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REACH

Phosphoric acid, 2-ethylhexyl ester, sodium salt

EC number: 269-044-8 | CAS number: 68186-64-1

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test: Negative;

HPRT Assay: Negative;

Chromosome Aberration Assay: Negative

Link to relevant study records

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-04-04 to 2019-06-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

Qualifier:

according to guideline

Guideline:

other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use, June 2012

Version / remarks:

2012

Deviations:

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– → trp+ reversions. The Escherichia coli WP2 uvrA strain detects mutagens that cause other base-pair substitutions (AT to GC).

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

induced rat liver S9

Test concentrations with justification for top dose:

At the preparation of the test item stock solution a correction (multiplier) factor of 1.953 (1/0.512=1.953) based on the purity of 51.2% was taken into consideration.

Based on the cytotoxicity and solubility results of the preliminary concentration range finding test (informatory toxicity test) and based on the recommendations in OECD 471 guideline, the following concentrations of the test item were prepared and investigated in the initial and confirmatory mutation tests:

3200; 1600; 800; 320; 128; 51.2 and 20.5 μg/plate.

Vehicle / solvent:

- Vehicle/solvent used: ultrapure water

- Justification for choice of solvent/vehicle: suitable vehicle according to the guideline and sufficient solubility of the test item.

- Justification for percentage of solvent in the final culture medium: as recommended in the guideline

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-Nitro-1,2-phenylenediamine, NPD (TA 98; without S9; 4 µg/plate); 2-aminoanthracene, 2AA (all strains, with S9; 2 µg/plate (S. typhimurium), 50 µg/plate (E.coli)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 20 min at 37 °C (bacterial culture and the S9 Mix or phosphate buffer)
- Exposure duration: 48 hours in the dark at 37 °C

NUMBER OF REPLICATIONS:
3

DETERMINATION OF CYTOTOXICITY
A dose level is considered toxic if:
- the reduced revertant colony numbers are observed as compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or
- the reduced revertant colony numbers are below the historical control data range and / or
- pinpoint colonies appear and / or
- reduced background lawn development occurs
- other: The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment. 7 concentrations were tested for toxicity and mutation induction with 3 plates each. The experimental conditions in this pre-experiment were the same as for the main experiment I (plate incorporation test) and included non-activated and S9 activated test conditions with appropriate positive and negative controls. The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate. In the toxicity test the concentrations examined were: 5000, 1600, 500, 160, 50, 16 and 5 μg/plate.

Rationale for test conditions:

According to guidelines.

Evaluation criteria:

The colony numbers on the control, positive control and the test plates were determined, the mean values, standard deviations and the mutation rates were calculated.

Mutation Rate = Mean revertants at the test item (or control) treatments / Mean revertrants of vehicle control

A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
According to the guidelines, the biological relevance of the results was the criterion for the interpretation of results, a statistical evaluation of the results was not regarded as necessary.
Criteria for a Negative Response:
A test article is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

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

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

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

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

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

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

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

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: yes
- Data on osmolality: yes
- Possibility of evaporation from medium: no
- Water solubility: sufficient soluble
- Precipitation and time of the determination: no

RANGE-FINDING/SCREENING STUDIES: yes

STUDY RESULTS
- Concurrent vehicle negative and positive control data

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value

Ames test:
- Signs of toxicity : please refer to "any other information on results including tables"
- Individual plate counts: please refer to "any other information on results including tables"
- Mean number of revertant colonies per plate and standard deviation : please refer to "any other information on results including tables"

HISTORICAL CONTROL DATA
- Positive historical control data: please refer to "any other information on results including tables"
- Negative (solvent/vehicle) historical control data: please refer to "any other information on results including tables"

Table 1: Summary of Signs of Cytotoxicity in the Plate Incorporation and Pre-Incubation Tests

Plate Incorporation Test
Concentrations (µg/plate)	*Salmonella typhimurium*								Escherichia coliWP2uvrA
	TA98		TA100		TA1535		TA1537		Escherichia coliWP2uvrA
	‑S9	+S9	‑S9	+S9	‑S9	+S9	‑S9	+S9	‑S9	+S9
3200	B<<	B<<	B<<	B<<	B<<	SB<	B0	B0	B<<	SB<<
1600	*	–	SB<<	<<	–	–	SB<	*	*	<<
800	–	*	<<	–	–	–	*	–	*	–
320	–	*	–	–	–	–	–	–	*	–
128	–	–	*	<<*	–	–	–	–	–	–
51.2	–	–	–	–	–	–	–	–	*	–
20.5	–	*	–	–	–	–	–	–	*	–
Pre-Incubation Test
Concentrations (µg/plate)	*Salmonella typhimurium*								Escherichia coliWP2uvrA
	TA98		TA100		TA1535		TA1537		Escherichia coliWP2uvrA
	‑S9	+S9	‑S9	+S9	‑S9	+S9	‑S9	+S9	‑S9	+S9
3200	A	A	A	A	A	A	A	A	B<<	SB, PP<<
1600	B0	B0	B0	B0	B0	B0	B0	B<<	SB<<	<
800	B<<	SB<<	B0	B<<	SB<	SB<	B<	SB0	*	–
320	SB<<	–	SB#	–	SB<	–	B<	SB	–	–
128	–	–	–	–	–	–	–	–	–	–
51.2	–	–	–	–	–	*	–	–	–	–
20.5	–	–	–	–	–	–	–	–	–	–

A: Absent background lawn development;

B0: No revertant growth and reduced background lawn development;

B<<: Reduced background lawn development and revertant colony numbers below the vehicle and historical control data ranges;

B<: Reduced background lawn development and revertant colony numbers below the vehicle control data range;

SB0: No revertant growth and slightly reduced background lawn development;

