polymeric zinc propylenebis(dithiocarbamate);propineb (ISO)

Phototoxicity in vitro

Link to relevant study record(s)

Reference

Endpoint:

phototoxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2014/04/14 to 2014/05/23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Type of study:

in vitro 3T3 NRU phototoxicity test

Remarks:

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

Qualifier:

according to guideline

Guideline:

OECD TG 432 (In Vitro 3T3 NRU Phototoxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.41 ( In vitro 3T3 NRU Phototoxicity Test)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EMEA, CPMP/SWP/3 98/01

Version / remarks:

2002

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species / strain / cell type:

BALB/c 3T3

Details on mammalian cell type (if applicable):

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 c31 was isolated first by Aaronson and Todaro.

Negative solvent / vehicle controls:

no

Positive controls:

yes

Positive control substance:

other: The solvent control for the positive control was EBSS.

Details on test system and experimental conditions:

Large stocks (Master Cell Stock) of the BALB/c 3T3 c31 cell line (supplied by Dr. Liebsch, ZEBET, Berlin, Germany) are stored in liquid nitrogen in the cell bank of Harlan CCR. The master cell stock has been characterized by Harlan CCR. A working cell stock is produced by multiplying from the master cell stock.

Thawed stock cultures were propagated at 37 ± 1.5 °C in 75 cm plastic flasks. Seeding was done with about 1 x 106 cells per flask in 15 mL of Dulbecco's Minimal Essential Medium (DMEM), supplemented with 10% NCS. The cells were sub-cultured twice weekly. The cell cultures were incubated at 37 ± 1.5 °C in a 7.5 ± 0.5% carbon dioxide atmosphere.

The irradiation was performed with a Dr. Hönle Sol 500 solar simulator. The filter HI was used to keep the UVB irradiation as low as possible. The produced wavelength of the solar simulator with the filter was > 320 nm. Due to the heterogenous distribution of irradiation intensity the UVA intensity was measured for the complete area with a UV-meter.

The homogeneous irradiation area was marked and the cultures were irradiated in this area. The solar simulator was switched on about 30 minutes prior to the start of the experiment. The absorption spectrum of the test item was determined in the range from 270-800 nm. The test item showed absorption maxima at 279.9 and at 300.0 nm. The medium was removed and 0.1 mL serum free medium containing 50 µg Neutral Red / mL was added to each well. The plates were returned to the incubator for another 3 hours to allow uptake of the vital dye into the lysosomes of viable cells.

Thereafter, the medium was removed completely and the cells were washed with EBSS. Then 0.15 mL of a solution of 49% (v/v) deionized water, 50% (v/v) ethanol and 1% (v/v) acetic acid were added to each well to extract the dye. After an additional approximately 10 minutes at room temperature and a brief agitation, the plates were transferred to a microplate reader equipped with a 540 nm filter to determine the absorbance of the extracted dye. This absorbance showed a linear relationship with the number of surviving cells.

Vehicle:

not specified

Vehicle / solvent:

The solvent control for the test item was EBSS containing 1% (v/v) DMSO.

Evaluation criteria:

If PIF < 2 or MPE <0.1: no phototoxic potential predicted.
If PIF > 2 and < 5 or MPE >0.1 and <0.15 a probable phototoxic potential is predicted.
If PIF > 5 or MPE > 0.15 a phototoxic potential predicted

The assay meets the acceptance criteria:
- if after irradiation with a UVA dose of 5 J/cm² the cell viability of the solvent control is > 80% of non irradiated cells.
- if for the positive control CPZ the factor (PIF) between the two ED50 values is > 6.
- if the mean OD540 of solvent controls is > 0.4.

Statistics:

The data generated were recorded in the laboratory raw data file. The results are presented in tabular form, including experimental groups with the test item, solvent, and positive control. Arithmetic means ± standard deviation were calculated for every test group. The ED50 values, the Photo-Irritancy-Factor (PIF), as well as the Mean Phototoxic Effect (MPE), were calculated using the software Phototox (Version 2.0) (distributed by ZEBET, 12277 Berlin, Germany, and recommended by the OECD guideline). The ED50 values (effective dose where only 50% of the cells survived) were determined by curve-fitting software.

Key result

Results:

The study was performed to assess the phototoxic potential of Propineb Technical. The test was performed using BALB/c 3T3 c3 1 cells. Two experiments were performed. The first experiment served as range finder (RFE), the second experiment (ME) was the confirming experiment. 125 µg/mL of the test item, dissolved in DMSO (further diluted in EBSS, final concentration of DMSO in EBSS was 1% (v/v)) was applied as the highest concentration in the RFE. According to the results in the RFE, the concentration range in the ME was chosen closer.

