Reaction mass of N-(2-hydroxyethyl)alkyl] alkylamide and glycerol

Acute Toxicity: other routes

Administrative data

Endpoint:

acute toxicity: other routes

Remarks:

Cytotoxicity Assay in vitro with BALB/c 3T3 Cells: Neutral Red Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 23 November 2016 Experimental completion date: 16 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: • OECD Guideline for Testing of Chemicals: Guideline: 432; In vitro 3T3 NRU phototoxicity test (Revised and approved by the National Co-ordinators in May 2002, approved by Council April 2004).

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 440/2008 B 41", dated May 30, 2008.

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Committee for Proprietary Medicinal Products (CPMP) Note for Guidance on Photosafety testing, EMEA, CPMP/SWP/398/01, adopted 27 June 2002, into operation in Dec 2002.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

liquid

Details on test material:

Chemical Name: N-[2-(2-Hydroxyethoxy)ethyl]acetamide
CAS No.: 118974-46-2
Batch: 27191705
Purity: 82% (dose calculation was adjusted to puriy)
Appearance: Pale to yellow liquid
Expiry Date: 16 June 2017
Storage Conditions: At room temperature
Stability in Solvent: Stable in water (not quantified)
Purpose of Use: Industrial chemical

Test animals

Species:

other: BALB/c 3T3 cell line

Details on test animals or test system and environmental conditions:

Details on mammalian cell line
The BALB/c 3T3 cell line was isolated from the muscle tissue of the mouse embryo. This fibroblast cell line has a high proliferation rate (doubling time 16 - 20 hours in stock cultures) and a high plating efficiency of untreated cells (as a rule more than 70%) both necessary for the appropriate performance of the study. The cell line BALB/c 3T3 was isolated first by Aaronson and Todaro.

Administration / exposure

Route of administration:

other: In vitro exposure of cells

Vehicle:

other: EBSS

Details on exposure:

Test concentrations with justification for top dose
The following concentrations were tested (each concentration of the test item was tested in six replicates: with and without irradiation in both experiments 7.81 15.6 31.3 62.5 125 250 500 1000 µg/mL of the test item

Doses:

The following concentrations were tested (each concentration of the test item was tested in six replicates: with and without irradiation in both experiments 7.81 15.6 31.3 62.5 125 250 500 1000 µg/mL of the test item

Control animals:

not specified

Details on study design:

The neutral red (NR) test was developed by Borenfreund & Puerner and is used for the investigation of the cytotoxic potential of a test item. In this study the toxicity of the test item after irradiation with artificial sunlight is determined. The test protocol was developed in 1991 at Beiersdorf AG, Hamburg and validated in an ECVAM, COLIPA, and ZEBET coordinated ring trial. The study was initiated involving 11 laboratories.
The NR assay is based on the uptake of neutral red, a vital dye, and its accumulation in the lysosomes of viable uninjured cells. After storing the neutral red in the lysosomes of the viable uninjured cells, they are lysed with a mixture of deionised water (49% (v/v)), ethanol (50% (v/v)) and glacial acetic acid (1% (v/v)). The extracted neutral red is quantified photometrically at 540 nm.
For the determination of a phototoxic potential the cells were treated with various concentrations of the test item with and without irradiation with artificial sunlight (wave length > 320 nm). The cytotoxic response curves of the test groups were compared. The IC50-values were determined and compared to measure a possible phototoxicity

Results and discussion

Effect levels

Dose descriptor:

other: No cytotoxicity observed

Effect level:

> 1 000 other: µg/mL

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Any other information on results incl. tables

