Bis(2-hydroxyethyl)ammonium acetate

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

2010-03-17 to 2010-04-06

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP certified guideline study on a read-across compound.

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

yes

Remarks:

Temporary deviations from the minimum level of relative humidity occurred. This deviation was not considered to have a detrimental effect on study outcome.

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

yes

Remarks:

emporary deviations from the minimum level of relative humidity occurred. This deviation was not considered to have a detrimental effect on study outcome.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

yes

Remarks:

emporary deviations from the minimum level of relative humidity occurred. This deviation was not considered to have a detrimental effect on study outcome.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Number of animals for main study: 20 female
Number of animals for pretest: 2 female
Age: 9 - 11 weeks
Body weight at start of test: 19-24 g
Source: Charles River France, L'Arbresle Cedex, France
Acclimatisation period: At least 5 days
Diet: SM R/M-Z from SSNIFF Spezialdiaten GmbH, Soest, Germany ad libitum
Water: Tap water, ad libitum
Housing: Individually housed in Macrolon cages with sterilised sawdust as bedding
Environmental conditions -
Temperature: 18 - 24 deg C
Humidity: 40 - 70 %
Photoperiod: 12-hour light/dark cycle

Vehicle:

dimethylformamide

Concentration:

Pretest: 25 % and 50 % concentration in dimethyl formamide

Main study-
Group 1 (control): dimethyl formamide
Group 2: 10 % in dimethyl formamide
Group 3: 25 % in dimethyl formamide
Group 4: 50 % in dimethyl formamide

No. of animals per dose:

Pretest: 1 animal per dose
Main study: 5 animals per dose

Details on study design:

In order to obtain homogeneity, the test substance formulations were heated in a water bath with a maximum temperature of 39 deg C for a maximum of 11 minutes. The test substance formulations were then allowed to cool to a maximum temperature of 38 deg C and were vortexed thoroughly prior to dosing.

On study days 1, 2 and 3, 25µl of the test substance was applied to the dorsal surface of both ears, at approximately the same time each day. The control animals were treated in the same manner as the experimental animals, except that vehicle alone was used.

Six days after initiation of the study, each animal was given an i.v. injection of 0.25 ml of 20 µCi of 3H-methyl thymidine in PBS. Approximately 5 hours after administration of the radioactive dose, all animals were sacrificed by intraperitoneal injection with Euthasol 20 %. The draining lymph node of each ear was excised. The relative size of the lymph nodes from each group was compared with control and any abnormalities of the nodes and surrounding area were recorded. The lymph nodes from all animals in each group were pooled in 3 ml of PBS.

The lymph nodes were passes through a stainless steel gauze filter (diameter 125 µm) to generate a single cell suspension. The lymph node cells were washed twice with PBS by centrifugation at 200 g for 10 minutes at 4 deg C. To precipitate the DNA, the lymph node cells were exposed overnight to 5 % trichloroacetic acid (TCA) at 4 deg C. Precipitates were recovered by centrifugation, resuspended in 1 ml TCA and transferred to 10 ml of scintillation fluid. Radioactive measurements were taken using a scintillation counter and expressed as disintegrations per minute (DPM)

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

N/A

Positive control results:

A positive control test was carried out within 6 months of the start of this study (performed in September/October 2009). In this study, females were checked for their sensitivity to alpha-hexylcinnamaldehyde.

The stimulation index (SI) values calculated for the substance at concentrations of 5, 10 and 25 % were 1.4, 1.2 and 5.1, respectively. As the 50 % concentration of the positive control substance gave an SI of greater than 3, it was concluded that this assay can detect substances classed as skin sensitisers.

Parameter:

SI

Remarks on result:

other: The mean SI values calculated for the substance concentrations of 10, 25 and 50 % were 1.3, 0.7 and 0.9, respectively.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM/animal values for the experimental groups treated with test substance concentrations of 10, 25 and 50 % were 280, 154 and 195 DPM, respectively. The mean DPM/animal value for the control group was found to be 214 DPM.

No mortality, clinical signs or irritation of the ears were observed in any of the animals examined following treatment with the test substance. The auricular lymph nodes of the treated animals were considered to be of normal size and were comparable in size with those of the control group. No macroscopic abnormalities were noted in the area surrounding the treatment site in any of the animals. The body weights and body weight gain of the animals treated with the test substance were not significantly different from controls over the study period.