SB<<: Slightly reduced background lawn development and revertant colony numbers below the vehicle and historical control data ranges;

SB<: Slightly reduced background lawn development and revertant colony numbers below the vehicle control data range;

SB#: Slightly reduced background lawn development and revertant colony numbers within the vehicle control data range; however, below the historical control data range;

SB: Slightly reduced background lawn development and revertant colony numbers within the historical control data range and within the vehicle control data range;

PP: Pinpoint colonies;

<<: Revertant colony numbers below the vehicle and historical control data ranges;

<: Revertant colony number significantly lightly below the corresponding vehicle control range; evaluated as inhibition;

‑: No inhibition

*: Revertant colony number slightly below the corresponding vehicle control range; however, within the biological variability range of the applied test system, evaluated as no inhibition

Table 2 Summary table of the results of the initial mutation test

Initial Mutation Test (Plate Incorporation Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains																*Escherichia coli*
	TA 98				TA 100				TA 1535				TA 1537				WP2uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	13.0	0.78	25.3	1.01	82.3	1.04	96.3	1.02	9.7	0.91	11.0	1.14	4.3	0.76	7.7	1.28	30.7	0.92	25.3	0.84
*DMSO Control*	22.0	1.00	23.7	1.00	–	–	97.7	1.00	–	–	11.0	1.00	4.3	1.00	6.0	1.00	–	–	39.0	1.00
Ultrapure Water Control	16.7	1.00	25.0	1.00	79.0	1.00	94.0	1.00	10.7	1.00	9.7	1.00	5.7	1.00	6.0	1.00	33.3	1.00	30.0	1.00
3200	1.0	0.06	7.3	0.29	10.0	0.13	4.0	0.04	1.7	0.16	5.3	0.55	0.0	0.00	0.0	0.00	9.7	0.29	13.3	0.44
1600	11.0	0.66	22.3	0.89	29.0	0.37	59.0	0.63	8.7	0.81	9.7	1.00	2.7	0.47	4.7	0.78	21.3	0.64	11.3	0.38
800	18.3	1.10	15.0	0.60	59.0	0.75	84.0	0.89	9.7	0.91	8.3	0.86	4.3	0.76	6.7	1.11	21.3	0.64	24.7	0.82
320	15.7	0.94	13.0	0.52	74.7	0.95	85.3	0.91	9.3	0.88	10.0	1.03	7.0	1.24	7.7	1.28	21.0	0.63	31.7	1.06
128	15.3	0.92	20.0	0.80	60.0	0.76	74.0	0.79	9.3	0.88	14.3	1.48	4.7	0.82	5.7	0.94	27.0	0.81	28.3	0.94
51.2	14.3	0.86	20.0	0.80	65.3	0.83	78.3	0.83	9.0	0.84	12.7	1.31	4.7	0.82	6.3	1.06	24.0	0.72	32.0	1.07
20.5	14.7	0.88	19.0	0.76	69.3	0.88	90.7	0.96	10.0	0.94	10.3	1.07	5.3	0.94	7.0	1.17	21.7	0.65	31.7	1.06
*NPD (4mg/plate)*	404.0	18.36	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
*SAZ (2mg/plate)*	–	–	–	–	567.3	7.18	–	–	965.3	90.50	–	–	–	–	–	–	–	–	–	–
*9AA (50mg/plate)*	–	–	–	–	–	–	–	–	–	–	–	–	1835.3	423.54	–	–	–	–	–	–
*MMS (2mL/plate)*	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	594.7	17.84	–	–
*2AA (2mg/plate)*	–	–	1517.3	64.11	–	–	781.3	8.00	–	–	116.7	10.61	–	–	85.3	14.22	–	–	–	–
*2AA (50mg/plate)*	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	207.0	5.31

MR: Mutation Rate; NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene
Remarks: Ultrapure water was applied as solvent of the test item and the positive control substance: SAZ and MMS; the DMSO was applied as solvent for positive control substances NPD, 9AA and 2AA. The mutation rate of the test item, SAZ, MMS and untreated control is given referring to the ultrapure water; the mutation rate of NPD, 9AA and 2AA is given referring to DMSO.

Table 3 Summary table of the results of the confirmatory mutation test

Confirmatory Mutation Test (Pre-Incubation Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains																*Escherichia coli*
	TA 98				TA 100				TA 1535				TA 1537				WP2uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	17.3	0.96	23.3	0.93	91.0	1.41	102.3	1.04	7.7	0.88	12.0	1.09	6.3	0.83	6.3	1.27	43.3	1.01	51.3	0.95
*DMSO Control*	18.0	1.00	22.3	1.00	–	–	93.3	1.00	–	–	12.7	1.00	7.3	1.00	6.0	1.00	–	–	51.3	1.00
Ultrapure Water Control	18.0	1.00	25.0	1.00	64.3	1.00	98.3	1.00	8.7	1.00	11.0	1.00	7.7	1.00	5.0	1.00	43.0	1.00	54.0	1.00
3200	0.0	0.00	0.0	0.00	0.0	0.00	0.0	0.00	0.0	0.00	0.0	0.00	0.0	0.00	0.0	0.00	3.3	0.08	14.3	0.27
1600	0.0	0.00	0.0	0.00	0.0	0.00	0.0	0.00	0.0	0.00	0.0	0.00	0.0	0.00	0.7	0.13	4.7	0.11	22.0	0.41
800	0.3	0.02	12.3	0.49	0.0	0.00	47.7	0.48	3.3	0.38	8.3	0.76	2.0	0.26	0.0	0.00	25.7	0.60	46.7	0.86
320	6.7	0.37	20.7	0.83	52.7	0.82	86.3	0.88	6.3	0.73	12.0	1.09	4.3	0.57	4.7	0.93	43.3	1.01	57.7	1.07
128	19.7	1.09	27.7	1.11	60.7	0.94	106.0	1.08	9.3	1.08	15.0	1.36	9.3	1.22	5.7	1.13	43.7	1.02	53.0	0.98
51.2	20.7	1.15	27.0	1.08	73.0	1.13	98.7	1.00	9.7	1.12	8.0	0.73	9.3	1.22	7.3	1.47	48.3	1.12	58.7	1.09
20.5	17.7	0.98	24.3	0.97	71.3	1.11	88.0	0.89	11.3	1.31	14.3	1.30	6.3	0.83	5.0	1.00	45.7	1.06	50.7	0.94
*NPD (4mg/plate)*	505.3	28.07	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
*SAZ (2mg/plate)*	–	–	–	–	744.0	11.56	–	–	1424.0	164.31	–	–	–	–	–	–	–	–	–	–
*9AA (50mg/plate)*	–	–	–	–	–	–	–	–	–	–	–	–	690.7	94.18	–	–	–	–	–	–
*MMS (2mL/plate)*	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	1456.0	33.86	–	–
*2AA (2mg/plate)*	–	–	1477.3	66.15	–	–	988.0	10.59	–	–	156.0	12.32	–	–	81.0	13.50	–	–	–	–
*2AA (50mg/plate)*	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	211.3	4.12

