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Description of key information

2017 LLNA  pre-test: The observations in the OECD 429 LLNA pretests are indicative of the substances being corrosive.

Scores of =/> 3 were observed at concentrations 50, 25 and 10%

Ear swelling was greater than 25% at 25 and 50%

Body weight loss (>5%) was observed possibly indicative of systemic toxicity.

The main argument for not continuing with the main LLNA studies is the interpretation of the study director with regard to eschar formation at all concentrations tested on day 6.

Based on this observation, and in light of experience with the substance it was decided to apply a precautionary labelling of Corrosive 1C to Hydroxypropyl, 2-, trimethylammonium formate.

In a non-guideline eyey irritation study in rabbits (1976) a preparation of 75% Hydroxypropyl, 2-, trimethylammonium formate in ethylene glycol was found to be a mild ocular irritant with effects fully reversible after 7 days.

However, based on observed skin corrosiveness and irritancy, and industrial experience, the substance must be considered as severly irritating to the eye, with the potential to cause severe eye damage.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation / corrosion, other
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Results of OECD 429 (LLNA) pre-test indicating corrosivity
Justification for type of information:
The OECD 429 (LLNA) test guideline states that

“Dose and vehicle selection should be based on the recommendations given in references (3) and (5). Consecutive doses are normally selected from an appropriate concentration series such as 100%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5%, etc. Adequate scientific rationale should accompany the selection of the concentration series used. All existing toxicological information (e.g. acute toxicity and dermal irritation) and structural and physicochemical information on the test substance of interest (and/or structurally related test substances) should be considered where available, in selecting the three consecutive concentrations so that the highest concentration maximises exposure while avoiding systemic toxicity and/or excessive local skinirritation(3)(25).”


.” Excessive local skin irritation is indicated by an erythema score ≥3 and/or an increase in ear thickness of≥25% on any day of measurement(26)(27). The highest dose selected for the main LLNA study will be the next lower dose in the pre-screen concentration series (see paragraph18)that does not induce systemic toxicity and/or excessive local skin irritation.”

Scores of =/> 3 were observed at concentrations 50, 25 and 10%

Ear swelling was greater than 25% in substance 1833500 at 25 and 50%

Body weight loss (>5%) was observed possibly indicative of systemic toxicity.


The main argument for not continuing with the main LLNA studies is the interpretation of the study director with regard to eschar formation at all concentrations tested on day 6.

The definition of corrosivity is not met formally “Corrosivity is defined as irreversible (14 days) damage of the epidermis and dermis following exposure to a substance up to 4 hours on skin”, because observation in the pretests ended on day 6 in the pretests.

However, the observations in the pretests are indicative of the substances being corrosive, which may well have an influence on the proliferation of lymphocytes which in turn would result in a false positive result.

Based on this observation, and in light of experience with the substance it was decided to apply a precautionary labelling of Corrosive 1C to (2-hydroxypropyl)trimethylammonium 2-ethylhexanoate.
Qualifier:
according to
Guideline:
other: OECD 429 Local Lymph Node Assay
Deviations:
not applicable
Principles of method if other than guideline:
LLNA Pretests (x4)

The maximum concentration of test item to be investigated in the LLNA is determined as the dose that can be uniformly applied to the dorsal surface of the ears of the mice and which does at the same time not cause excessive skin irritation or clinical signs of toxicity after three consecutive daily applications.

In the absence of suitable acute toxicity and dermal irritation data or if the information suggests that irritation and/or toxicity is possible, a preliminary screening test will be conducted.

