Phosphoric acid, mixed decyl and octyl esters, compds. with diethanolamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base-pair changes or frameshifts in the genome of the strains used. Therefore, the test item is not considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

Mammalian Cell Gene Mutation AssayI:

n conclusion, in the described mutagenicity test under the experimental conditions reported, the test item is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Chromosome aberration test:

In conclusion, it can be stated that during the described in vitro chromosome aberration test and under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line.Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

S. typhimurium TA 1538

Principles of method if other than guideline:

Use of S. typhimurium strain TA 1538 instead of strain TA 102.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Name: Phosphoric acid, mixed decyl and octyl esters, compds. with diethanolamine (Phosphorsaeure (C8-C10-Alkyl)-ester, DEA-Salz)
CAS No.: 68425-57-0 (68186-45-8)
Physical state: white solid at 20 °C (turbid, non-pourable paste)

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The S. typhimurium strains are constructed to differentiate between base pair (TA 100, TA 1535) and frameshift (TA 98, TA 1537, TA 1538) mutations.

Species / strain / cell type:

S. typhimurium TA 98

Remarks:

S. typhimurium TA 98 his D3052 rfa- uvrB- R+

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 100

Remarks:

S. typhimurium TA 100 his G46 rfa- uvrB- R+

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1535

Remarks:

G46 rfa- uvrB-

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1537

Remarks:

his C3076 rfa- uvrB-

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1538

Remarks:

his D3052 rfa- uvrB-

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

The test substance was tested in two independent test series (plate incorporation assay) at concentration ranges of 8.0 - 5000 µg/plate (first test) and 6.25 - 1600 µg/plate (second test and repetition). Toxic effects were noted at concentrations of 200 µg/plate or higher.

Vehicle / solvent:

Aqua dest.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-aminoanthracene: TA 98 (-S9/+S9), TA 1535 (-S9/+S9), TA 100 (-S9/+S9); TA 1537 (-S9), TA 1538 (-S9/+S9); 4-Nitro-o-phenylendiamine: TA 98 (-S9/+S9), TA 1538 (-S9/+S9);

Details on test system and experimental conditions:

In order to investigate the potential of the test item for its ability to induce gene mutations the plate incorporation test (experiment I & II) was performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538. In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate.
Solutions of the test substance were prepared in aqua dest. and diluted in aqua dest. just before use. The following concentrations were tested:

1st test: 8, 40, 200, 1000 and 5000 pg/plate
2nd test: 6.25, 25, 100, 400 and 1600 pg/plate (and repetition)

Evaluation criteria:

A combination of the following criteria was considered as a positive result:

- The plate background of non-reverted bacteria did not show any growth reduction versus the respective negative controls.

- The spontaneous mutation rates of each tester strain per plate were within the characteristic spontaneous mutation range.

- As a rule, the positive controls showed mutation rates exceeding the control values of TA 100 at least by the factor 2.0 and those of the other tester strains at least by the factor 3.0.

- At more than one dose tested, the test substance caused at least a 2.0-fold increase in comparison with the negative controls in the tester strain TA 100. For the other tester strains, an increase in the mutation rate of more than 3.0 above the corresponding negative controls was considered positive.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Remarks:

Aqua dest.

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Sodium azide & 2-Aminoanthracene (-S9/+S9)

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Remarks:

Aqua dest.

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Sodium azide & 2-Aminoanthracene (-S9/+S9)

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Remarks:

Aqua dest.

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks:

9-Aminoacridine & 2-Aminoanthracene (-S9/+S9)

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks:

4-Nitro-o-phenylendiamine & 2-Aminoanthracene (-S9/+S9)

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Remarks:

Aqua dest.

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks:

4-Nitro-o-phenylendiamine & 2-Aminoanthracene (-S9/+S9)

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base-pair changes or frameshifts in the genome of the strains used.
Therefore, the test item is not considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

In order to investigate the potential of the test item for its ability to induce gene mutations two plate incorporation tests (experiment I & II) were performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. All criteria of validity were met.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base-pair changes or frameshifts in the genome of the strains used. Therefore, the test item is not considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Mammalian Cell Gene Mutation Assay

Specific details on test material used for the study:

Name: Phosphoric acid, mixed decyl and octyl esters, compds. with diethanolamine
CAS No.: 68425-57-0
Physical state: white solid at 20 °C (turbid, non-pourable paste)
Batch No.: PU61810016
Re-certification date of batch: 16 June 2018
Purity: 100 % (UVCB)
Moisture, % (KF) 1,2
pH, 10% in dW 7,2
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Target gene:

Thymidine Kinase Locus/TK+/-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Mouse Lymphoma L5178Y cells (clone TK+/- -3.7.2C). These cells are characterised by their high proliferation rate (10-12 h doubling time of the Eurofins Munich stock cultures) and their cloning efficiency, usually more than 50%. The cells obtain a near diploid karyotype (40 ± 2 chromosomes). They are heterozygous at the Thymidine Kinase (TK) locus in order to detect mutation events at the TK- locus.