MR: Mutation Rate; NPD:4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene
Remarks: Ultrapure water was applied as solvent of the test item and the positive control substance: SAZ and MMS; the DMSO was applied as solvent for positive control substances NPD, 9AA and 2AA. The mutation rate of the test item, SAZ, MMS and untreated control is given referring to the ultrapure water; the mutation rate of NPD, 9AA and 2AA is given referring to DMSO.

Table 4 Historical Control Values for Revertants/Plate (for the Period of 2016-2018)

			Bacterial strains
Historical control data of untreated control	‑S9		TA98	TA100	TA1535	TA1537	*E. coli*
		Average	18.9	88.7	10.7	7.9	28.0
		SD	2.0	10.5	0.6	0.7	3.7
		Minimum	8	62	4	2	12
		Maximum	36	128	21	18	50
	+S9		TA98	TA100	TA1535	TA1537	*E. coli*
		Average	24.0	106.5	11.2	8.3	35.1
		SD	1.7	10.5	0.3	0.7	3.9
		Minimum	11	71	3	2	16
		Maximum	40	152	20	17	59
			Bacterial strains
Historical control data of DMSO control	‑S9		TA98	TA100	TA1535	TA1537	*E. coli*
		Average	17.5	80.6	10.7	7.5	25.9
		SD	1.2	10.6	0.8	0.7	2.6
		Minimum	9	58	4	2	12
		Maximum	35	124	21	18	52
	+S9		TA98	TA100	TA1535	TA1537	*E. coli*
		Average	22.3	95.6	10.9	8.1	34.5
		SD	1.1	10.3	0.6	0.7	4.2
		Minimum	10	65	3	2	16
		Maximum	37	146	24	19	58
			Bacterial strains
Historical control data of Water control	‑S9		TA98	TA100	TA1535	TA1537	*E. coli*
		Average	18.6	87.0	11.1	7.9	29.8
		SD	1.9	11.5	0.8	0.5	4.7
		Minimum	10	60	3	2	13
		Maximum	31	120	24	17	55
	+S9		TA98	TA100	TA1535	TA1537	*E. coli*
		Average	23.6	105.0	11.3	8.1	36.8
		SD	1.8	11.7	0.7	0.6	4.0
		Minimum	13	76	5	3	18
		Maximum	41	148	19	17	63
			Bacterial strains
Historical control data of positive controls	‑S9		TA98	TA100	TA1535	TA1537	*E. coli*
		Average	364.6	1095.3	987.7	564.4	911.6
		SD	111.2	197.3	119.3	73.8	112.8
		Minimum	182	543	409	137	361
		Maximum	844	2200	2123	2265	1995
	+S9		TA98	TA100	TA1535	TA1537	*E. coli*
		Average	1419.2	1580.8	168.2	148.2	203.3
		SD	249.7	337.3	22.0	16.3	5.3
		Minimum	281	688	93	71	132
		Maximum	3421	3333	349	367	364

Conclusions:

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats. The study included a preliminary solubility tests, a preliminary concentration range finding test (informatory toxicity test), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test). Based on the results of the solubility tests and the concentration range finding test the test item was dissolved in ultrapure water (ASTM Type I). At the preparation of the test item stock solution a correction (multiplier) factor of 1.953 (1/0.512=1.953) based on the purity of 51.2% was taken into consideration. Based on the cytotoxicity and solubility results of the preliminary concentration range finding test (informatory toxicity test) and based on the recommendations in OECD 471 guideline, the following concentrations of the test item were prepared and investigated in the initial and confirmatory mutation tests: 3200; 1600; 800; 320; 128; 51.2 and 20.5 μg/plate.

In the preliminary concentration range finding test strong inhibitory effect of the test item was observed at the recommended maximum test concentration of 5000 μg/plate.

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9) throughout the study. In the initial and confirmatory mutation tests unequivocal inhibitory effect of the test item on bacterial growth was observed. The cytotoxicity was indicated by affected background lawn development (absent, reduced or slightly reduced background lawn), affected colony development (pinpoint colonies) and decreased revertant colony counts (absent revertants or revertants below the historical control data ranges and/or corresponding vehicle control data ranges). In general, 320 μg/plate (noticed following the pre-incubation procedure in S. typhimurium strains) was considered as lowest concentration showing unequivocal cytotoxicity.

