimisch 2). In addition to this, as supporting information, there is a well—documented guideline study on Chlormequat available, which is provided to allow correct interpretation of the results of the Key study. Both studies are considered reliable to assess the possible skin sensitising properties of Choline chloride 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 key study by Driscoll, 1998, was performed on an aqueous formulation of Choline Chloride (CC, 320 g/L) and Chlorocholine chloride (CCC, 460 g/L), and contains therefore to relevant parts but not only Choline chloride. It additionally contains CCC, which is a structural analogue for CC. In detail, the test item in the study by Driscoll, 1998, contains Choline chloride, i.e. 41 % of the solid ingredients, and the solvent (water). The results of this Key study showed no skin reactions or differences in body weight gain compared to control.

Regarding the requirements set in OECD guideline 406 to choose the test doses based i.a. on skin irritating effects, the following needs to be added. 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, the irritation study in IUCLID chapter 7.3.1 revealed Choline chloride as not irritating at all after the application of the undiluted compound for 20h on rabbits, 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.

Regarding the employed test method, the following remark is added. The guinea pig maximization test (GPMT) employed in the Key study Driscoll, 1998 is considered to be more sensitive than the alternative method by Buehler (employed in the supporting study), 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. However, since no skin reaction was seen in any of the test animals, this risk is not relevant anymore, and it can safely be concluded that the CC-CCC formulation does not exhibit skin-sensitizing properties.

The provided supporting study by Suresh, 1992, was conducted on the structural analogue for CC, chlormequat (CCC), only. This information is provided to further support the results derived of the Key study. The outcome of the study by Suresh on CCC only (very slight erythema in 2/8 animals after 72 hours) showed that also CCC is not a skin sensitiser. The observed and very slight results - only two of eight animals showed a very slight erythema only at one single timepoint (72h of challenge phase) and even no noted effects during the rechallenge phase – are expected to represent a worst-case (please also refer to arguments presented in the following paragraph).

Regarding possible mixture effects of the CC-CCC formulation, the following remarks are added: The CC-CCC formulation employed in the Key study did not induce any skin reactions in the Magnus-Kligmann test, CCC alone employed in the supporting study (Suresh, 1992) 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, 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, if there should any possible mixture effects.

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 in humans. First, CC 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.

So, since no skin sensitizing effects were seen and neither water nor CCC, was shown to be a dermal sensitizer, it can be concluded that every possible positive result obtained in the key study from the formulation must be attributed to Choline chloride. 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 (CC-CCC-formulation) did not exhibit any sensitizing properties, furthermore CCC alone was not a skin sensitizer as well. Consequently, Choline chloride is non-sensitizing, too and therefore does not need to be classified as a skin sensitiser according to Regulation 1272/2008/EC.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Both available in vivo sensitisation studies on guinea pigs - either on a formulation with Choline chloride (320 g/L) and Chlorocholine chloride (CCC, 460 g/L) or on Chlorocholine chloride, a structural analogue, alone - revealed negative results. The results were assessed as reliable and safely transferrable to Choline Chloride.

Hence, Choline chloride does not need to be classified as skin-sensitizer according to Regulation 1272/2008/EC.