One animal will be treated with the test item at the maximum concentration that is suitable for application to the dorsal surface of the ears and an additional animal will be treated with the next lower concentration of the concentration series mentioned above.
Alternatively, a lower concentration may be investigated first if information is available to suggest that higher concentrations would be irritant or toxic. The animals will be treated, as detailed in the main experiment procedures section, for three consecutive days (days 1, 2 and 3). Clinical signs of toxicity and/or irritation at the treatment sites will be recorded on days 1 to 6 and a score will be used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness will be determined using a micrometer. Additionally, for both animals, the ears will be punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2 ) and will be immediately pooled per animal and weighed using an analytical balance.
GLP compliance:
yes
Species:
mouse
Strain:
CBA
Type of coverage:
open
Preparation of test site:
other: Mice were treated by (epidermal) topical application to the dorsal surface of each ear
Vehicle:
not specified
Controls:
not required
Amount / concentration applied:
0.25% - 50%
Duration of treatment / exposure:
Animals were treated for three consecutive days (days 1, 2 and 3).
Observation period:
Animals sacrificed 3 days after final treatment (test duration 6 days)
Number of animals:
2 animals per pretest. 4 pretests (8 animals total)
Irritation parameter:
other: eschar formation
Basis:
animal #1
Time point:
other: Day 6
Reversibility:
not reversible
Remarks on result:
other: Eshar formation and ear swelling indicating corrosion at 50% concentration of test substance
Irritation parameter:
other: eschar formation
Basis:
animal #2
Time point:
other: Day 6
Reversibility:
not reversible
Remarks on result:
other: Eshar formation and ear swelling indicating corrosion at 25% concentration of test substance
Irritation parameter:
other: eschar formation
Basis:
animal #3
Time point:
other: Day 6
Reversibility:
not reversible
Remarks on result:
other: Eshar formation and ear swelling indicating corrosion at 10% concentration of test substance
Irritation parameter:
other: eshar formation
Basis:
animal #4
Time point:
other: Day 6
Reversibility:
not reversible
Remarks on result:
other: Eshar formation and ear swelling indicating corrosion at 5% concentration of test substance
Irritation parameter:
other: eschar formation
Basis:
animal #5
Time point:
other: Day 6
Reversibility:
not reversible
Remarks on result:
other: Eshar formation and ear swelling indicating corrosion at 2.5% concentration of test substance
Irritation parameter:
other: eschar formation
Basis:
animal #6
Time point:
other: Day 6
Reversibility:
not reversible
Remarks on result:
other: Eshar formation and ear swelling indicating corrosion at 1% concentration of test substance
Irritation parameter:
other: ear lesions
Basis:
animal: 7
Time point:
other: Day 6
Reversibility:
not reversible
Remarks on result:
other: Eshar formation and ear swelling indicating corrosion at 0.5% concentration of test substance
Irritation parameter:
other: lesions
Basis:
animal: 8
Time point:
other: Day 6
Reversibility:
not reversible
Remarks on result:
other: Eshar formation and ear swelling indicating corrosion at 0.25% concentration of test substance
Irritant / corrosive response data:
The LLNA study was terminated after the fourth pre-test, since eschar formation was observed at all tested concentrations (see details in attached results). This is an indication for corrosive properties of the test item.
After 3 consecutive days of treatment animals showed visible ear swelling, evidence of hardened and scaly skin and lesions that progressed to eschar formation at sacrifice on day 6.
Interpretation of results:
Category 1C (corrosive) based on GHS criteria
Conclusions:
The observations in the OECD 429 LLNA pretests are indicative of the substances being corrosive.
Executive summary:

The OECD 429 (LLNA) test guideline states that:

“Dose and vehicle selection should be based on the recommendations given in references (3) and (5). Consecutive doses are normally selected from an appropriate concentration series such as 100%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5%, etc. Adequate scientific rationale should accompany the selection of the concentration series used. All existing toxicological information (e.g. acute toxicity and dermal irritation) and structural and physicochemical information on the test substance of interest (and/or structurally related test substances) should be considered where available, in selecting the three consecutive concentrations so that the highest concentration maximises exposure while avoiding systemic toxicity and/or excessive local skinirritation(3)(25).”

.” Excessive local skin irritation is indicated by an erythema score ≥3 and/or an increase in ear thickness of≥25% on any day of measurement(26)(27). The highest dose selected for the main LLNA study will be the next lower dose in the pre-screen concentration series (see paragraph18)that does not induce systemic toxicity and/or excessive local skin irritation.”