The revertant colony numbers of solvent control ultrapure water (ASTM Type I) plates with and without S9 mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-04-02 to 2019-04-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

14th February 2017

Deviations:

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

29th July 2016

Deviations:

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1998

Deviations:

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79 Chinese hamster lung male, Lot. No. 15H003
Supplier: ECACC (European Collection of Cells Cultures)
- Suitability of cells:
The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for gene toxicity assays with low background aberrations. These cells were chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates (doubling time 12-14 h). The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male).

This cell line was purchased from ECACC (European Collection of Cells Cultures). The cell stocks were kept in liquid nitrogen and were routinely checked for mycoplasma infections. Trypsin-EDTA (0.25 % Trypsin, 1mM EDTA x 4 Na) solution was used for cell detachment to subculture. The laboratory cultures were maintained in 75 cm2 plastic flasks at 37 +/- 0.5 °C in a humidified atmosphere in an incubator, set at 5% CO2. The V79 cells for this study were grown in DME (Dulbecco’s Modified Eagle’s) medium supplemented with L-glutamine (2mM) and 1 % of Antibiotic-antimycotic solution (containing 10000 units/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphoptericin-B) and heat-inactivated bovine serum (final concentration 10%). During the 3 and 20 hours treatments with test item, negative and positive controls, the serum content was reduced to 5%.

Cytokinesis block (if used):

Cell cultures were treated with colchicine (0.2 μg/mL) 2.5 hours prior to harvesting.

Metabolic activation:

with and without

Metabolic activation system:

Rat Liver S9 Fraction
The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver was provided by Trinova Biochem GmbH (Rathenau Strasse 2, D-35394 Giessen, Germany; manufacturer: MOLTOX INC., P.O. BOX 1189, BOONE, NC 28607 USA). Certificate of Analysis was obtained from the supplier. The Certificate of Analysis of rat liver S9 mix is stored in the laboratory.

Test concentrations with justification for top dose:

In order to determine the treatment concentrations of test item in the main cytogenetic study a pre-test on cytotoxicity was performed. Based on the results of this pretest, the following concentrations were chosen for the main test:
Experiment A:
Without S9 Mix, 3-hour treatment:
0 µg/mL, 12.5 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL
Positive Control (Ethyl methanesulphonate): 1.0 μL/mL

With S9 Mix (50 μL/mL), 3-hour treatment
0 µg/mL, 12.5 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL
Positive Control (Cyclophosphamide): 50 μL/mL

Experiment B:
20-hour treatment, harvest 20 hours from the beginning of treatment
Without S9 Mix, 20-hour treatment:
0 µg/mL, 1.6 μg/mL, 3.2 μg/mL, 6.3 μg/mL, 12.5 μg/mL
Positive Control (Ethylmethane sulphonate): 0.4 μL/mL

20 (without S9 mix)- and 3-hour (with S9 mix) treatment, harvest 28 hours from the beginning of treatment
Without S9 Mix, 20-hour treatment:
0 µg/mL, 1.6 μg/mL, 3.2 μg/mL, 6.3 μg/mL, 12.5 μg/mL
Positive Control (Ethylmethane sulphonate): 0.4 μL/mL

With S9 Mix (50 μL/mL), 3-hour treatment
0 µg/mL, 12.5 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL
Positive Control (Cyclophosphamide): 5 μg/mL

Vehicle / solvent:

- Vehicle/solvent used: DME (Dulbecco’s Modified Eagle’s) medium
- Justification for choice of solvent/vehicle:
A non GLP Preliminary Solubility Test was performed February 27, 2019. The test item was dissolved in DME (Dulbecco’s Modified Eagle’s) medium. A clear solution was obtained up to a concentration of 100 mg/mL.

Untreated negative controls:

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 5 x 10^5 cells per culture
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Experiment A: 3 hours, Experiment B: 20 h and 3 hours
- Harvest time after the end of treatment:
Experiment A: 20 hours
Experiment B: 20-hour treatment: harvest 20 hours from the beginning of treatment; 20 (without S9 mix)- and 3-hour (with S9 mix) treatment: harvest 28 hours from the beginning of treatment

- Spindle inhibitor: Cell cultures were treated with colchicine (0.2 μg/mL) 2.5 hours prior to harvesting.
- Methods of slide preparation and staining technique used including the stain used: Cells were swollen with 0.075 M KCl hypotonic solution, then washed in fixative (approx. 10 min. in 3:1 mixture of methanol: acetic-acid until the preparation becomes free of cytoplasm) and dropped onto slides and air-dried. The preparation was stained with 5 % Giemsa for subsequent scoring of chromosome aberration frequencies.
- Number of cells spread and analysed per concentration: 300 well-spread metaphase cells containing 22 ± 2 chromosomes were scored per test item concentration, negative and positive controls and were equally divided among the duplicates (150 metaphases/slide).
- Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately. Additionally, the number of polyploid and endoreduplicated cells were scored.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Based on cell counts the Relative Increase in Cell Counts (RICC) was calculated, which is an indicator of cytotoxicity.

Rationale for test conditions:

According to Guidelines

Evaluation criteria:

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

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

Both biological and statistical significance should be considered together.

Statistics:

For statistical analysis CHI2 test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control.
The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too. The lower and upper 95 % confidence intervals of historical control were calculated with C-chart.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In both experiments, clear cytotoxicity between 54- 60% was observed at the highest concentration.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
-No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item.