Additional strain / cell type characteristics:

other: TK+/- -3.7.2C

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

The selection of the concentrations used in the main experiment was based on data from the pre-experiment. In the main experiment without metabolic activation 95 µg/mL and with metabolic activation 120 µg/mL were selected as the highest concentrations. The experiment without and with metabolic activation was performed as a 4 h short-term exposure assay.

Vehicle / solvent:

Treatment medium RPMI 1640 supplemented with 5 % Horse serum, 100 U/100 µg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B.

Untreated negative controls:

yes

Remarks:

RPMI cell culture medium

Negative solvent / vehicle controls:

yes

Remarks:

RPMI cell culture medium

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

methylmethanesulfonate

Evaluation criteria:

The test item is considered mutagenic if the following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126
mutants per 10^6 cells and
- a dose-dependent increase in mutant frequency is detected.

Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potentialclastogenic effects and/or chromosomal aberrations. According to the OECD guideline, the biological relevance is considered first for the interpretation of result. Statistical methods might be used as an aid in evaluation of the test result. A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend of the test is negative.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Remarks:

RPMI cell culture

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks:

MMS & EMS

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Remarks:

RPMI cell culture

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks:

MMS & EMS

Results

The test item was assessed for a possible potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The assay was conducted without and with metabolic activation. The experiment with metabolic activation was performed by including liver microsomes and NADP for efficient detection of a wide variety of carcinogens requiring metabolic activation. The selection of the concentrations used in the main experiment was based on data from the preexperiment according to the OECD guideline 490. In the main experiment without metabolic activation 95 µg/mL and with metabolic activation 120 µg/mL were selected as the highest concentrations. The experiment without and with metabolic activation was performed as a 4 h short-term exposure assay. The test item was investigated at the following concentrations:

without metabolic activation:

50, 60, 70, 80, 90 and 95 µg/mL

and with metabolic activation:

70, 80, 90, 95, 100, 110, 115 and 120 µg/mL

The pH-value detected with the test item was within the physiological range.

Precipitation:

No precipitation of the test item was noted in the experiment.

Toxicity:

Growth inhibition was observed in the main experiment without and with metabolic activation.In the main experiment without metabolic activation the relative total growth (RTG) was 13.5% for

the highest concentration (95 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 120 µg/mL with a RTG of 11.2%.

Mutagenicity:

The mutant frequencies obtained from all experiments were compared with the Global Evaluation Factor (GEF) and a statistical analysis was performed. The GEF is defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data are gathered from ten laboratories. For the microwell method the GEF was defined to be 126. Criterion for mutagenicity is the extension of the GEF by the induced mutant frequency as well as a dose-dependent increase in mutant frequency. The positive controls EMS (300 µg/mL), MMS (10 µg/mL) and B[a]P (2.5 µg/mL) showed distinct effects in mutation frequency, thus proving the ability of the test system to detect potential mutagenic effects. In the main experiment without metabolic activation all validity criteria were met. The negative controls showed mutant frequencies within the acceptance range of 50-170 mutants/10^6 cells, according to the IWGT criteria. The mutant frequencies of the negative control were 52.2 and 59.8 mutants/106 cells, the positive controls EMS and MMS induced a distinct increase in mutant frequency with 612.8 and 402.9 mutants/10^6 cells. The mutant frequencies induced by the test item did not show a biologically relevant increase. The GEF of 126 was not exceeded in any of the dose groups showing induced mutant frequencies between -4.4 and 44.5 mutants/106 cells (Table 5). A statistical analysis displayed that one of the mutant frequencies was significantly increased over those of the negative controls, but neither the GEF was exceeded nor evidence for a dose-response relationship was noted. Therefore this effect was considered as not biologically relevant. In the main experiment with metabolic activation all validity criteria were met. The negative controls showed mutant frequencies within the acceptance range of 50-170 mutants/10^6 cells, according to the IWGT criteria. The mutant frequencies of the negative control were 50.1 and 73.1 mutants/10^6  cells, the positive control B[a]P induced a distinct increase in mutant frequency with 604.2 mutants/10^6 cells. The mutant frequencies induced by the test item did not show a biologically relevant increase. The GEF of 126 was not exceeded in any of the dose groups showing induced mutant frequencies between -11.6 and 26.5 mutants/10^6 cells. None of the observed mutant frequencies was statistically significantly increased over those of the negative controls. All mutant frequencies for negative and positive controls of the main experiment were found within the historical range of the test facility Eurofins Munich.