A dose dependent cytotoxicity was observed after treatment of cells with the test item in the presence and absence of irradiation with artificial sunlight in both experiments. Both calculated MPE values and the PIF value of the ME indicated a non-phototoxic potential of the test item, however the PIF value of the RFE suggested a probable phototoxic effect. Since the concentration range tested in the ME was narrowed to calculate more precise ED50 and PIF, the results observed in the ME are considered to be more accurate than those observed in the RFE. Therefore, the test item can be stated as non-phototoxic.

Results with reference substance (positive control):

The concurrent positive control Chlorpromazine showed a marked decrease of survival after irradiation with artificial sun light with an ED50 value with artificial sunlight of 0.18 µg/mL vs. an ED50 value without artificial sunlight of 16.71 µg/mL, PIF = 91.81 and MPE = 0.717.

Table one

summary of results

	Substance	ED50 (+UV) [μg/mL]	ED50 (-UV) [μg/mL]	PIF	MPE	%viability of solvent control of irradiated versus non-irradiated plate
RFE	Propineb technical	6.74	30.66	4.55	0.095	93.8
	Positive control	0.18	16.71	91.81	0.717	97.0
ME	Propineb technical	7.21	5.01	0.70	0.005	92.1
	Positive control	0.39	11.44	29.31	0.606	119.0

 

 

Table two

Treatment of BALB/c 3T3 with Propineb in the RFE

With artificial sunlight				Without artificial sunlight
Conc. [µg/mL]	O.D.540 nm Mean Value	Standard Deviation	% of Solv. Control	Conc. [µg/mL]	O.D.540 nm Mean Value	Standard Deviation	% of Solv. Control
Solvent Control	0.5552*	0.0299	100.00	Solvent Control	0.5918*	0.0312	100.00
0.98	0.6092	0.0178	109.72	0.98	0.6596	0.0237	111.45
1.95	0.5974	0.0179	107.60	1.95	0.5974	0.0150	100.94
3.91	0.5298	0.0394	95.42	3.91	0.5593	0.0237	94.50
7.81	0.1922	0.0429	34.62	7.81	0.5344	0.0239	90.30
15.6	0.0778	0.0057	14.00	15.6	0.4548	0.0297	76.84
31.3	0.0735	0.0056	13.23	31.3	0.3466	0.0457	58.57
62.5	0.0675	0.0041	12.15	62.5	0.0770	0.0074	13.01
125	0.0679	0.0031	12.23	125	0.0608	0.0040	10.27

* mean O.D._{540 nm} out of 12 wells
ED₅₀ value (with artificial sunlight) = 6.74 µg/mL
ED₅₀ value (without artificial sunlight) = 30.66 µg/mL
PIF = 4.55
MPE = 0.095
Mean OD_{540 nm} solvent control value (viability) irradiated versus non-irradiated group: 93.8%

 

 

Table three

Treatment of BALB/c 3T3 with the positive control (Chlorpromazine) in the RFE

With artificial sunlight				Without artificial sunlight
Conc. [µg/mL]	O.D.540 nm Mean Value*	Standard Deviation	% of Solv. Control	Conc. [µg/mL]	O.D.540 nm Mean Value*	Standard Deviation	% of Solv. Control
Solvent Control	0.6011*	0.0434	100.00	Solvent Control	0.6195*	0.0275	100.00
0.125	0.4381	0.0225	72.88	6.25	0.5985	0.0065	96.60
0.250	0.1465	0.0584	24.38	12.50	0.4786	0.0155	77.26
0.500	0.0909	0.0165	15.11	25.00	0.0999	0.0071	16.13
0.750	0.0704	0.0039	11.70	37.50	0.0631	0.0048	10.18
1.000	0.0718	0.0086	11.95	50.00	0.0569	0.0034	9.18
1.500	0.0748	0.0043	12.44	75.00	0.0590	0.0054	9.52
2.000	0.0729	0.0048	12.12	100.00	0.0553	0.0038	8.93
4.000	0.0747	0.0036	12.43	200.00	0.0586	0.0069	9.46

* mean O.D._{540 nm} out of 12 wells
ED₅₀ value (with artificial sunlight) = 0.18 µg/mL
ED₅₀ value (without artificial sunlight) = 16.71 µg/mL
PIF = 91.81
MPE = 0.717
Mean OD540 nm solvent control value (viability) irradiated versus non-irradiated group: 97.0%

 

 