STUDY RESULTS
No relevant increases in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation.
In experiment A in the absence and in the presence of metabolic activation, one - one value (5 aberrant cells excluding gaps/150 cells) were slightly above the 95% control limits of the historical control data (upper limit approximately 4 aberrant cells excluding gaps/150 cells). However, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent as well as the historical control groups. In addition, no dose-response relationship was observed and therefore, the findings were not considered as being biologically relevant. No increase in the rate of polyploid and endoreduplicated metaphases was found after treatment with the different concentrations of the test item. The number of aberrations found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 μL/mL) and cyclophosphamide (5 μg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

TABLE 1: MEAN NUMBER OF CELLS WITH STRUCTURAL CHROMOSOME ABERRATIONS, EXPERIMENT A

Concentration (µg/mL)	S9 mix	Treatment time	Harvesting time	Mean aberrant cells/150cells
Concentration (µg/mL)	S9 mix	Treatment time	Harvesting time	incl. gaps	excl. gaps
Negative (Solvent) control	-	3 h	20 h	7	4
Test item
12.5 µg/mL	-	3 h	20 h	10	4
25 µg/mL	-	3 h	20 h	9	4
50 µg/mL	-	3 h	20 h	8	3
100 µg/mL	-	3 h	20 h	10	5
Pos. Control (Ethyl methanesulphonate)	-	3 h	20 h	39**	34**
Negative (Solvent) control	+	3 h	20 h	8	4
Test item
12.5 µg/mL	+	3 h	20 h	8	5
25 µg/mL	+	3 h	20 h	10	4
50 µg/mL	+	3 h	20 h	9	3
100 µg/mL	+	3 h	20 h	9	4
Pos. Control (Cyclophosphamide)	+	3 h	20 h	44**	41**

Positive control (-S9): Ethyl methanesulphonate (1.0L/mL)

Positive control (+S9): Cyclophosphamide (5.0g/mL)

** = p < 0.01 to the concurrent negative control and to the historical control

TABLE 2: MEAN NUMBER OF CELLS WITH STRUCTURAL CHROMOSOME ABERRATIONS EXPERIMENT B

Concentration
(µg/mL)

S9 mix

Treatment
time

Harvesting time

Mean aberrant cells/150cells

incl. gaps

excl. gaps

Negative (Solvent) control

20 h

Test item

1.6 µg/mL

20 h

3.2 µg/mL

20 h

6.3 µg/mL

20 h

12.5 µg/mL

20 h

Pos. Control
(Ethyl methanesulphonate)

20 h

42**

38**

Negative (Solvent) control

20 h

28 h

Test item

1.6 µg/mL

20 h

28 h

3.2 µg/mL

20 h

6.3 µg/mL

20 h

28 h

12.5 µg/mL

20 h

28 h

Pos. Control
(Ethyl methanesulphonate)

20 h

28 h

44**

37**

Concentration
(µg/mL)

S9 mix

Treatment
time

Harvesting time

Mean aberrant cells/150cells

incl. gaps

excl. gaps

Negative (Solvent) control

3 h

28 h

Test item

12.5 µg/mL

3 h

28 h

25 µg/mL

3 h

28 h

50 µg/mL

3 h

28 h

100 µg/mL

3 h

28 h

Pos. Control (Cyclophosphamide)

3 h

28 h

42**

39**

Positive control (-S9): Ethyl methanesulphonate (0.4L/mL) (-S9 mix)

Cyclophosphamide: 5.0g/mL (+ S9 mix)

** = p < 0.01 to the concurrent negative control and to the historical control

TABLE 3: NUMBER OF POLYPLOID CELLS AND ENDOREDUPLICATED CELLS, EXPERIMENT A

Concentration (µg/mL)	S9 mix	Treatment/Harvesting time	Polyploid Cells (mean)	Endoredup-lication (mean)
Negative (Solvent) control	-	3/20 h	0.0	0.0
Test item
12.5 µg/mL	-	3/20 h	0.0	0.0
25 µg/mL	-	3/20 h	0.0	0.0
50 µg/mL	-	3/20 h	0.0	0.0
100 µg/mL	-	3/20 h	0.0	0.0
Pos. Control (Ethyl methanesulphonate)	-	3/20 h	0.0	0.0
Negative (Solvent) control	+	3/20 h	0.0	0.0
Test item
12.5 µg/mL	+	3/20 h	0.0	0.0
25 µg/mL	+	3/20 h	0.0	0.0
50 µg/mL	+	3/20 h	0.0	0.0
100 µg/mL	+	3/20 h	0.0	0.0
Pos. Control (Cyclophosphamide)	+	3/20 h	0.0	0.0

Ethyl methanesulphonate: 1.0mL/mL

Cyclophosphamide: 5.0g/mL

TABLE 4: NUMBER OF POLYPLOID CELLS AND ENDOREDUPLICATED CELLS, EXPERIMENT B

Concentration (µg/mL)			S9 mix	Treatment/Harvesting time	Polyploid Cells (mean)	Endoredup-lication (mean)
Negative (Solvent) control			-	20/20 h	0.0	0.0
Test item
1.6 µg/mL			-	20/20 h	0.0	0.0
3.2 µg/mL			-	20/20 h	0.0	0.0
6.3 µg/mL			-	20/20 h	0.0	0.0
12.5 µg/mL			-	20/20 h	0.0	0.0
Pos. Control			-	20/20 h	0.0	0.0
Negative (Solvent) control			-	20/28 h	0.0	0.0
Test item
1.6 µg/mL			-	20/28 h	0.0	0.0
3.2 µg/mL			-	20/28 h	0.0	0.0
6.3 µg/mL			-	20/28 h	0.0	0.0
12.5 µg/mL			-	20/28 h	0.0	0.0
Pos. Control			-	20/28 h	0.0	0.0
	Concentration (µg/mL)	S9 mix		Treatment/Harvesting time	Polyploid Cells (mean)	Endoredup-lication (mean)
	Negative (Solvent) control	+		3/28 h	0.0	0.0
	Test item
	12.5 µg/mL	+		3/28 h	0.0	0.0
	25 µg/mL	+		3/28 h	0.0	0.0
	50 µg/mL	+		3/28 h	0.0	0.0
	100 µg/mL	+		3/28 h	0.0	0.0
	Pos. Control	+		3/28 h	0.0	0.0

Positive control (-S9): Ethyl methanesulphonate (0.4L/mL) (-S9 mix)

Cyclophosphamide: 5.0g/mL (+ S9 mix)

Conclusions:

The test item did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation.