Clastogenicity:

Colony sizing was performed for the highest concentrations of the test item and for the negative and positive controls. An extension of the GEF by the induced mutant frequency in combination with an increased occurrence of small colonies (defined by slow growth and/or morphological alteration of the cell clone) is an indication for potential clastogenic effects and/or chromosomal aberrations. The positive controls MMS (10 µg/mL) and B[a]P (2.5 µg/mL) induced a significant increase in mutant frequency and a biologically significant increase of small colonies (≥40%), thus proving the ability of

the test system to indicate potential clastogenic effects. In the main experiment without metabolic activation the percentage of small colonies in the negative controls was found to be 8.6% and 6.4%. The percentage of small colonies in the positive control MMS was found to be 49.4%. In the highest dose groups 6.8% (80 µg/mL), 19.4% (90 µg/mL) and 8.3% (95 µg/mL) of small colonies were found (Table 6). As none of the values exceeded 40%, all dose groups were considered as not clastogenic. With metabolic activation the percentage of small colonies in the negative controls was found to be 6.7% and 11.1%. The percentage of small colonies of the positive control B[a]P was found to be 57.8%. In the highest dose groups 8.5% (110 µg/mL), 12.1% (115 µg/mL) and 7.0% (120 µg/mL) of small colonies were found. As none of the values exceeded 40%, all dose groups were considered as not clastogenic.

Tables

Pre-Experiment for Toxicity, without metabolic activation

Test Group	Concentration [µg/mL]	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	Suspension Growth (SG) a	Relative Suspension Growth (RSG) [%] b
C1	0	299000	911000	1560000	14.2	100.0
C2	0	309000	922000	1600000	14.8	100.0
1	10	267000	871000	1570000	13.7	94.4
2	20	313000	947000	1580000	15.0	103.3
3	50	308000	999000	1510000	15.1	104.2
4	150	30800	18500	48100	0.1	1.0
5	500	42600	†	†	n.d.	n.d.
6	1000	2130	†	†	n.d.	n.d.

C: Negative control

a: Suspension Growth, SG = [((value 24h x 30) / 1x10^7) x ((value 48 h x 20) / (value 24 h*x20))];

* : for value 24 h > 3x10^5 then value 24 h = 3x10^5

b: Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]

†: no viable cells

n.d.: no data

Pre-Experiment for Toxicity, with metabolic activation

Test Group	Concentration [µg/mL]	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	Suspension Growth (SG) a	Relative Suspension Growth (RSG) [%] b
C1	0	312000	1020000	1560000	15.9	100.0
C2	0	323000	981000	1570000	15.4	100.0
1	10	311000	913000	1580000	14.4	92.1
2	20	331000	997000	1510000	15.1	96.2
3	50	305000	946000	1550000	14.7	93.7
4	150	28600	33700	163000	0.5	3.1
5	500	2690	†	†	n.d.	n.d.

C: Negative control

a: Suspension Growth, SG = [((value 24h x 30) / 1x10^7) x ((value 48 h x 20) / (value 24 h*x20))];

* : for value 24 h > 3x10^5 then value 24 h = 3x10^5

b: Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]

†: no viable cells

n.d.: no data

Main Experiment - Toxicity Data, without metabolic activation

Test Group	Concentration [µg/mL]	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	Suspension Growth (SG) a	Relative Suspension Growth (RSG) [%] b	Relative Cloning Efficiency (RCE) c	Relative Total Growth (RTG) d
C1	0	318000	938000	1480000	13.9	100.0	100.0	100.0
C2	0	337000	1010000	1430000	14.4	100.0	100.0	100.0
1	50	293000	900000	1540000	13.9	97.9	97.0	94.9
2	60	350000	996000	1580000	15.7	111.1	108.9	121.0
3	70	276000	707000	1630000	11.5	81.4	89.7	73.0
4	80	282000	800000	1520000	12.2	85.9	92.5	79.4
5	90	182000	351000	756000	5.2	36.4	87.0	31.7
6	95	111000	137000	756000	2.3	16.0	84.4	13.5
EMS	300	327000	781000	1430000	11.2	78.9	73.0	57.5
MMS	10	315000	782000	1390000	10.9	76.7	69.9	53.9

