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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
- In vivo Guinea pig Bühler test 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 formulation of Choline chloride with structural analogue
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 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 chloride only / itself, they could be only classified as Klimisch 2. Nevertheless, they are still reliable enough 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 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 Choline chloride, i.e. 41 % of the solid ingredients, but it additionally performed on CCC which can serve as a read-across substance for CC.

The study by Suresh, 1992, was conducted on the read-across substance for CC, 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 body 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 Choline chloride. Since only two of eight animals showed a very slight erythema only at one single timepoint (72h of challenge phase) and even no effects were noted during the rechallenge phase, it can be safely concluded that Chlormequat and hence Choline chloride 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 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 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 chloride 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, 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.

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 chloride 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, 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.

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 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.

Since no skin sensitizing effects were seen anyway and the risk for humans is even lower, it can safely be concluded that Choline chloride 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.

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 Choline chloride with structural analogue (Chlorocholine chloride), whereas the study by Suresh was only performed on the read-across substance Chlorocholine chloride. Hence, the formulation is more similar to the registered substance Choline chloride. 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.

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