Isotridecanol, ethoxylated

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay (Ames test, OECD 471): negative with and without metabolic activation

In vitro mammalian cell micronucleus test (cultured peripheral human lymphocytes, OECD 487): negative with and without metabolic activation

In vitro mammalian cell gene mutation test using the Hprt gene (Chinese hamster lung fibroblasts (V79), OECD 476): negative with and without metabolic activation

Conclusions based on data obtained with isotridecanol, ethoxylated, < 2.5 EO (CAS No. 69011-36-5, EC No. 500-241-6).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 May - 10 Oct 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted in 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
For lymphocytes:
- Sex, age and number of blood donors: Female, 35 and 33 years, two (Main Assay I); female, 27 and 28 years, two (Main Assay II); male, 30 years, one (Main Assay III)
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: pooled (Main Assays I and II), not pooled (Main Assay III)
- Mitogen used for lymphocytes: phytohaemagglutinin (PHA)

MEDIA USED
- Type and composition of media:
Culture medium:
RPMI 1640 1x (Dutch modification): 500 mL
Foetal Calf Serum (FCS): 100 mL
L-Glutamine (200 mM): 6.25 mL
Antibiotic solution (not further specified): 1.25 mL

Treatment medium (3 h exposure):
+S9:
Test item solution or solvent/vehicle: 0.05 mL
S9 mix: 1.00 mL
Culture medium (without PHA): 3.95 mL
-S9:
Test item solution or solvent/vehicle: 0.05 mL
Culture medium (without PHA): 4.95 mL

Treatment medium (continous exposure):
Test item as supplied, test item solution or solvent/vehicle: 0.025 mL
Culture medium (without PHA): 4.975 mL

Cytokinesis block (if used):

Cytochalasin B (6 μg/mL)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone.
- Species: rat
- Strain: Sprague Dawley
- Sex: male
- Age: 5 - 6 weeks
- Weight: 175 - 199 g
- Tissue: liver
- Inducing Agents: phenobarbital and 5,6-benzoflavone
- Producer: TRINOVA BIOCHEM GmbH, Giessen, Germany
- Batch Number: 3971
- Quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): yes

Test concentrations with justification for top dose:

Main Assay I:
3 h exposure (with and without metabolic activation): 0.195, 0.293, 0.439, 0.658, 0.988, 1.48, 2.22, 3.33, 5.00 μL/mL
31 h exposure (without metabolic activation): 0.130, 0.195, 0.293, 0.439, 0.658, 0.988, 1.48, 2.22, 3.33, 5.00 μL/mL
Haemolysis was observed at all doses in all experiments.

Main Assay II:
3 h exposure (with and without metabolic activation): 0.00780, 0.0117, 0.0176, 0.0263, 0.0395, 0.0593, 0.0889, 0.133, 0.200 µL/mL
31 h exposure (without metabolic activation): 0.000520, 0.000780, 0.00117, 0.00176, 0.00263, 0.00395, 0.00593, 0.00889, 0.0133, 0.0200 µL/mL
Cytotoxicity was observed in the 3 h experiments in the presence of metabolic activation.

Main Assay III:
3 h exposure (with metabolic activation): 0.0654, 0.0752, 0.0865, 0.0994, 0.114, 0.132, 0.151, 0.174, 0.200 µL/mL

Vehicle / solvent:

- Vehicle/solvent used: dimethylsulfoxide (DMSO)

- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle. This vehicle was selected since it is compatible with the survival of cells and the S9 metabolic activation system.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Test material added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 48 h
- Exposure duration/duration of treatment: 3 h (3 h exposure experiments) and 31 h (continous exposure experiments)
- Harvest time after the end of treatment (sampling/recovery times): 32 h in 3 h exposure experiments and 31 h in continous exposure experiments

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: cytB 6 µL/mL medium, added 3 h after start of treatment
- Methods of slide preparation and staining technique used including the stain used: The lymphocyte cultures were centrifuged and the supernatant was removed. The cells were resuspended in hypotonic solution and fresh methanol/acetic acid fixative was added. The fixative was changed several times by centrifugation and resuspension. A few drops of the cell suspension were transfered to glass slides which were allowed to air dry before staining with acridine Orange in phosphate buffered saline (PBS).
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 2000 binucleated cells per cell culture were scored
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
1. The micronucleus diameter was less than 1/3 of the nucleus diameter
2. The micronucleus diameter was greater than 1/16 of the nucleus diameter
3. No overlapping with the nucleus was observed
4. The aspect was the same as the chromatin

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index (CBPI), 1000 cells counted per concentration

Evaluation criteria:

EVALUATION CRITERIA
A test item is considered clearly positive if the following criteria are met:
1. Significant increases in the proportion of micronucleated cells over the concurrent controls occur at one or more concentrations.
2. The proportion of micronucleated cells at such data points exceeds the normal range based on historical control values (95% control limits).
3. There is a significant dose-effect relationship.

A item is considered clearly negative if the following criteria are met:
1. None of the dose levels shows a statistically significant increase in the incidence of micronucleated cells.
2. There is no concentration-related increase when evaluated with the Cochran-Armitage trend test.
3. All the results are inside the distribution of the historical control data (95% control limits).

ACCEPTANCE CRITERIA
The assay is considered valid if the following criteria are met:
1. The incidence of micronucleated cells of the negative control is within the distribution range of our historical control values.
2. Concurrent positive controls induce responses that are compatible with those generated in our historical positive control database and produce a statistically significant increase compared with the concurrent negative control.
3. Adequate cell proliferation is observed in solvent control cultures.
4. The appropriate number of doses and cells is analysed.