C: Negative control

a: Suspension Growth, SG = [((value 24hx30)/1x10^7) x ((value 48 h x 20)/(value 24 h*x20))];

* : for value 24h > 3x10^5 then value 24h = 3x10^5

b: Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]

c: Relative Cloning Efficiency, RCE = [(CE dose group / CE of corresponding controls) x 100]

Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x 100)

d: Relative Total Growth, RTG = (RSG x RCE)/100

EMS: Ethylmethanesulfonate

MMS: Methylmethanesulfonate

Main Experiment - Mutagenicity Data, without metabolic activation

		Cloning Efficiency (CE)			Mutagenicity Data
Test Group	Concentration [µg/mL]	Plate 1 e	Plate 2 e	Cloning Efficiency (CE) f	Number of cultures / 96 wells					MF      [mutants/ 10^6 cells]  g	IMF      [mutants/ 10^6 cells]  h
					Plate 1 e	Plate 2 e	Plate 3 e	Plate 4  e	Mean
C1	0	73	75	92.1	13	7	5	10	8.8	52.2	/
C2	0	78	81	110.1	7	12	18	10	11.8	59.8	/
1	50	81	71	98.0	10	9	13	7	9.8	54.8	-1.2
2	60	79	80	110.1	8	7	15	11	10.3	51.6	-4.4
3	70	72	75	90.7	9	17	15	14	13.8	85.6	29.6
4	80	74	75	93.5	13	11	11	9	11.0	65.1	9.1
5	90	70	75	88.0	20	13	17	12	15.5	100.6	44.5
6	95	74	69	85.4	12	10	15	11	12.0	78.4	22.3
EMS	300	66	67	73.7	59	52	60	57	57.0	612.8	556.7
MMS	10	63	67	70.6	40	35	46	45	41.5	402.9	346.9

C: Negative control

e: Number of cultures with cell growth.

f: Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x 100)

g: Mutant frequency,

MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800

h: Induced mutant frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls

EMS: Ethylmethanesulfonate

MMS: Methylmethanesulfonate

Main Experiment - Colony Sizing, without metabolic activation

Test group	Concentration [µg/mL]	Wells with at least   1 colony	Large colonies	Small colonies	% small colonies
C1	0	35	32	3	8.6
C2	0	47	44	3	6.4
4	80	44	41	3	6.8
5	90	62	50	12	19.4
6	95	48	44	4	8.3
MMS	10	166	84	82	49.4

C: Negative control

MMS: Methylmethanesulfonate

Main Experiment - Toxicity Data, with metabolic activation

Test Group	Concentration [µg/mL]	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	Suspension Growth (SG) a	Relative Suspension Growth (RSG) [%] b	Relative Cloning Efficiency (RCE) c	Relative Total Growth (RTG) d
C1	0	287000	768000	1570000	12.1	100.0	100.0	100.0
C2	0	298000	785000	1530000	12.0	100.0	100.0	100.0
3	70	278000	768000	1620000	12.4	103.4	107.0	110.6
4	80	299000	821000	1630000	13.4	111.2	97.8	108.7
5	90	248000	726000	1580000	11.5	95.3	100.7	96.0
6	95	283000	753000	1580000	11.9	98.9	110.3	109.0
7	100	219000	528000	1620000	8.6	71.1	119.3	84.8
8	110	222000	374000	1580000	5.9	49.1	123.3	60.5
9	115	139000	154000	883000	2.6	22.0	112.0	24.7
10	120	123000	95200	446000	1.3	11.1	100.7	11.2
B[a]P	2.5	242000	369000	1390000	5.1	42.6	75.5	32.2

C: Negative control

a: Suspension Growth, SG = [((value 24hx30)/1x10^7) x ((value 48 h x 20)/(value 24 h*x20))];

* : for value 24h > 3x10^5 then value 24h = 3x105

b: Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]

c: Relative Cloning Efficiency, RCE = [(CE dose group / CE of corresponding controls) x 100)

Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x 100

d: Relative Total Growth, RTG = (RSG x RCE)/100

B[a]P: Benzo[a]pyrene

Main Experiment - Mutagenicity Data, with metabolic activation

		Cloning Efficiency (CE)			Mutagenicity Data
Test Group	Concentration [µg/mL]	Plate 1 e	Plate 2 e	Cloning Efficiency (CE) f	Number of cultures / 96 wells					MF      [mutants/ 10^6 cells]  g	IMF      [mutants/ 10^6 cells]  h
					Plate 1 e	Plate 2 e	Plate 3 e	Plate 4  e	Mean
C1	0	64	76	81.6	6	9	4	11	7.5	50.1	/
C2	0	69	74	85.4	12	9	13	11	11.3	73.1	/
3	70	71	75	89.3	6	10	13	14	10.8	66.9	5.3
4	80	70	70	81.6	10	6	5	9	7.5	50.0	-11.6
5	90	73	69	84.1	13	5	16	8	10.5	69.6	8.0
6	95	74	74	92.1	14	13	12	11	12.5	75.8	14.2
7	100	79	74	99.6	10	14	13	13	12.5	70.1	8.5
8	110	77	78	102.9	13	14	9	11	11.8	63.6	2.0
9	115	76	73	93.5	14	20	10	14	14.5	88.1	26.5
10	120	65	77	84.1	14	11	10	8	10.8	70.8	9.2
B[a]P	2.5	60	62	63.1	49	48	49	58	51.0	604.2	542.6

C: Negative control

e: Number of cultures with cell growth.

f: Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x 100)

g: Mutant frequency,

MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800

h: Induced mutant frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls

B[a]P: Benzo[a]pyrene

Main Experiment - Colony Sizing, with metabolic activation

Test group	Concentration [µg/mL]	Wells with at least   1 colony	Large colonies	Small colonies	% small colonies
C1	0	30	28	2	6.7
C2	0	45	40	5	11.1
8	110	47	43	4	8.5
9	115	58	51	7	12.1
10	120	43	40	3	7.0
B[a]P	2.5	204	86	118	57.8

C: Negative control

B[a]P: Benzo[a]pyrene

Statistical significance at the 5% level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Biometry - Main Experiment without metabolic activation

Test group	Concentration [µg/mL]	mean mutant frequency	mean induced mutant frequency	p-value	statistical significance
C1	0	52.2	/	/	/
C2	0	59.8	/	/	/
1	50	54.8	-1.2	> 0.999	-
2	60	51.6	-4.4	0.715	-
3	70	85.6	29.6	0.109	-
4	80	65.1	9.1	0.346	-
5	90	100.6	44.5	0.028	+
6	95	78.4	22.3	0.109	-
EMS	300	612.8	556.7	0.004	+
MMS	10	402.9	346.9	0.004	+

C: Negative control

EMS: Ethylmethanesulfonate

MMS: Methylmethanesulfonate

+: significant

-: not significant

Statistical significance at the 5% level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Biometry - Main Experiment with metabolic activation

Test group	Concentration [µg/mL]	mean mutant frequency	mean induced mutant frequency	p-value	statistical significance
C1	0	50.1	/	/	/
C2	0	73.1	/	/	/
3	70	66.9	5.3	0.570	-
4	80	50.0	-11.6	0.348	-
5	90	69.6	8.0	0.683	-
6	95	75.8	14.2	0.214	-
7	100	70.1	8.5	0.655	-
8	110	63.6	2.0	0.849	-
9	115	88.1	26.5	0.141	-
10	120	70.8	9.2	0.683	-
B[a]P	2.5	604.2	542.6	0.004	+

C: Negative control

B[a]P: Benzo[a]pyrene

+: significant

-: not significant

Conclusions:

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Executive summary:

The test item was assessed for a possible potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.The assay was conducted without and with metabolic activation. The experiment with metabolic activation was performed by including liver microsomes and NADP for efficient detection of a wide variety of carcinogens requiring metabolic activation. The selection of the concentrations used in the main experiment was based on data from the pre-experiment according to the OECD guideline 490. In the main experiment without metabolic activation 95 µg/mL and with metabolic activation 120 µg/mL were selected as the highest concentrations. The experiment without and with metabolic activation was performed as a 4 h short-term exposure assay. In the described mutagenicity test under the experimental conditions reported, the test item is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro Mammalian Chromosome Aberration Test

Specific details on test material used for the study:

Name: Phosphoric acid, mixed decyl and octyl esters, compds. with diethanolamine
CAS No.: 68425-57-0
Physical state: white solid at 20 °C (turbid, non-pourable paste)
Batch No.: PU61810016
Re-certification date of batch: 16 June 2018
Purity: 100 % (UVCB)
Moisture, % (KF) 1,2
pH, 10% in dW 7,2
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Target gene:

Induction of structural chromosome aberrations in Chinese hamster V79 cells.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Cell bank of Eurofins Munich

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

A pre-experiment was conducted under identical conditions as described for the main experiment. The following concentrations were tested without and with S mix: 5, 10, 20, 50, 100, 200, 500, 1000, 2000 and 5000 µg/mL
Cytotoxicity was characterised by the percentages of the relative increase in cell count (RICC) in comparison with the controls.