 Results AND DISCUSSION

Table2   RFE: Treatment of BALB/c 3T3 withElfaMoist AC

With artificial sunlight				Without artificial sunlight
Conc. [µg/mL]	O.D.540 nmMean Value	Standard Deviation	% of Solv. Control	Conc. [µg/mL]	O.D.540 nmMean Value	Standard Deviation	% of Solv. Control
Solvent Control	0.7028*	0.0311	100.00	Solvent Control	0.7372*	0.0459	100.00
7.81	0.7067	0.0121	100.56	7.81	0.7258	0.0161	98.45
15.6	0.7078	0.0198	100.71	15.6	0.7017	0.0304	95.17
31.3	0.7011	0.0174	99.75	31.3	0.7132	0.0428	96.73
62.5	0.7221	0.0181	102.75	62.5	0.7201	0.0231	97.67
125	0.7058	0.0139	100.43	125	0.7119	0.0439	96.56
250	0.6832	0.0098	97.21	250	0.6820	0.0370	92.51
500	0.7209	0.0274	102.57	500	0.7053	0.0381	95.66
1000	0.6912	0.0191	98.34	1000	0.7015	0.0281	95.15

* mean O.D._{540 nm}out of 12 wells

IC₅₀values = could not be determined, since the viability of the cells was not reduced with and without irradiation

PIF = could not be determined, since no IC₅₀values could be calculated

MPE = -0.044

Mean OD_{540 nm}solvent control value (≙viability) irradiated versus non-irradiated group: 95.3%

 

Table3   RFE:Treatmentof BALB/c 3T3with the Positive Control (chlorpromazine)

With artificial sunlight				Without artificial sunlight
Conc.[µg/mL]	O.D.540 nmMean Value	Standard Deviation	% of Solv.Control	Conc. [µg/mL]	O.D.540 nmMean Value	Standard Deviation	% of Solv. Control
Solvent Control	0.6892*	0.0590	100.00	Solvent Control	0.7584*	0.0768	100.00
0.125	0.6597	0.0627	95.72	6.25	0.7020	0.0177	92.56
0.250	0.4959	0.0534	71.95	12.5	0.3755	0.0226	49.51
0.500	0.3743	0.0403	54.31	25.0	0.0628	0.0050	8.28
0.750	0.3494	0.0429	50.69	37.5	0.0585	0.0029	7.72
1.000	0.2653	0.0669	38.49	50.0	0.0561	0.0011	7.39
1.500	0.0667	0.0106	9.68	75.0	0.0578	0.0025	7.62
2.000	0.0602	0.0032	8.73	100	0.0594	0.0021	7.83
4.000	0.0623	0.0037	9.04	200	0.0601	0.0037	7.93

* mean O.D._{540 nm}out of 12 wells

IC₅₀value (with artificial sunlight) = 0.60 µg/mL

IC₅₀value (without artificial sunlight) = 11.88 µg/mL

PIF = 20.04

MPE = 0.540

Mean OD₅₄₀nm solvent control value (≙viability) irradiated versus non-irradiated group: 90.9%

Table4   ME: Treatment of BALB/c 3T3 withElfaMoist AC

With artificial sunlight				Without artificial sunlight
Conc. [µg/mL]	O.D.540 nmMean Value	Standard Deviation	% of Solv. Control	Conc. [µg/mL]	O.D.540 nmMean Value	Standard Deviation	% of Solv. Control
Solvent Control	0.8461*	0.0291	100.00	Solvent Control	0.8195*	0.0356	100.00
7.81	0.8595	0.0234	101.58	7.81	0.8027	0.0173	97.95
15.6	0.8571	0.0348	101.30	15.6	0.7830	0.0264	95.55
31.3	0.8404	0.0282	99.33	31.3	0.7726	0.0179	94.28
62.5	0.8437	0.0138	99.72	62.5	0.7847	0.0227	95.75
125	0.8310	0.0365	98.22	125	0.8018	0.0214	97.84
250	0.8239	0.0252	97.38	250	0.7753	0.0201	94.60
500	0.8073	0.0223	95.41	500	0.7865	0.0266	95.97
1000	0.8264	0.0186	97.68	1000	0.7695	0.0288	93.89

* mean O.D._{540 nm}out of 12 wells

IC₅₀values = could not be determined, since the viability of the cells was not reduced with and without irradiation

PIF = could not be determined, since no IC₅₀values could be calculated

MPE = -0.029

Mean OD_{540 nm}solvent control value (≙viability) irradiated versus non-irradiated group: 103.2%

 