Executive summary:

The test item dissolved in DME (Dulbecco’s Modified Eagle’s) medium, was tested in a chromosome aberration assay in V79 cells in two independent experiments. For the cytogenetic experiments five concentrations were selected on the basis of a pre-test on (without and with metabolic activation using rodent S9 mix) in accordance with the current OECD Guideline 473.

Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 μg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture). Clear cytotoxicity of about 50% was observed after test item treatment in all experimental parts for the highest test concentrations. No relevant increases in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 μL/mL) and cyclophosphamide (5 μg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

Reference 3

Endpoint:: in vitro gene mutation study in mammalian cells
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 2019-04-09 to 2019-04-25
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Qualifier:: according to guideline
Guideline:: OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:: 29 July 2016
Deviations:: no
Qualifier:: according to guideline
Guideline:: EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:: May 2008
Deviations:: yes
Remarks:: There is a deviation from the guidelines regarding the confirmation of negative results. Negative results were not confirmed as the confirmation of negative results is not required by the most current Guideline (OECD 476, 29 July 2016).
GLP compliance:: yes (incl. QA statement)
Type of assay:: in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:: The objective of this study was to determine whether the test item or its metabolites can induce forward mutation at the hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) in cultured Chinese hamster cells.
Species / strain / cell type:: Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):: CELLS USED
CHO K1: Sub-line (K1) of Chinese hamster ovary cell line CHO
Lot. No.: 16H036
Supplier: ECACC (European Collection of Cell Cultures)

The CHO cell line was originally derived from the ovary of a female Chinese hamster (Kao and Puck, 1967). The CHO K1 is a sub-line of CHO cell line and was purchased from ECACC (European Collection of Cell Cultures).
Metabolic activation:: with and without
Metabolic activation system:: The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver was provided by Trinova Biochem GmbH (Rathenau Strasse 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).
Test concentrations with justification for top dose:: Treatment concentrations for the mutation assay were selected on the basis of the result of a pre-test on toxicity.
Without S9-mix:
10, 20, 30, 40, 50, 60, 70 µg/mL

With S9-mix:
5, 10, 20, 30, 40 µg/mL
Vehicle / solvent:: - Solvent used: Ham's F12 medium

- Justification for choice of solvent/vehicle: This solvent was chosen based on the results of the preliminary solubility test and its suitability is confirmed with the available laboratory’s historical database.
Untreated negative controls:: no
Negative solvent / vehicle controls:: yes
Remarks:: Ham's F12 medium
True negative controls:: no
Positive controls:: yes
Positive control substance:: 7,12-dimethylbenzanthracene; ethylmethanesulphonate
Details on test system and experimental conditions:: NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 5*10^6 cells
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 5 hours
- Harvest time after the end of treatment: Following the exposure period the cells were washed with F12-5 medium and incubated in fresh F12-10 medium for 19 hours. After the 19-hour incubation period, cells were washed twice with F12-10 medium and suspended by treatment with trypsin-EDTA solution and counted using a Bürker chamber.

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 19 h
- At the end of the expression period, cultures from each dose level were adjusted to 2 x 10^5 cells / dish ( 4 x five dishes) in selection medium (hypoxanthine Ham's F12-SEL medium) containing 3.4 µg/mL of thioguanine (6-TG).
- The mutation frequency was calculated by dividing the total number of mutant colonies by the number of cells selected (10^6 cells: 5 plates at 2 x 10^5 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection (viability), and was expressed as 6-TG resistant mutants per 10^6 clonable cells.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- In order to determine cytotoxicity, survivals were assessed by comparing the colony forming ability of the treated groups to the negative (solvent) control.
Rationale for test conditions:: according to Guidelines
Evaluation criteria:: Acceptance Criteria
The assay was considered valid as all the following criteria were met:
-The mutant frequency of concurrent negative controls is within the 95% control limits of the distribution of the laboratory’s historical negative control database.
-The positive control chemicals induced a statistically significant and biologically relevant increase in mutant frequency compared to the concurrent negative control. The increases are compatible with the laboratory historical positive control data base.
-Adequate number of cells and concentrations were analysable.
-Two experimental conditions with and without metabolic activation were tested.
-The highest concentration is adequate.
-The cloning efficiency of the negative controls is between the range of 60 % to 140 % on Day 1 and 70 % to 130 % on Day 8.

Evaluation of Results
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
-at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
-any of the results are outside the distribution of the laboratory historical negative control data (based 95% control limit),
-the increase of mutant frequency is concentration-related when evaluated with an appropriate trend test.
Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if, in all experimental conditions examined:
-none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
-there is no concentration-related increase when evaluated with an appropriate trend test,
-all results are inside the distribution of the historical negative control data (based 95% control limit).
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
: Key result
Species / strain:: Chinese hamster Ovary (CHO)
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Remarks:: In the absence and presence of metabolic activation clear cytotoxicity of the test item was observed at the highest concentration applied (60 µg/mL in the absence and 40 µg/mL in the presence of S9 mix).
Vehicle controls validity:: valid
Untreated negative controls validity:: not applicable
True negative controls validity:: not applicable
Positive controls validity:: valid
Additional information on results:: The test item was dissolved in Ham's F12 medium. A clear solution of the test item was obtained up to a concentration of 50 mg/mL. For the examined test item concentrations, no precipitation in the medium was noted.