Statistics:

A modified χ2 test was used to compare the number of cells with micronuclei in control and treated cultures.
Cochran-Armitage Trend Test (one-sided) was performed for the assessment of the concentration-response relationship.

Key result

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Strong cytotoxicity was observed at and above 0.130 μL/mL in the absence and presence of metabolic activation.

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
- Data on pH: Treatment with the test item did not change the pH at any dose level in any experiment.
- Data on osmolality: Treatment with the test item did not increases the osmolality at any dose level in any experiment.
- Precipitation and time of the determination: Following treatment with the test item, no opacity or precipitation of the medium was observed at the beginning or by the end of treatment in any experiment.

STUDY RESULTS
- Concurrent vehicle negative and positive control data are reported in tabular form under 'Any other information on results incl. tables'.

HISTORICAL CONTROL DATA
- The incidence of micronucleated cells of the negative controls was within the distribution range of our historical control values, data are presented in tabular form under 'Any other information on results incl. tables'.

Table 1: Proliferation index

Main Assay I
Treatment (µL/mL)	Culture No.	MonoN	BiN	PoliN	Total cells	CBPI	Mean Value	Cytotoxicity (%)
Treatment: 3 h, - S9
Test Item*	19	Only mononucleated cells
0.195	20
Treatment: 3 h, +S9
Solvent*	21	124	219	157	500	2.066	2.052	0
1%	22	123	235	142	500	2.038
Test Item*	39	378	122	0	500	1.244	1.235	78
0.195	40	388	111	1	500	1.226
Test Item*	37	Only mononucleated cells
0.293	38
Test Item*	35	Only mononucleated cells
0.439	36
Treatment: 31 h, - S9
Test Item*	65	Only mononucleated cells
0.130	66
Main Assay II
Treatment (µL/mL)	Culture No.	MonoN	BiN	PoliN	Total cells	CBPI	Mean Value	Cytotoxicity (%)
Treatment: 3 h, -S9
Solvent*	71	122	275	103	500	1.962	1.953	0
1%	72	138	252	110	500	1.944
Test Item*	85	121	256	123	500	2.004	1.983	-3
0.0176	86	143	233	124	500	1.962
Test Item*	83	123	274	103	500	1.960	1.925	3
0.0263	84	155	245	100	500	1.890
Test Item*	81	154	248	98	500	1.888	1.897	6
0.0395	82	139	269	92	500	1.906
Test Item*	79	120	294	86	500	1.932	1.878	8
0.0593	80	154	280	66	500	1.824
Test Item*	77	305	177	18	500	1.426	1.445	53
0.0889	78	289	190	21	500	1.464
Test Item*	75	Very few mononucleated cells recovered onto slides
0.133	76
Test Item*	73	No cells recovered onto slides
0.200	74
Treatment: 3 h, +S9
Solvent*	91	144	217	139	500	1.990	1.802	0
1%	92	243	207	50	500	1.614
Test Item*	103	165	211	124	500	1.918	1.911	-14
0.0263	104	166	216	118	500	1.904
Test Item*	101	162	249	89	500	1.854	1.857	-7
0.0395	102	169	232	99	500	1.860
Test Item*	99	154	242	104	500	1.900	1.832	-4
0.0593	100	204	210	86	500	1.764
Test Item*	97	179	237	84	500	1.810	1.861	-7
0.0889	98	134	276	90	500	1.912
Test Item*	95	276	209	15	500	1.478	1.469	42
0.133	96	290	190	20	500	1.460
Test Item*	93	Very few mononucleated cells recovered onto slides
0.200	94
Cyclophosphamide	111	310	182	8	500	1.396	1.394	51
20.0 µg/mL	112	311	182	7	500	1.392
Cyclophosphamide	113	316	176	8	500	1.384	1.392	51
15.0 µg/mL	114	306	188	6	500	1.400
Treatment: 31 h, - S9
Solvent*	115	118	273	109	500	1.982	1.951	0
0.5%	116	140	260	100	500	1.920
Test Item*	129	172	266	62	500	1.780	1.796	16
0.00176	130	164	266	70	500	1.812
Test Item*	127	147	293	60	500	1.826	1.782	18
0.00263	128	188	255	57	500	1.738
Test Item*	125	168	298	34	500	1.732	1.733	23
0.00395	126	170	293	37	500	1.734
Test Item*	123	187	289	24	500	1.674	1.656	31
0.00593	124	206	269	25	500	1.638
Test Item*	121	228	265	7	500	1.558	1.535	44
0.00889	122	251	242	7	500	1.512
Test Item*	119	316	183	1	500	1.370	1.393	59
0.0133	120	293	206	1	500	1.416
Test Item*	117	391	109	0	500	1.218	1.225	76
0.0200	118	384	116	0	500	1.232
Colchicine	137	495	5	0	500	1.010	1.007	99
0.0800 µg/mL	138	498	2	0	500	1.004
Colchicine	139	487	13	0	500	1.026	1.028	97
0.0400 µg/mL	140	485	15	0	500	1.030
Main Assay III
Treatment (µL/mL)	Culture No.	MonoN	BiN	PoliN	Total cells	CBPI	Mean Value	Cytotoxicity (%)
Treatment: 3 h, -S9
Solvent*	141	297	164	39	500	1.484	1.482	0
1%	142	308	144	48	500	1.480
Test Item*	159	344	110	46	500	1.404	1.484	0
0.0654	160	290	138	72	500	1.564
Test Item*	157	324	128	48	500	1.448	1.436	10
0.0752	158	325	138	37	500	1.424
Test Item*	155	319	140	41	500	1.444	1.441	9
0.0865	156	321	139	40	500	1.438
Test Item*	153	333	138	29	500	1.392	1.389	19
0.0994	154	335	137	28	500	1.386
Test Item*	151	380	103	17	500	1.274	1.266	45
0.114	152	379	113	8	500	1.258
Test Item*	149	805	190	5	1000	1.200	1.213	56
0.132	150	783	208	9	1000	1.226
Test Item*	147	428	72	0	500	1.144	1.134	72
0.151	148	438	62	0	500	1.124
Test item*	145	Few cells recovered onto slides
0.174	146
Test Item*	143	396	82	22	500	1.252
0.200	144	Few cells recovered onto slides
Cyclophosphamide	161	356	142	2	500	1.292	1.280	42
20.0 µg/mL	162	366	134	0	500	1.268
Cyclophosphamide	163	343	150	7	500	1.328	1.310	36
15.0 µg/mL	164	358	138	4	500	1.292
*: 2 replicates
MonoN:	Mononucleated cells
BiN:	Binucleated cells
PoliN:	Polinucleated cells
CBPI:	Cytokinesis block proliferation index
% Cytotoxicity:	Relative to vehicle/solvent control cultures
Mn:	Micronucleus/micronuclei