Table 1: Mean Disintegrations per Minute (DPM) and Stimulation Index (SI)

Test substance (% w/w)	Mean
	DPM ± SEM	SI ± SEM
0 (vehicle control)	280 ± 42	1.3 ± 0.3
10	154 ± 40	0.7 ± 0.2
25	195 ± 40	0.9 ± 0.3
50	214 ± 40	1.0 ± 0.3

Table 2: Skin reactions, body weights and relative size of auricular lymph nodes

	Animal Number	Day 1	Day 6	Day 3
				Skin reactions on dorsal surface of ear
				Left		Right
Test substance (% w/w)		Body weight (g)	Body weight (g)	Erythema	Oedema	Erythema	Oedema
0 (vehicle control)	1	21	21	0	0	0	0
	2	22	20	0	0	0	0
	3	21	22	0	0	0	0
	4	20	21	0	0	0	0
	5	22	22	0	0	0	0
10	6	22	21	0	0	0	0
	7	21	22	0	0	0	0
	8	22	23	0	0	0	0
	9	24	23	0	0	0	0
	10	19	19	0	0	0	0
25	11	19	17	0	0	0	0
	12	23	23	0	0	0	0
	13	20	21	0	0	0	0
	14	22	22	0	0	0	0
	15	22	23	0	0	0	0
50	16	22	22	0	0	0	0
	17	21	22	0	0	0	0
	18	19	18	0	0	0	0
	19	22	22	0	0	0	0
	20	22	22	0	0	0	0

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In this study, none of the animals treated with TEA acetate had a stimulation index ≥ 3 when tested at concentrations of up to 50 % (w/w). Furthermore, the test substance had no effect on body weights, local lymph node size and did not induce any clinical signs or mortality in any of the animals tested.

Therefore, TEA acetate applied at a concentration of 50 % in dimethyl formamide is not considered to be a skin sensitiser.

Executive summary:

In order to assess the cutaneous allergenic potential of TEA acetate, three experimental groups of five CBA/J mice were treated with the test article at concentrations of 10, 25 or 50 % in dimethylformamide. On study days 1, 2 and 3, 25 µl of the test substance was applied to the dorsal surface of both ears, at approximately the same time each day. A control group of five mice was treated with vehicle alone.

None of the animals treated with TEA acetate had a stimulation index ≥ 3 when tested at concentrations of up to 50 % (w/w). Furthermore, the test substance had no effect on body weights, local lymph node size and did not induce any clinical signs or mortality in any of the animals tested. Thus under the conditions of this test, TEA acetate does not warrant classification as a skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

No studies are available on the skin sensitising potential of DEA acetate, but three reliable studies are available on structurally-related read-across compounds.

 

A local lymph node assay (LLNA) has been conducted on TEA acetate at concentrations of up to 50% (w/w). None of the treated mice had a stimulation index of 3 or more, therefore RA2 was not considered to be a skin sensitiser (Stitzinger, 2010).

 

Guinea pig maximisation tests (GPMTs) for skin sensitisation have been conducted using RA and RA2. No treatment-related reactions indicative of sensitisation were seen for either substance (Arcelin, 1997, 2000b).

Migrated from Short description of key information:
No data were available on the skin sensitising potential of DEA acetate. One LLNA and two GPMTs showing a lack of sensitising potential are, however, available on structurally-related read-across compounds. All were negative for skin sensitisation potential.

Justification for selection of skin sensitisation endpoint:
GLP, OECD guideline study (reliability 2).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

This study was not conducted as no standards test method is available, it is not a data requirement according to REACH Regulation 1907/2006, and inhalation exposure to DEA acetate is not expected to occur.

Migrated from Short description of key information:
No study available.

Justification for classification or non-classification

According to Regulation (EC) No. 1272/2008, classification is applicable when positive results are seen in an appropriate animal test (e.g. a response rate of at least 30% for guinea pigs in an adjuvant test method, or at least 15% in a non-adjuvant test method). Based on three read-across studies showing no evidence for skin sensitisation (Arcelin 1997, 2000b; Stitzinger, 2010), DEA acetate does not require classification as a skin sensitiser.