Vehicle / solvent:

A solubility test was performed with different solvents up to the maximum recommended concentration of 5 mg/mL. Due to the nature of the test item, it was not possible to prepare a solution of the test item with an appropriate solvent. Based on the results of the solubility test, the best suited vehicle was MEM cell culture medium. For osmolality 331 mOsmol/kg was measured in the highest concentration of 5 mg/mL and the pH was within the physiological range. Different test item concentrations were prepared by dilution of the 5 mg/mL test item suspension in cell culture medium. The solvent was compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

yes

Remarks:

MEM medium

Negative solvent / vehicle controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data

When all of these criteria are met, the test chemical is then considered able to induce chromosomal aberrations in cultured mammalian cells in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data.

The test chemical is then considered unable to induce chromosomal aberrations in cultured mammalian cells in this test system.

Statistics:

Statistical significance at the 5% level (p < 0.05) was evaluated by the Fischer´s exact test. The p value was used as a limit in judging for significance levels in comparison with the corresponding negative control. Aberrant cells without gaps were only used for the calculation. Gaps are recorded separately and reported but generally not included in the total aberration frequency calculation according to the guideline.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Remarks:

MEM medium

Untreated negative controls validity:

valid

Remarks:

MEM medium

Positive controls validity:

valid

Remarks:

EMS, ethylmethanesulfonate(-S9);

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Remarks:

MEM medium

Untreated negative controls validity:

valid

Remarks:

MEM medium

Positive controls validity:

valid

Remarks:

CPA, cyclophosphamide (+S9);

Results

Exposure Concentrations

On the basis of the data and the observations from the pre experiment and taking into account the recommendations of the guidelines, the following concentrations were selected for the main experiments I and II. The dose group selection for microscopic analyses of chromosomal aberrations was based in accordance with the recommendations of the guidelines.

4h exposure, -S9: 5, 10, 20 µg/ml

4h exposure, +S9: 40, 60, 70 µg/ml

21h exposure, -S9: 20, 30, 50 (P) µg/ml

P = Precipitation was observed at the end of treatment

Discussion

In an in vitro chromosome aberration assay, the test item was investigated for the potential to induce structural chromosomal aberrations in Chinese hamster V79 cells in the absence and presence of metabolic activation with S9 homogenate. The selection of the concentrations used in experiment I and II was based on data from the solubility test and the pre-experiment which were performed according to the guidelines. In experiment I without metabolic activation, 20 µg/mL and with metabolic activation, 70 µg/mL were selected as highest concentrations for the microscopic analysis of chromosomal aberrations. In experiment II without metabolic activation, 50 µg/mL was selected as highest test item concentration for the microscopic analysis of chromosomal aberrations. The chromosomes were prepared 21 h after start of treatment with the test item. The treatment intervals were 4 h without and with metabolic activation (experiment I) and 21 h without metabolic activation (experiment II). Duplicate cultures were set up. 150 metaphases per culture were scored for structural chromosomal aberrations.

Experiment I:

without metabolic activation: 5, 10 and 20 µg/mL

with metabolic activation: 40, 60 and 70 µg/mL

Experiment II:

without metabolic activation: 20, 30 and 50 µg/mL

Precipitation

Precipitation was noted in experiment I without metabolic activation at a concentration of 50 µg/mL and with metabolic activation at 150 µg/mL. In experiment II without metabolic activation, precipitation was observed at a concentration of 50 µg/mL.

Toxicity

Cytotoxic effects of the test item (decrease below 70% rel. RICC) were noted in experiment I without metabolic activation at a concentration of 20 µg/mL (RICC 63%). At a concentration of 30 µg/mL and higher almost each cell was destroyed. In experiment I with metabolic activation, no cytotoxic effects were determined at a concentration of 70 µg/mL (RICC 76%). However, at the test item concentration of 80 µg/mL and higher, the RICC was decreased to 0%. Because of the steep increase in cytotoxicity despite closely spaced concentrations, the concentrations 20 µg/mL and 70 µg/mL were evaluated for chromosomal aberrations. In experiment II without metabolic activation, no cytotoxic effects were detected up to a concentration of 50 µg/mL (RICC 91%).