Table 2: Scoring of micronuclei

Main Assay II
Dose level (μL/mL)	Culture No.	Cells scored (BiN cells)	BiN cells with 1 Mn	BiN cells with 2 Mn	BiN cells with >2 Mn	BiN cells with Mn
Treatment: 3 h, -S9
Solvent 1%	71	1000	1	0	0	1
	72	1000	3	0	0	3
0.0395	81	1000	3	0	0	3
	82	1000	2	0	0	2
0.0593	79	1000	3	0	0	3
	80	1000	2	0	0	2
0.0889	77	1000	4	0	0	4
	78	1000	1	0	0	1
Cyclophosphamide 15.0 µg/mL	113	1000	20	0	0	20
	114	1000	45	1	0	46
Treatment: 31 h, -S9
Solvent 0.5%	115	1000	7	0	0	7
	116	1000	2	0	0	2
0.00263	127	1000	3	0	0	3
	128	1000	4	0	0	4
0.00593	123	1000	7	0	0	7
	124	1000	1	0	0	1
0.0133	119	1000	3	0	0	3
	120	1000	4	0	0	4
Colchicine 0.0400 µg/mL	139	1000	21	5	0	26
	140	1000	16	6	0	22
Main Assay III
Dose level (μL/mL)	Culture No.	Cells scored (BiN cells)	BiN cells with 1 Mn	BiN cells with 2 Mn	BiN cells with >2 Mn	BiN cells with Mn
Treatment: 3 h, +S9
Solvent 1%	141	1000	9	1	0	10
	142	1000	7	3	0	10
0.0752	157	1000	9	0	0	9
	158	1000	9	0	0	9
0.0994	153	1000	5	0	0	5
	154	1000	7	0	0	7
0.132	149	1000	8	2	0	10
	150	1000	7	0	0	7
Cyclophosphamide 20.0 µg/mL	161	1000	28	4	0	32
	162	1000	35	4	0	39
MonoN:	Mononucleated cells
BiN:	Binucleated cells
PoliN:	Polinucleated cells
CBPI:	Cytokinesis block proliferation index
% Cytotoxicity:	Relative to vehicle/solvent control cultures
Mn:	Micronucleus/micronuclei

Table 3: Historical control data

UNTREATED AND SOLVENT CONTROLS
	-S9		+S9
	Short treatment time (3 h)	Long treatment time (31-32 h)	Short treatment time (3 hours)
Mean	0.33	0.39	0.36
SD	0.21	0.30	0.24
n	64	61	64
UCL	0.75	0.96	0.85
LCL	0.00	0.00	0.00

POSITIVE CONTROL
	+S9	-S9
	Short treatment time (3 h)	Long treatment time (31-32 h)
	Cyclophosphamide	Colchicine
Mean	2.97	3.10
SD	1.40	1.30
n	51	46
Minimum	0.95	1.10
Maximum	7.65	8.50

SD:	Standard deviation
UCL:	Upper Confidence Limit (Mean value + 2SD)
LCL:	Lower Confidence Limit (Mean value - 2SD)

Conclusions:

Interpretation of results: negative

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 June - 11 Oct 2019

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 using the Hprt and xprt genes)

Version / remarks:

adopted in 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr. J. Thacker, MRC Radiobiology Unit, Harwell, UK.
- Suitability of cells: Cell type selected is listed as one of the recommended cell types in OECD guideline 476.

For cell lines:
- Absence of Mycoplasma contamination: checked periodically
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature:

Eagle’s Minimal Essential Medium (EMEM) Minimal medium:
EMEM (10X): 58.7 mL
L-glutamine (200 mM): 5.9 mL
Sodium bicarbonate (7.5%): 15.7 mL
Non-essential amino acids (100X): 5.9 mL
Streptomycin sulphate (50000 IU/mL) + Penicillin G (50000 IU/mL): 1.2 mL
Sterile distilled water: 500 mL

EMEM Complete medium:
EMEM Minimal medium: 900 mL
Foetal Calf Serum (FCS): 100 mL

- Incubation conditions: 37 °C in a 5% CO2 atmosphere (100% nominal relative humidity)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone.
- Species: rat
- Strain: Sprague Dawley
- Sex: male
- Age: 5 - 6 weeks
- Weight: 175 - 199 g
- Tissue: liver
- Inducing Agents: phenobarbital and 5,6-benzoflavone
- Producer: TRINOVA BIOCHEM GmbH, Giessen, Germany
- Batch Number: 3971
- Quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): yes

Test concentrations with justification for top dose:

Cytotoxicity test (+/-S9): 0.00328, 0.00819, 0.0205, 0.0512, 0.128, 0.320, 0.800, 2.00, 5.00 µL/mL
No cells survived at and above 0.0512 µL/mL in the absence of metabolic activation and at and above 0.320 µL/mL in the presence of metabolic activation.