Scores of =/> 3 were observed at concentrations 50, 25 and 10%

Ear swelling was greater than 25% at 25 and 50%

Body weight loss (>5%) was observed possibly indicative of systemic toxicity.

The main argument for not continuing with the main LLNA studies is the interpretation of the study director with regard to eschar formation at all concentrations tested on day 6.

The definition of corrosivity is not met formally “Corrosivity is defined as irreversible (14 days) damage of the epidermis and dermis following exposure to a substance up to 4 hours on skin”, because observation in the pretests ended on day 6 in the pretests.

However, the observations in the pretests are indicative of the substances being corrosive, which may well have an influence on the proliferation of lymphocytes which in turn would result in a false positive result.

Based on this observation, and in light of experience with the substance it was decided to apply a precautionary labelling of Corrosive 1C to Hydroxypropyl, 2-, trimethylammonium formate (TMR2)

Endpoint:
skin irritation: in vitro / ex vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin irritation study does not need to be conducted because adequate data from an in vivo skin irritation study are available
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 18, 1976 - December 29, 1976
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Justification for type of information:
The method employed was similar to that described in Section 1500.41. - Hazardous Substances and Articles , Administration and Enforcement Regulations, Federal Register, Vol. 38, No. 187, P. 27019, 27 September 1973.
Qualifier:
equivalent or similar to
Guideline:
other: The method employed was similar to that described in Section 1500.41. - Hazardous Substances and Articles , Administration and Enforcement Regulations, Federal Register, Vol. 38, No. 187, P. 27019, 27 September 1973.
Deviations:
not specified
Principles of method if other than guideline:
A group of six albino rabbits were clipped over a wide area. One side of the animals' backs was abraded at one site with a lancet sufficiently deep to penetrate the stratum corneum but not enter the derma t o produce bleeding.
The skin of the other side was allowed to remain intact . A 0.5 ml portion of material was applied to an abraded and an intact skin site on the same rabbit.
Gauze patches were then placed over the treated areas and an impervious material was wrapped snugly around the trunks o f the animals t o hold the patches i n place.
The wrapping was removed a t the end o f the twenty-four hour period and the treated areas were examined. Readings were also made after seventy-two hours. The Draize method of scoring was employed.
GLP compliance:
not specified
Specific details on test material used for the study:
DABCO TMR-2
Species:
rabbit
Strain:
not specified
Remarks:
Albino
Type of coverage:
occlusive
Preparation of test site:
other: One side of the animals' backs was abraded at one site with a lancet sufficiently deep to penetrate the stratum corneum but not enter the derma to produce bleeding. The skin of the other side was allowed to remain intact.
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
0.5 ml
Duration of treatment / exposure:
24 hours
Observation period:
72 hours
Number of animals:
6 rabbits
Irritation parameter:
erythema score
Basis:
mean
Time point:
24 h
Score:
1
Max. score:
1
Reversibility:
not specified
Remarks on result:
no indication of irritation
Remarks:
both intact and abraded skin sections
Irritation parameter:
erythema score
Basis:
mean
Time point:
72 h
Score:
1
Max. score:
1
Reversibility:
not specified
Remarks on result:
no indication of irritation
Remarks:
both intact and abraded skin sections
Irritation parameter:
edema score
Basis:
mean
Time point:
24 h
Score:
0
Max. score:
0
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Remarks:
both intact and abraded skin sections
Irritation parameter:
edema score
Basis:
mean
Time point:
72 h
Score:
0
Max. score:
0
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Remarks:
both intact and abraded skin sections
Irritant / corrosive response data:
No indications of irritation on both intact and abraded skin sections in all animals.
Other effects:
None reported.
Interpretation of results:
GHS criteria not met
Conclusions:
The tested product is not a primary irritant on intact or abraded rabbit skin at applied concentrations of 0.5 ml undiluted test material, occluded for 24 hours.

Additional information

Justification for classification or non-classification