Clastogenicity

There are several criteria for determining a positive result, such as a concentration-related increase or a reproducible increase in the number of cells with chromosome aberrations for at least one of the dose groups, which is higher than the laboratory negative control range. In experiment I without metabolic activation the aberration rates of the negative control (2.7%) and all concentrations treated with the test item (5 µg/mL: 1.7%; 10 µg/mL: 2.3% and 20 µg/mL: 3.0%) were within the historical control data of the testing facility (0.0% – 4.0%). With metabolic activation, the aberration rates of the negative control (1.7%) and all test item concentrations (40 µg/mL and 60 µg/mL: 1.3%; 70 µg/mL: 2.0%) were within the historical control data of the testing facility (0.0% – 4.3%).

In experiment II without metabolic activation, the aberration rates of the negative control (2.3%) and the following concentrations treated with the test item (20 µg/mL: 1.7% and 50 µg/mL: 1.3%) were within the historical control data of the testing facility (0.0% – 3.0%). An increase of aberrant cells was noted at a concentration of 30 µg/mL (4.0%). However, this increase was considered as not biologically relevant as the next higher concentration was within the historical control range. Moreover, the induction was neither statistically significant nor a concentration-related increase was detected. The Fisher´s exact test was performed to verify the results in the experiment. No statistically significant increase (p < 0.05) of cells with chromosomal aberrations was noted in the dose groups of the test item evaluated in experiment I and II without and with metabolic activation.The chi square Test for trend was performed to test whether there is a concentration-related increase in chromosomal aberrations. No statistically significant increase was observed in experiment I without and with metabolic activation and in experiment II without metabolic activation.

EMS (600 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the ability of the test system to indicate potential clastogenic effects.

Polyploid Cells

No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item.

Tables:

Experiment I - Summary of Cytotoxicity Data

Cell count
Dose group	Concentration [µg/ml]	Culture			RICC [%]	Precipitate (+/-)
without metabolic activation		1	2	mean
C	0	154	139	147	100	-
1	5	128	113	121	81	-
2	10	142	132	137	93	-
3	20	103	90	97	63	-
4	30	5	6	6	0	-
5	40	0	0	0	0	-
6	50	0	0	0	0	+
7	60	n.d.	n.d.	-	-	+
8	80	n.d.	n.d.	-	-	+
9	100	n.d.	n.d.	-	-	+
EMS	600					-
with metabolic activation
C	0	148	170	159	100	-
1	40	148	120	134	83	-
2	60	146	154	150	94	-
3	70	112	134	123	76	-
4	80	12	5	9	0	-
5	100	1	4	3	0	-
6	120	0	0	0	0	-
7	150	n.d.	n.d.	-	-	+
8	200	n.d.	n.d.	-	-	+
CPA	0.83	120	131	126	78	-

RICC: Relative Increase in Cell Count, calculated by the increase in cell number of the test concentrations compared to the control concentration. The cell count was determined by a cell counter per culture for each test group.

C: Negative Control (Culture Medium)

EMS: Positive Control (without metabolic activation: Ethylmethanesulfonate)

CPA: Positive Control (with metabolic activation: Cyclophosphamide)

n.d: not determined

Experiment I – Summary of Aberration Rates

Dose Group	Concentration [µg/ml]	Treatment time [h]	Fixation Interval [h]	mean abberant cells		Precipitation
without metabolic activation				incl. gaps	excl. gaps
C	0	4	21	5.3	2.7	-
1	5	4	21	2.3	1.7	-
2	10	4	21	3.3	2.3	-
3	20	4	21	5.3	3.0	-
EMS	600	4	21	12.8	9.2	-
with metabolic activation
C	0	4	21	3.3	1.7	-
1	40	4	21	3.0	1.3	-
2	60	4	21	3.3	1.3	-
3	70	4	21	3.7	2.0	-
CPA	0.83	4	21	9.7	7.0	-

300 cells were evaluated for each concentration, except for the positive control EMS without metabolic activation (250 cells), because of a clearly positive result.

C: Negative Control (Culture Medium)

EMS: Positive Control (without metabolic activation: Ethylmethanesulfonate)

CPA: Positive Control (with metabolic activation: Cyclophosphamide)

Experiment II - Summary of Cytotoxicity Data

Cell count
Dose group	Concentration [µg/ml]	Culture			RICC [%]	Precipitate (+/-)
without metabolic activation		1	2	mean
C	0	232	174	203	100	-
1	0.1	204	216	210	104	-
2	0.2	184	183	184	90	-
3	0.5	201	186	194	95	-
4	1	148	197	173	84	-
5	2	174	193	184	90	-
6	5	228	230	229	113	-
7	10	183	173	178	87	-
8	20	205	222	214	105	-
9	30	231	250	241	119	-
10	50	213	158	186	91	+
EMS	600	153	167	160	78	-

RICC: Relative Increase in Cell Count, calculated by the increase in cell number of the test concentrations compared to the control concentration. The cell count was determined by a cell counter per culture for each test group.