Mutation assay (-S9): 0.00205, 0.00410, 0.00819, 0.0102, 0.0128, 0.0160, 0.0200 µL/mL
Mutation assay (+S9): 0.0307, 0.0614, 0.123, 0.154, 0.192, 0.240, 0.300 µL/mL
The selection of the concentrations used in the main experiments was based on data from the cytotoxicity test.

Vehicle / solvent:

- Vehicle/solvent used: dimethylsulfoxide (DMSO)

- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle. This vehicle was selected since it is compatible with the survival of cells and the S9 metabolic activation system.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

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 : single

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 2 x 10E6
- Test material added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 h

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 8 days
- Selection time (if incubation with a selective agent): 10 - 15 days
- Selective agent used: 6-thioguanine, 7.5 µg/mL
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 1 x 10E5

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative survival (RS)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Method: Mutant frequency (MF) per million surviving cells

Evaluation criteria:

EVALUATION CRITERIA
A test item is considered to be clearly positive if:
– At least one of the test concentrations exhibits a statistically significant increase, compared with the concurrent solvent/vehicle control.
– The increase is concentration-related.
– Any of the results are outside the distribution of the historical negative control data (95% confidence limits).

A test item is considered to be clearly negative if:
– None of the test concentrations exhibits a statistically significant increase, compared with the concurrent solvent/vehicle control.
– There is no concentration-related increase.
– All results are inside the distribution of the historical negative control data (95% confidence limits).

ACCEPTANCE CRITERIA
The assay was considered valid if the following criteria were met:
1. The mutant frequency of the solvent/vehicle control is within the 95% control limits of the distribution of the laboratory’s historical control database.
2. The positive controls induce responses that are compatible with those generated in the historical control database and produce a statistically significant increase in mutant frequency, compared with the concurrent solvent/vehicle control.
3. Two experimental conditions are tested (i.e. with and without metabolic activation), unless one gives positive results.
4. Adequate number of cells (i.e. at least 20 x 10E6 at treatment time and at least 2 x 10E6 during the expression period) and concentrations (at least 4 with appropriate cytotoxicity) are analysable.
5. The selection of dose levels is consistent with those indicated in § 4.3 of the Study Protocol.

Statistics:

Individual mutation frequencies were transformed to induce homogeneous variance and normal distribution. The mutant frequency in the solvent control and treated cultures was compared using the Dunnett’s test (one-tailed). For each experimental point, the corrected sum of squares of transformed mutation frequencies was calculated.For each experimental point, the t value was calculated. For each comparison of treatment with control, the calculated t value was compared with tabulated critical values for the one tailed Dunnett’s test. The results of the experiment were subjected to an Analysis of Variance in which the effect of replicate culture and dose level in explaining the observed variation were examined.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Strong cytotoxicity was observed at and above a test substance concentration of 0.0205 µL/mL in cytotoxicity test (RS < 10%); concentrations in main assays were selected accordingly.

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
- Data on pH: Treatment with the test item did not change the pH at any dose level in any experiment.
- Data on osmolality: Treatment with the test item did not increases the osmolality at any dose level in any experiment.
- Precipitation and time of the determination: No precipitation of the test item was noted at any dose level.

RANGE-FINDING/SCREENING STUDIES
A preliminary cytotoxicity assay was performed using a single culture at each test point both in the presence and absence of metabolic activation. No positive controls were included. The test item was assayed at concentrations of 0.00328, 0.00819, 0.0205, 0.0512, 0.128, 0.320, 0.800, 2.00 and 5.00 µL/mL. In the absence of S9 mix, no cell survived treatment at and above concetrations of 0.0512 µL/mL while in the presence of S9 mix, no cell survived treatment at and above 0.320 μL/mL. Based on these findings the test item concentrations in the main assay were selected.

STUDY RESULTS
- Treatment with the positive control items gave marked responses that were compatible with those generated in the historical control database and produced a statistically significant increase in mutant frequency, compared with the concurrent solvent/vehicle control, indicating the correct functioning of the test system. An adequate number of cells and concentrations was analysed; concurrent vehicle negative and positive control data are reported in tabular form under 'Any other information on results incl. tables'.

HISTORICAL CONTROL DATA
- The mutant frequencies in the negative control cultures fell within the 95% control limits of the distribution of the laboratory’s historical control data; data are presented in tabular form under 'Any other information on results incl. tables'.

Table 1: Summary
Without metabolic activation				With metabolic activation
Dose level (µL/mL)	%RS	MF	Sig	Dose level (µL/mL)	%RS	MF	Sig
0.00	100	4.26		0.00	100	14.5
0.00205	82	15.3	*	0.0307	88	9.04	NS
0.00410	99	11.3	NS	0.0614	105	6.02	NS
0.00819	65	15.0	*	0.123	63	16.8	NS
0.0102	62	9.40	NS	0.154	91	13.9	NS
0.0128	23	9.66	NS	0.192	64	6.05	NS
0.0160	£	£		0.240	29	14.7	NS
0.0200	£	£		0.300	£	£
EMS 10.0 mM	57	2681	**	DMBA 10.0 µg/mL	51	241	**