RANGE-FINDING/SCREENING STUDIES
The concentrations for the main mutation assay were selected on the basis of preliminary cytotoxicity investigations (without and with metabolic activation using S9-mix). Toxicity was determined by comparing the colony forming ability of the treated groups to the negative (solvent) control. Results were noted as percentage of cells in relation to the negative control.
Conclusions:: The test item was not mutagenic in the in vitro mammalian cell gene mutation test performed with Chinese hamster ovary cells.
Executive summary:: The test item, dissolved in Ham's F12 medium, was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The following concentrations were selected on the basis of a pre-test on cytotoxicity without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver: Mutation Assay 5-hour treatment period without S9-mix: 10, 20, 30, 40, 50, 60 and 70 µg/mL Mutation Assay 5-hour treatment period with S9-mix: 5, 10, 20, 30 and 40 µg/mL. This concentration was tested but was very toxic and there were not enough cells to start the phenotypic expression period after the treatment.In the performed mutation assay the concentration levels were chosen mainly based on the cytotoxicity.In the absence and presence of metabolic activation clear cytotoxicity (survival between 15-17 %) of the test item was observed at the highest concentration applied (60 µg/mL in the absence and 40 µg/mL in the presence of S9 mix). Phenotypic expression was evaluated up to 8 days following exposure. There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested. In both experimental parts, there were no increases in mutation frequency when compared to the concurrent solvent control and the laboratory historical control data at any concentration tested in the absence and presence of metabolic activation. All results were inside the distribution of the historical negative control data (based 95% control limit). The mutation frequency found in the solvent controls was in the 95 % confidence interval of the historical control data. The concurrent positive controlsethyl methanesulfonate (1.0 µL/mL) and 7, 12-dimethyl benzanthracene (20 µg/mL) caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.The test itemtested up to cytotoxicconcentrationswith and without metabolic activationover a 5 hour treatment period did not induce statistically significant and biologically relevant increases in mutant frequency. It is concluded that the test item was not mutagenic in this in vitro mammalian cell gene mutation test performed with Chinese hamster ovary cells.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:: no study available

Additional information

Chromosme Aberrration Assay:

Experiment A with 3/20 h treatment/sampling time

without S9 mix: 0,12.5, 25, 50 and 100 μg/mL test item

with S9 mix: 0,12.5, 25, 50 and 100 μg/mL test item

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 0,1.6, 3.2, 6.3 and 12.5 μg/mL test item

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 0,1.6, 3.2, 6.3 and 12.5 μg/mL test item

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 0,12.5, 25, 50 and 100 μg/mL test item

HPRT Assay:

The test item, dissolved in Ham's F12 medium, was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The following concentrations were selected on the basis of a pre-test on cytotoxicity without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver:

Mutation Assay 5-hour treatment period without S9-mix: 10, 20, 30, 40, 50, 60 and 70 µg/mL

Mutation Assay 5 -hour treatment period with S9-mix: 5, 10, 20, 30 and 40 µg/mL.

This concentration was tested but was very toxic and there were not enough cells to start the phenotypic expression period after the treatment. In the performed mutation assay the concentration levels were chosen mainly based on the cytotoxicity. In the absence and presence of metabolic activation clear cytotoxicity (survival between 15-17 %) of the test item was observed at the highest concentration applied (60 µg/mL in the absence and 40 µg/mL in the presence of S9 mix). Phenotypic expression was evaluated up to 8 days following exposure. There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested. In both experimental parts, there were no increases in mutation frequency when compared to the concurrent solvent control and the laboratory historical control data at any concentration tested in the absence and presence of metabolic activation. All results were inside the distribution of the historical negative control data (based 95% control limit). The mutation frequency found in the solvent controls was in the 95 % confidence interval of the historical control data. The concurrent positive controlsethyl methanesulfonate(1.0 µL/mL) and 7, 12 -dimethyl benzanthracene (20 µg/mL) caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.The test itemtested up to cytotoxicconcentrationswith and without metabolic activationover a 5 hour treatment period did not induce statistically significant and biologically relevant increases in mutant frequency. It is concluded that the test item was not mutagenic in this in vitro mammalian cell gene mutation test performed with Chinese hamster ovary cells.

Ames Test:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.

In the preliminary concentration range finding test strong inhibitory effect of the test item was observed at the recommended maximum test concentration of 5000 μg/plate.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genotoxicity, the test item does not require classification as mutagenic according to Regulation (EC) No 1272/2008 (CLP).

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Test group	Dose µg/ml	S9-mix	Treatment/ time/ hour	Number of colonies/200cells/dish			Mean	Relative^a survival in percent
Test group	Dose µg/ml	S9-mix	Treatment/ time/ hour	dish 1	dish 2	dish 3	Mean	Relative^a survival in percent
Solvent Control (Ham's F12 medium)		–	5	200	195	194	196.3	100
Test item	12.5	–	5	185	186	187	186.0	95
	25	–	5	170	170	171	170.3	87
	50	–	5	93	88	92	91.0	46
	100	–	5	0	0	0	0.0	0
	150	–	5	0	0	0	0.0	0
	200	–	5	0	0	0	0.0	0
Solvent Control (Ham's F12 medium)	–	+	5	195	194	196	195.0	100
Test item	12.5	+	5	147	142	142	143.7	74
	25	+	5	134	130	129	131.0	67
	50	+	5	6	7	10	7.7	4
	100	+	5	0	0	0	0.0	0
	150	+	5	0	0	0	0.0	0
	200	+	5	0	0	0	0.0	0