Table 5   ME:Treatmentof BALB/c 3T3with the Positive Control (chlorpromazine)

With artificial sunlight				Without artificial sunlight
Conc.[µg/mL]	O.D.540 nmMean Value	Standard Deviation	% of Solv.Control	Conc. [µg/mL]	O.D.540 nmMean Value	Standard Deviation	% of Solv. Control
Solvent Control	0.7766*	0.0172	100.00	Solvent Control	0.8160*	0.0193	100.00
0.125	0.7573	0.0271	97.50	6.25	0.8228	0.0171	100.84
0.250	0.7221	0.0339	92.98	12.5	0.1285	0.0103	15.75
0.500	0.5974	0.0373	76.92	25.0	0.0675	0.0056	8.27
0.750	0.4479	0.0609	57.67	37.5	0.0677	0.0096	8.29
1.000	0.1292	0.0133	16.63	50.0	0.0666	0.0069	8.16
1.500	0.0882	0.0070	11.36	75.0	0.0689	0.0107	8.44
2.000	0.0839	0.0083	10.80	100	0.0679	0.0075	8.33
4.000	0.0919	0.0088	11.83	200	0.0720	0.0161	8.83

* mean O.D._{540 nm}out of 12 wells

IC₅₀value (with artificial sunlight) = 0.77 µg/mL

IC₅₀value (without artificial sunlight) = 10.72 µg/mL

PIF = 13.89

MPE = 0.548

Mean OD₅₄₀nm solvent control value (≙viability) irradiated versus non-irradiated group: 95.2%

Applicant's summary and conclusion

Conclusions:

Reaction mass of N-[2-(2-hydroxyethoxy)ethyl]acetamide and glycerol did not have any phototoxic effects on BALB/c 3T3 cells, and showed no cytotoxicity up to the highest tested concentration of 1000 µg/mL.

Executive summary:

The study was performed to assess the phototoxic potential of reaction mass of N-[2-(2-hydroxyethoxy)ethyl]acetamide and glycerol. The test was performed using BALB/c 3T3 cells clone 31.

1000 µg/mL of the test item, dissolved in EBSS were applied as the highest concentration.

The experiment was performed twice. The first experiment served as a range finding experiment (RFE), the second experiment was the main experiment (ME).

The following concentrations of the test item were tested with and without irradiation in both experiments:

7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 µg/mL

As solvent control EBSS was used.

Chlorpromazine was used as positive control. The following concentrations were applied:

without irradiation	6.25, 12.5, 25.0, 37.5, 50, 75, 100, 200 µg/mL
with irradiation	0.125, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 4.0 µg/mL

In both experiments one test group of cells treated with the test item was irradiated withartificial sunlight for 50 minutes with 1.65 mW/cm²UVA, resulting in an irradiation dose of~ 5 J/cm²UVA. Another test group of test item treated cells were kept in the dark for 50 minutes.

Table1            Summary of Results

	Test Item	IC50(+UV) [µg/mL]	IC50(-UV) [µg/mL]	PIF	MPE	% viability of solvent control of irradiated versus non irradiated plate
RFE	ElfaMoist AC	-	-	-	-0.044	95.3
	Positive control	0.60	11.88	20.04	0.540	90.9
ME	ElfaMoist AC	-	-	-	-0.029	103.2
	Positive control	0.77	10.72	13.89	0.548	95.2

In both experiments no cytotoxic effects were observed after treatment of cells with reaction mass of N-[2-(2-hydroxyethoxy)ethyl]acetamide and glycerol, neither in the presence nor in the absence of irradiation with artificial sunlight. Therefore, IC₅₀-values or PIFs could not be calculated. The resulting MPE was -0.044 or -0.029, respectively,and therefore, the test item is classified as not phototoxic.

The positive control chlorpromazine induced phototoxicity in the expected range after irradiation with artificial sunlight.

In conclusion, it can be stated that in the study described and under the experimental conditions reported Reaction mass of N-[2-(2-hydroxyethoxy)ethyl]acetamide and glycerol did not have any phototoxic effects on BALB/c 3T3 cells.