Table 2: Survival after treatment
SURVIVAL AFTER TREATMENT WITHOUT METABOLIC ACTIVATION
Treatment	Dose Level (µL/mL)		106 cells post treatment	Plate counts			Mean	CE	Adjusted CE	Mean	RS (%)
Sovent control	A	0.00	20.2	116	108	115	113	0.57	0.47	0.55	100
	B		20.4	158	144	152	151	0.76	0.63
Test Item	A	0.00205	19.6	89	93	93	92	0.46	0.37	0.45	82
	B		16.2	164	160	157	160	0.80	0.53
Test Item	A	0.00410	17.4	104	104	107	105	0.53	0.37	0.54	99
	B		17.6	199	203	191	198	0.99	0.71
Test Item	A	0.00819	11.4	131	138	137	135	0.68	0.32	0.36	65
	B		12.1	155	160	163	159	0.80	0.40
Test Item	A	0.0102	12.6	133	130	137	133	0.67	0.34	0.34	62
	B		11.9	131	135	145	137	0.69	0.33
Test Item	A	0.0128	7.4	89	89	90	89	0.45	0.14	0.13	23
	B		7.7	76	76	78	77	0.38	0.12
Test Item	A	0.0160	1.3
	B		1.3	£	£	£
Test item	A	0.0200	1.3
	B		1.7	£	£	£
EMS		10.0 mM	17	88	90	90	89	0.45	0.31	0.31	57
Baseline count			24.4
SURVIVAL AFTER TREATMENT WITH METABOLIC ACTIVATION
Treatment	Dose Level (µL/mL)		106 cells post treatment	Plate counts			Mean	CE	Adjusted CE	Mean	RS (%)
Sovent control	A	0.00	30	144	152	152	149	0.75	1.07	1.00	100
	B		30.3	129	131	124	128	0.64	0.93
Test Item	A	0.0307	24.9	160	150	152	154	0.77	0.92	0.88	88
	B		26.7	136	136	125	132	0.66	0.85
Test Item	A	0.0614	26.4	170	167	165	167	0.84	1.06	1.05	105
	B		29.7	147	146	144	146	0.73	1.04
Test Item	A	0.123	21	125	118	112	118	0.59	0.60	0.63	63
	B		21.8	120	131	127	126	0.63	0.66
Test Item	A	0.154	21.6	154	152	154	153	0.77	0.79	0.91	91
	B		25.2	172	174	165	170	0.85	1.03
Test Item	A	0.192	23.6	125	138	139	134	0.67	0.76	0.64	64
	B		21.9	100	100	102	101	0.50	0.53
Test Item	A	0.240	11	116	113	118	116	0.58	0.31	0.29	29
	B		10	114	119	114	116	0.58	0.28
Test item	A	0.300	0.5
	B		0.4	£	£	£
DMBA		10.0 µg/mL	25.2	80	82	90	84	0.42	0.51	0.51	51
Baseline count			20.9

Table 3: Cloning efficiency
CLONING EFFICIENCY WITHOUT METABOLIC ACTIVATION
Treatment		Dose Level (µL/mL )	Plate counts			Mean	%CE
Solvent control	A	0.00	134	131	140	118	59
	B		99	96	105
Test Item	A	0.00205	86	80	86	88	44
	B		92	95	90
Test Item	A	0.00410	160	160	160	185	93
	B		211	211	209
Test Item	A	0.00819	140	142	140	120	60
	B		103	100	97
Test Item	A	0.0102	151	150	159	149	75
	B		145	144	145
Test Item	A	0.0128	197	200	200	166	83
	B		131	134	132
EMS		10.0 mM	70	70	79	73	37
CLONING EFFICIENCY WITH METABOLIC ACTIVATION
Treatment		Dose Level (µL/mL)	Plate counts			Mean	%CE
Solvent control	A	0.00	108	112	101	111	55
	B		110	122	110
Test Item	A	0.0307	130	128	123	144	72
	B		159	156	167
Test Item	A	0.0614	113	111	108	108	54
	B		101	106	109
Test Item	A	0.123	104	101	93	98	49
	B		96	97	99
Test Item	A	0.154	104	96	97	101	50
	B		105	104	97
Test Item	A	0.192	98	98	102	108	54
	B		112	113	122
Test Item	A	0.240	122	120	112	136	68
	B		151	146	165
DMBA		10.0 µg/mL	131	155	148	145	72