C: Negative Control (Culture Medium)

EMS: Positive Control (without metabolic activation: Ethylmethanesulfonate)

Experiment II – Summary of Aberration Rates

Dose Group	Concentration [µg/ml]	Treatment time [h]	Fixation Interval [h]	mean abberant cells		Precipitation
without metabolic activation				incl. gaps	excl. gaps
C	0	21	21	4.0	2.3	-
8	20	21	21	2.3	1.7	-
9	30	21	21	5.0	4.0	-
10	50	21	21	2.3	1.3	+
EMS	600	21	21	33.3	32.0	-

300 cells were evaluated for each concentration, except for the positive control EMS (150 cells), because of a clearly positive result.

C: Negative Control (Culture Medium)

EMS: Positive Control (without metabolic activation: Ethylmethanesulfonate)

Biometry

Statistical significance at the 5% level (p < 0.05) was evaluated by the Fischer´s exact test. The p value was used as a limit in judging for significance levels in comparison with the corresponding negative control. Aberrant cells without gaps were only used for the calculation. Gaps are recorded separately and reported but generally not included in the total aberration frequency calculation according to the guideline.

Biometry - Experiment I, without metabolic activation

Negative Control versus Test Group	Concentration [µg/ml]	Treatment time [h]	abberant cells (excl. gaps)	Significance	p value
C	0	4	8	/	/
1	5	4	5	-	0.5786
2	10	4	7	-	1.0000
3	20	4	9	-	1.0000
EMS	600	4	23	+	0.0013

+: significantly increased

-: not significant

EMS: Positive Control (Ethylmethanesulfonate)

Biometry - Experiment I, with metabolic activation

Control versus Test Group	Concentration [µg/ml]	Treatment time [h]	abberant cells (excl. gaps)	Significance	p value
C	0	4	5	/	/
1	40	4	4	-	0.5786
2	60	4	4	-	1.0000
3	70	4	6	-	1.0000
CPA	0.83	4	21	+	0.0020

+: significantly increased

-: not significant

CPA: Positive Control (Cyclophosphamide)

Biometry - Experiment II, without metabolic activation

Control versus Test Group	Concentration [µg/ml]	Treatment time [h]	abberant cells (excl. gaps)	Significance	p value
C	0	21	7	/	/
1	20	21	5	-	0.7721
2	30	21	12	-	0.3515
3	50	21	4	-	0.5450
EMS	600	21	48	+	< 0.0001

+: significantly increased

-: not significant

EMS: Positive Control (Ethylmethanesulfonate)

Statistical significance at the 5% level (p < 0.05) was evaluated by the chi square test for trend. The p value was used as a limit in judging for significance levels.

Biometry – Trend test

Experiment	Treatment time [h]	Significance	p value
Exp. I without metabolic activation	4	-	0.2794
Exp. I with metabolic activation	4	-	0.5094
Exp. II without metabolic activation	21	-	0.7868

+: significant

-: not significant

Statistical significance: statistical significant concentration-related increase in cells with chromosomal aberrations (chi squaretest for trend, p < 0.05).

No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item. Therefore, the test item the test item is considered to be non-clastogenic in this chromosome aberration test.

Conclusions:

In conclusion, it can be stated that during the described in vitro chromosome aberration test and under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line.
Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test.

Executive summary:

In an in vitro chromosome aberration assay, the test item was investigated for the potential to induce structural chromosomal aberrations in Chinese hamster V79 cells in the absence and presence of metabolic activation with S9 homogenate. The selection of the concentrations used in experiment I and II was based on data from the solubility test and the pre-experiment which were performed according to the guidelines.

In experiment I without metabolic activation, 20 µg/mL and with metabolic activation, 70 µg/mL were selected as highest concentrations for the microscopic analysis of chromosomal aberrations. In experiment II without metabolic activation, 50 µg/mL was selected as highest test item concentration for the microscopic analysis of chromosomal aberrations. The chromosomes were prepared 21 h after start of treatment with the test item. The treatment intervals were 4 h without and with metabolic activation (experiment I) and 21 h without metabolic activation (experiment II). Duplicate cultures were set up. 150 metaphases per culture were scored for structural chromosomal aberrations. It can be stated that during the described in vitro chromosome aberration test and under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line. No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item.

Therefore, the test item the test item is considered to be non-clastogenic in this chromosome aberration test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available