Table four

Treatment of BALB/c 3T3 with Propineb in the ME

With artificial sunlight				Without artificial sunlight
Conc. [µg/mL]	O.D.540 nm Mean Value	Standard Deviation	% of Solv. Control	Conc. [µg/mL]	O.D.540 nm Mean Value	Standard Deviation	% of Solv. Control
Solvent Control	1.0717*	0.0938	100.00	Solvent Control	1.1641*	0.0661	100.00
1.56	0.9007	0.0852	84.04	1.0	1.0150	0.0648	87.19
3.13	0.7629	0.0457	71.18	2.5	0.9078	0.0278	77.98
6.25	0.6317	0.0439	58.94	5.0	0.3748	0.0918	32.20
12.5	0.5305	0.0451	49.50	10	0.1205	0.0423	10.35
25.0	0.2825	0.0316	26.36	15	0.0689	0.0062	5.92
50.0	0.0716	0.0048	6.68	20	0.0627	0.0019	5.39
75.0	0.0615	0.0019	5.74	25	0.0644	0.0036	5.53
100	0.0622	0.0041	5.81	30	0.0658	0.0031	5.65

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

ED₅₀ value (with artificial sunlight) = 7.21 µg/mL
ED₅₀ value (without artificial sunlight) = 5.01 µg/mL
PIF = 0.70
MPE = 0.005
Mean OD_{540 nm} solvent control value (viability) irradiated versus non-irradiated group: 92.1%

 

 

Table five

 

Treatment of BALB/c 3T3 with the positive control (Chlorpromazine) in the ME

With artificial sunlight				Without artificial sunlight
Conc. [µg/mL]	O.D.540 nm Mean Value*	Standard Deviation	% of Solv. Control	Conc. [µg/mL]	O.D.540 nm Mean Value*	Standard Deviation	% of Solv. Control
Solvent Control	1.1218*	0.0610	100.00	Solvent Control	0.9428*	0.0841	100.00
0.125	0.9862	0.1025	87.91	6.25	0.7969	0.0893	84.53
0.250	0.8035	0.0568	71.62	12.50	0.4056	0.0642	43.02
0.500	0.3973	0.0463	35.41	25.00	0.0905	0.0149	9.60
0.750	0.0636	0.0022	5.67	37.50	0.0552	0.0010	5.86
1.000	0.0838	0.0507	7.47	50.00	0.0559	0.0020	5.93
1.500	0.0695	0.0062	6.19	75.00	0.0547	0.0021	5.81
2.000	0.0867	0.0186	7.73	100.00	0.0540	0.0017	5.72
4.000	0.1213	0.0193	10.81	200.00	0.0577	0.0026	6.12

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

ED₅₀ value (with artificial sunlight) = 0.39 µg/mL
ED₅₀ value (without artificial sunlight) = 11.44 µg/mL
PIF = 29.31
MPE = 0.606
Mean OD₅₄₀ nm solvent control value (viability) irradiated versus non-irradiated group: 119.0%

 

Validity criteria fulfilled:

yes

Conclusions:

The study was performed to assess the phototoxic potential of Propineb Technical. The test was
performed using BALB/c 3T3 c31 cells. The experiment was performed twice. The first experiment served as a range finding experiment (RFE), the second one was the main experiment (ME). The ED50 value of the test item under irradiation was 7.21
(µg/mL (similar to the RFE) but only 5.01 (µg/mL in the absence of the artificial sunlight. The PIF
of the test item was, therefore, 4.55 in the RFE, and 0.70 in the ME (no phototoxic potential). The MPE values were calculated as 0.095 and 0.005, respectively. Both MPE values indicate a lack of
phototoxicity. Since the refined concentration selection in the ME is more precise than in the
RFE, it can be stated that in the study described and under the experimental conditions reported
Propineb Technical did not have a phototoxic effects on BALB/c 3T3 cells.

Executive summary:

A dose dependent cytotoxicity was observed after treatment of cells with Propineb Technical in the presence and absence of irradiation with artificial sunlight in both experiments. In the range finding experiment the ED50 value of the test item under irradiation was 6.74 μg/mL and 30.66 μg/mL in the absence of the artificial sunlight. In the main experiment selected the concentration range of Propineb Technical tested in absence of irradiation with artificial sunlight was narrowed around the ED50 values observed in the RFE in order to calculate the ED50 values and therefore, the PIF more precisely. The main experiment confirmed the cytotoxic effects both in presence and absence of the artificial sunlight. The ED50 value of the test item under irradiation was 7.21 μg/mL (similar to the RFE) but only 5.01 μg/mL in the absence of the artificial sunlight.

 

The PIF of the test item was, therefore, 4.55 in the RFE and 0.70 in the ME (no phototoxic potential). The MPE values were calculated as 0.095 and 0.005, respectively. Both MPE values indicate a lack of phototoxicity. Since the refined concentration selection in the ME is more precise than in the RFE, it can be stated that in the study described and under the experimental conditions reported. Propineb Technical did not have a phototoxic effects on BALB/c 3T3 cells.

Description of key information

No evidence of phototoxicity is observed in an in vitro phototoxicity study according to OECD 432 and GLP.

Key value for chemical safety assessment

Results:

no phototoxicity