Table 4: Mutation frequency
MUTATION FREQUENCY WITHOUT METABOLIC ACTIVATION
Treatment		Dose Level (µL/mL)	Plate counts										Tot.	Mean	SD	MF	MF Pooled cultures	t	Sig.
Solvent control	A	0.00	1	0	1	0	0	0	0	0	1	0	7	0.4	0.67	5.19	4.26
			0	0	0	2	0	0	0	0	0	2
	B		0	0	0	0	0	1	0	0	0	0	3	0.2	0.37	3.00
			0	1	1	0	0	0	0	0	0	0
Test item	A	0.00205	0	0	0	2	0	0	1	0	0	1	11	0.6	0.76	13.10	15.31	3.060	*
			0	0	1	2	1	2	1	0	0	0
	B		1	1	1	0	3	0	0	0	3	0	16	0.8	1.01	17.33
			0	0	1	0	0	2	1	0	2	1
Test item	A	0.00410	4	0	2	1	0	1	2	1	0	1	21	1.05	1.00	13.13	11.34	2.335	NS
			1	0	2	0	2	1	0	1	1	1
	B		0	2	2	1	0	2	0	2	1	0	21	1.05	0.89	9.98
			3	0	1	1	1	1	1	2	1	0
Test item	A	0.00819	1	0	0	1	1	1	0	0	1	0	11	0.55	0.69	7.82	14.96	2.976	*
			0	0	1	1	0	0	0	2	0	2
	B		2	2	0	2	0	2	1	1	2	1	25	1.3	0.97	25.00
			1	1	4	0	1	1	2	1	1	0
Test item	A	0.0102	2	1	0	3	0	2	0	0	0	1	14	0.7	1.03	9.13	9.40	1.841	NS
			3	1	0	0	0	0	0	1	0	0
	B		2	0	2	0	1	0	1	0	1	0	14	0.7	0.73	9.68
			1	1	1	1	2	0	0	0	1	0
Test item	A	0.0128	2	1	0	0	1	2	1	2	3	0	19	1.0	1.05	9.55	9.66	1.913	NS
			2	1	0	0	0	3	1	0	0	0
	B		0	2	0	0	1	0	0	2	2	0	13	0.7	0.99	9.82
			0	0	0	0	0	0	0	3	1	2
EMS		10.0 mM	88	79	98	94	96	103	134	123	121	128	1957	97.9	18.39	2681		28.190	**
			120	82	85	85	104	86	83	79	71	98
MUTATION FREQUENCY WITH METABOLIC ACTIVATION
Treatment		Dose Level (µL/mL)	Plate counts										Tot.	Mean	SD	MF	MF Pooled cultures	t	Sig.
Solvent control	A	0.00	1	0	1	0	1	1	0	0	1	0	11	0.6	0.51	10.28	14.48
			0	1	1	0	1	1	1	0	1	0
	B		1	1	3	0	0	0	1	0	3	0	21	1.1	1.10	18.42
			1	0	2	3	1	1	0	2	2	0
Test item	A	0.0307	0	2	0	1	0	1	1	1	0	1	9	0.5	0.60	7.09	9.04	-1.360	NS
			0	0	1	0	0	0	1	0	0	0
	B		1	2	1	1	2	0	0	1	1	0	17	0.9	0.75	10.58
			2	1	0	1	0	0	1	1	0	2
Test item	A	0.0614	1	0	1	0	1	0	0	1	0	1	8	0.4	0.50	7.23	6.02	-2.365	NS
			0	0	1	0	0	0	0	0	1	1
	B		0	1	0	0	0	1	0	0	0	1	5	0.25	0.44	4.75
			0	0	1	0	0	1	0	0	0	0
Test item	A	0.123	0	0	1	1	2	1	0	0	0	3	15	0.75	0.79	15.10	16.78	0.583	NS
			1	1	1	0	1	1	1	1	0	0
	B		1	3	1	0	0	0	1	0	3	1	18	0.9	0.97	18.49
			1	0	0	1	2	2	1	1	0	0
Test item	A	0.154	2	1	2	1	1	0	2	1	0	1	19	1.0	0.89	19.19	13.93	-0.144	NS
			1	0	1	0	2	0	3	1	0	0
	B		0	0	0	0	0	1	1	0	1	1	9	0.45	0.51	8.82
			0	1	1	0	0	0	0	1	1	1
Test item	A	0.192	1	0	0	0	0	0	0	1	0	0	4	0.2	0.41	4.03	6.05	-2.454	NS
			0	1	0	0	1	0	0	0	0	0
	B		0	2	1	1	0	0	1	0	0	0	9	0.5	0.60	7.78
			0	0	0	1	1	1	0	1	0	0
Test item	A	0.240	2	1	0	0	0	0	1	1	0	0	17	0.9	0.93	14.41	14.71	0.162	NS
			3	1	2	1	2	0	0	1	0	2
	B		3	1	1	0	0	3	2	0	0	2	23	1.2	1.09	14.94
			3	2	1	1	2	0	1	0	1	0
DMBA		10.0 µg/mL	15	17	16	19	16	15	20	16	17	15	348	17.4	2.01	240.6		10.909	**
			20	12	16	20	18	18	16	20	21	18

CE = Cloning efficiency

RS = Relative survival

SD = Standard deviation

Tot = Total number of mutant colonies

MF = Mutant frequency per million survival cells

£ = Insufficient number of cells recovered after treatment period

Sig = Significance level

NS = Not statistically significant

* = Statistically significant at p < 0.05

** = Statistically significant at p < 0.01

*** = Statistically significant at p > 0.001

Table 5: Historical control data
HISTORICAL DATA OF NEGATIVE/SOLVENT CONTROL mutation frequencies per million surviving cells (up to May 2019)
WITHOUT METABOLIC ACTIVATION	WITH METABOLIC ACTIVATION
Day 8 / Day 9	Day 8 / Day 9
Mean value
9.49	11.19
Standard deviation
8.65	8.82
Number of experiments
72	73
Upper confidence limit (5%)
26.8	28.8
Observed range
1.45 - 65.14	1.48 - 59.72
HISTORICAL DATA OF POSITIVE CONTROL mutation frequencies per million surviving cells (up to November 2018)
WITHOUT METABOLIC ACTIVATION	WITH METABOLIC ACTIVATION
Day 8 / Day 9	Day 8 / Day 9
Mean value
1147.38	503.30
Standard deviation
401.15	282.27
Number of experiments
72	73
Observed range
391 - 2631	109.86 - 1670.18

Conclusions:

Interpretation of results: negative

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 May - 24 June 2019

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:

adopted in 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (S. typhimurium strains) and trp operon (E. coli strain)

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:

Type and composition of metabolic activation system:
- source of S9: cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone.
- Species: rat
- Strain: Sprague Dawley
- Sex: male
- Age: 5 - 6 weeks
- Weight: 175 - 199 g
- Tissue: liver
- Inducing Agents: phenobarbital and 5,6-benzoflavone
- Producer: TRINOVA BIOCHEM GmbH, Giessen, Germany
- Batch Number: 4009
- Quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): yes

Test concentrations with justification for top dose:

Main assay I (plate incorporation) and main assay II (pre-incubation):
TA1535, WP2 uvrA, TA98 (+/-S9): 0.313, 0.625, 1.25, 2.50, 5.00 µL/plate
TA1537 (+/-S9) and TA100 (+S9): 0.00977, 0.0195, 0.0391, 0.0781, 0.156, 0.313 µL/plate
TA100 (-S9): 0.00244, 0.00488, 0.00977, 0.0195, 0.0391, 0.0781 µL/plate

Main assay III (pre-incubation):
TA1535 (-S9): 0.00977, 0.0195, 0.0391, 0.0781, 0.156, 0.313 µL/plate
TA1537 (-S9): 0.000610, 0.00122, 0.00244, 0.00488, 0.00977, 0.0195 µL/plate
WP2 uvrA (-S9): 0.0391, 0.0781, 0.156, 0.313, 0.625, 1.25 µL/plate
TA100 (-S9): 0.000305, 0.000610, 0.00122, 0.0024, 0.00488, 0.00977 µL/plate

The test concentrations were selected based on the results of a toxicity test with all 5 strains in which no precipitation occured. However, severe cytotoxicity at most of the concentrations tested with TA1537 and TA100 strains (both in the absence and presence of S9) was observed up to and including the highest concentration of 5 µL/plate.

Vehicle / solvent:

- Vehicle used: dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: DMSO was selected based on the solubility of the test substance and its compatibility with the survival of the bacteria and the S9 metabolic activity. The test item was found to be miscible at 100 μL/mL. This allowed a maximum concentration of 5.00 μL/plate.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-Aminoanthracene (2-AA), +S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : three

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation, main assay I) and preincubation (main assays II and III)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 30 min at 37 °C (main assays II and III)
- Exposure duration/duration of treatment: 72 h at 37 °C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation

Evaluation criteria:

The test material may be considered positive in this test system if the following criteria are met:
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.

Statistics:

Mean values and standard deviation were calculated.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Plate incorporation method: no cytotoxicity +/- S9; preincubation method: at and above 0.156 µL/plate +S9, no cytotoxicity -S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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

Remarks:

Plate incorporation method: at and above 0.156 µL/plate +/- S9; preincubation method: at and above 0.00977 µL/plate -S9, at and above 0.156 µL/plate +S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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

Remarks:

Plate incorporation method: at and above 0.313 µL/plate +S9 and 0.0781 µg/plate -S9; preincubation method: at and above 0.313 µL/plate +S9 and 0.00488 µL/plate -S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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

Remarks:

Plate incorporation method: no cytotoxicity +/s S9; preincubation method: at and above 0.625 µL/plate -S9, no cytotoxicity +S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: No precipitation of the test item was observed at the end of the incubation period at any concentration in any experiment.

RANGE-FINDING/SCREENING STUDY:
Concentrations of 0.0500, 0.158, 0.500, 1.58 and 5.00 μL/plate of the test item in the presence and absence of S9 mix in S. typhimurium TA1935, TA1937, TA98, TA100 and E. coli WP2 uvr A were tested in an plate incorporation experiment. Severe toxicity, as indicated by thinning of the background lawn and/or reduction in revertant numbers, was observed at all or almost all concentrations with TA1537 and TA100 tester strains both in the absence and presence of S9 mix. The highest concentration analysed was selected based on the solubility of the test substance.

STUDY RESULTS
- Concurrent vehicle negative and positive control data are presented in tabular form under 'Any other information on results incl. tables'.

Ames test:
- Individual plate counts are presented in tabular form under 'Any other information on results incl. tables'.
- Mean number of revertant colonies per plate and standard deviation are presented in tabular form under 'Any other information on results incl. tables'.

HISTORICAL CONTROL DATA:
- Mean plate counts for untreated and positive control plates fell within the normal range based on historical control data. Data are presented in tabular form under 'Any other information on results incl. tables'.