Test Group a and b	Concentration µg/mL			S9-mix			Treatment time/ hour			Number of colonies/200cells/dish											Cloning efficiency (%)		% of Control
										dish 1				dish 2			dish 3		Mean
Negative (Solvent Control)	-			–			5			199				202			200		200.3		100		100
Test item- Group a	10			–			5			198				197			199		198.0		99		99
	20			–			5			189				188			190		189.0		95		94
	30			–			5			167				162			163		164.0		82		82
	40			–			5			118				116			116		116.7		58		58
	50			–			5			95				98			97		96.7		48		48
	60			–			5			30				29			33		30.7		15		15
	EMS (1µL/mL)			–			5			56				53			55		54.7		27		27
Negative (Solvent Control)	-			–			5			200				200			200		200.0		100		100
Test item - Group b	10			–			5			199				196			200		198.3		99		99
	20			–			5			188				187			189		188.0		94		94
	30			–			5			166				163			163		164.0		82		82
	40			–			5			117				118			115		116.7		58		58
	50			–			5			96				97			98		97.0		49		49
	60			–			5			29				30			31		30.0		15		15
	EMS (1µL/mL)			–			5			55				54			52		53.7		27		27
Test Group c and d	Concentration µg/mL			S9-mix			Treatment time/ hour			Number of colonies/200cells/dish											Cloning efficiency (%)		% of Control
										dish 1				dish 2			dish 3		Mean
Negative (Solvent Control)	-			–			5			203				201			200		201.3		101		100
Test item- Group c	10			–			5			199				200			201		200.0		100		99
	20			–			5			192				193			190		191.7		96		95
	30			–			5			159				160			162		160.3		80		80
	40			–			5			120				116			118		118.0		59		59
	50			–			5			97				94			93		94.7		47		47
	60			–			5			36				34			32		34.0		17		17
	EMS (1µL/mL)			–			5			53				52			51		52.0		26		26
Negative (Solvent Control)	-			–			5			202				200			200		200.7		100		100
Test item - Group d	10			–			5			198				199			200		199.0		100		99
	20			–			5			190				191			191		190.7		95		95
	30			–			5			158				159			161		159.3		80		79
	40			–			5			119				117			117		117.7		59		59
	50			–			5			96				95			94		95.0		48		47
	60			–			5			31				33			34		32.7		16		16
	EMS (1µL/mL)			–			5			52				55			53		53.3		27		27
Test group a and b		Concentration µg/mL			S9-mix			Treatment time/ hour			Number of colonies/200cells/dish										Cloning efficiency (%)		% of Control
											dish 1		dish 2			dish 3			Mean
Negative (Solvent Control)		-			+			5			200		197			200			199.0		100		100
Test item- Group a		5			+			5			192		195			190			192.3		96		97
		10			+			5			159		159			157			158.3		79		80
		20			+			5			143		148			144			145.0		73		73
		30			+			5			106		105			105			105.3		53		53
		40			+			5			32		35			33			33.3		17		17
		DMBA (20µg/mL)			+			5			121		118			118			119.0		60		60
Negative (Solvent Control)		-			+			5			198		200			199			199.0		100		100
Test item-Groupb		5			+			5			193		195			194			194.0		97		97
		10			+			5			160		158			158			158.7		79		80
		20			+			5			145		147			145			145.7		73		73
		30			+			5			107		104			103			104.7		52		53
		40			+			5			30		32			33			31.7		16		16
		DMBA (20µg/mL)			+			5			120		119			117			118.7		59		60
Test group c and d			Concentration µg/mL			S9-mix			Treatment time/ hour			Number of colonies/200cells/dish										Cloning efficiency (%)		% of Control
												dish 1			dish 2			dish 3		Mean
Negative (Solvent Control)			-			+			5			200			198			196		198.0		99		100
Test item- Group c			5			+			5			190			194			192		192.0		96		97
			10			+			5			163			162			161		162.0		81		82
			20			+			5			142			141			138		140.3		70		71
			30			+			5			102			101			99		100.7		50		51
			40			+			5			36			33			31		33.3		17		17
			DMBA (20µg/mL)			+			5			114			113			110		112.3		56		57
Negative (Solvent Control)			-			+			5			199			197			198		198.0		99		100
Test item-Groupd			5			+			5			191			193			193		192.3		96		97
			10			+			5			161			160			164		161.7		81		82
			20			+			5			141			142			139		140.7		70		71
			30			+			5			101			101			102		101.3		51		51
			40			+			5			35			32			30		32.3		16		16
			DMBA (20µg/mL)			+			5			116			115			115		115.3		58		58
Test group c and d			Concentration µg/mL			S9-mix			Treatment time/ hour			Number of colonies/200cells/dish										Cloning efficiency (%)		% of Control
												dish 1			dish 2			dish 3		Mean
Negative (Solvent Control)			-			+			5			200			198			196		198.0		99		100
Test item- Group c			5			+			5			190			194			192		192.0		96		97
			10			+			5			163			162			161		162.0		81		82
			20			+			5			142			141			138		140.3		70		71
			30			+			5			102			101			99		100.7		50		51
			40			+			5			36			33			31		33.3		17		17
			DMBA (20µg/mL)			+			5			114			113			110		112.3		56		57
Negative (Solvent Control)			-			+			5			199			197			198		198.0		99		100
Test item-Groupd			5			+			5			191			193			193		192.3		96		97
			10			+			5			161			160			164		161.7		81		82
			20			+			5			141			142			139		140.7		70		71
			30			+			5			101			101			102		101.3		51		51
			40			+			5			35			32			30		32.3		16		16
			DMBA (20µg/mL)			+			5			116			115			115		115.3		58		58

Registration Dossier

Registration Dossier

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Diss Factsheets

Phosphoric acid, 2-ethylhexyl ester, sodium salt

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Link to relevant study records

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Reference 1

Reference 2

Reference 3

Endpoint conclusion

Genetic toxicity in vivo

Endpoint conclusion

Additional information

Justification for classification or non-classification

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