Table 1: Detailed results of Main Assay I
MAIN ASSAY I	Plate incorporation method
Strain: TA1535
Dose-level [µL/plate]	Without metabolic activation			Mean	S. E.	With metabolic activation			Mean	S. E.
	Plate counts					Plate counts
Untreated	14	20	14	16	2.0	16	14	18	16	1.2
0.00	15	17	14	15	0.9	18	20	17	18	0.9
0.313	14	16	19	16	1.5	18	17	18	18	0.3
0.625	17	14	15	15	0.9	17	20	17	18	1.0
1.25	17	17	16	17	0.3	18	14	13	15	1.5
2.50	17	16	21	18	1.5	14	14	16	15	0.7
5.00	15	20	19	18	1.5	15	16	14	15	0.6
Controls		S9	Plate counts			Mean	S. E.
Sodium Azide	1 µg/plate	-	532	489	507	509	12.5
DMSO	100 µL/plate	+	18	20	17	18	0.9
2-Aminoanthracene	1 µg/plate	+	161	172	154	162	5.2
Strain: TA1537
Dose-level [µL/plate]	Without metabolic activation			Mean	S. E.	With metabolic activation			Mean	S. E.
	Plate counts					Plate counts
Untreated	17	17	18	17	0.3	24	20	17	20	2.0
0.00	19	22	16	19	1.7	20	20	21	20	0.3
0.00977	16	17	22	18	1.9	21	24	24	23	1.0
0.0195	17	22	18	19	1.5	23	25	20	23	1.5
0.0391	15	19	14	16	1.5	19	23	21	21	1.2
0.0781	12	14	16	14	1.2	25	23	22	23	0.9
0.156	14	16	16	15*	0.7	22	18	16	19*	1.8
0.313	12	12	10	11**	0.7	20	24	21	22**	1.2
Controls		S9	Plate counts			Mean	S. E.
DMSO	100 µL/plate	-	19	22	16	19	1.7
9-Aminoacridine	50 µL/plate	-	207	163	182	184	12.7
DMSO	100 µL/plate	+	20	20	21	20	0.3
2-Aminoanthracene	1 µL/plate	+	117	121	129	122	3.5
* : Slight thinning of the background lawn
**: Moderate thinning of the background lawn
Strain: WP2 uvrA
Dose-level [µL/plate]	Without metabolic activation			Mean	S. E.	With metabolic activation			Mean	S. E.
	Plate counts					Plate counts
Untreated	27	31	25	28	1.8	37	31	42	37	3.2
0.00	26	31	34	30	2.3	33	28	34	32	1.9
0.313	29	27	24	27	1.5	31	36	32	33	1.5
0.625	29	28	31	29	0.9	32	35	35	34	1.0
1.25	26	28	34	29	2.4	38	36	35	36	0.9
2.50	26	26	29	27	1.0	24	29	32	28	2.3
5.00	27	24	24	25	1.0	25	29	30	28	1.5
Controls		S9	Plate counts			Mean	S. E.
MMS	500 µg/pl	-	144	152	153	150	2.8
DMSO	100 µL/pl	+	33	28	34	32	1.9
2-Aminoanthracene	10 µg/pl	+	130	127	131	129	1.2
Strain: TA98
Dose-level [µL/plate]	Without metabolic activation			Mean	S. E.	With metabolic activation			Mean	S. E.
	Plate counts					Plate counts
Untreated	28	30	26	28	1.2	39	32	39	37	2.3
0.00	27	30	26	28	1.2	40	37	41	39	1.2
0.313	24	27	21	24	1.7	34	43	37	38	2.6
0.625	24	21	28	24	2.0	38	33	39	37	1.9
1.25	26	31	25	27	1.9	30	32	33	32	0.9
2.50	26	27	25	26	0.6	38	34	37	36	1.2
5.00	28	26	26	27	0.7	38	37	37	37	0.3
Controls		S9	Plate counts			Mean	S. E.
DMSO	100 µL/plate	-	27	30	26	28	1.2
2-Nitrofluorene	2 µL/plate	-	140	166	152	153	7.5
DMSO	100 µL/plate	+	40	37	41	39	1.2
2-Aminoanthracene	1 µL/plate	+	317	356	406	360	25.8
Strain: TA100
Dose-level [µL/plate]	Without metabolic activation			Mean	S. E.	With metabolic activation			Mean	S. E.
	Plate counts					Plate counts
Untreated	117	113	113	114	1.3	114	127	127	123	4.3
0.00	126	127	121	125	1.9	115	108	102	108	3.8
0.00244	140	141	136	139	1.5	NT
0.00488	140	145	133	139	3.5	NT
0.00977	136	140	140	139	1.3	108	109	110	109	0.6
0.0195	133	133	120	129	4.3	102	106	113	107	3.2
0.0391	126	127	122	125	1.5	107	106	116	110	3.2
0.0781	90	91	83	88*	2.5	129	121	124	125	2.3
0.156	NT					109	109	108	109	0.3
0.313	NT					98	97	86	94*	3.8
Controls		S9	Plate counts			Mean	S. E.
Sodium Azide	1 µg/plate	-	621	655	724	667	30.3
DMSO	100 µL/plate	+	115	108	102	108	3.8
2-Aminoanthracene	1 µg/plate	+	918	1020	1083	1007	48.1
* : Slight thinning of the background lawn
NT: Not tested

Table 2: Detailed results of Main Assay II
MAIN ASSAY II	Pre-incubation method
Strain: TA1535
Dose-level [µL/plate]	Without metabolic activation			Mean	S. E.	With metabolic activation			Mean	S. E.
	Plate counts					Plate counts
Untreated	21	20	17	19	1.2	13	14	14	14	0.3
0.00	18	18	21	19	1.0	14	16	13	14	0.9
0.313	13	15	17	15**	1.2	17	13	15	15	1.2
0.625	13	15	16	15**	0.9	15	14	14	14	0.3
1.25	14	16	15	15**	0.6	16	15	15	15	0.3
2.50	17	15	19	17**	1.2	12	15	16	14	1.2
5.00	12	12	11	12**	0.3	12	11	12	12	0.3
Controls		S9	Plate counts			Mean	S. E.
Sodium Azide	1 µg/plate	-	382	416	384	28.1	11.0
DMSO	50 µg/plate	+	14	16	13	14	0.9
2-Aminoanthracene	1 µg/plate	+	113	113	110	21.4	1.0
**: Moderate thinning of the background lawn
Strain: TA1537
Dose-level [µL/plate]	Without metabolic activation			Mean	S. E.	With metabolic activation			Mean	S. E.
	Plate counts					Plate counts
Untreated	20	16	15	17	1.5	23	22	21	22	0.6
0.00	18	18	21	19	1.0	21	20	20	20	0.3
0.00977	15	14	12	14*	0.9	25	20	23	23	1.5
0.0195	10	11	12	11**	0.6	23	20	20	21	1.0
0.0391	8	8	7	8**	0.3	18	23	15	19	2.3
0.0781	4	5	7	5**	0.9	17	18	21	19	1.2
0.156	8	6	9	8***	0.9	17	17	15	16*	0.7
0.313	5	7	5	6***	0.7	14	12	18	15**	1.8
Controls		S9	Plate counts			Mean	S. E.
DMSO	50 µL/plate	-	18	18	21	19.1	1.0
9-Aminoacridine	50 µL/plate	-	92	86	94	31.3	2.4
DMSO	50 µL/plate	+	21	20	20	20.0	0.3
2-Aminoanthracene	1 µL/plate	+	78	78	85	20.3	2.3
*: Slight thinning of the background lawn
** : Moderate thinning of the background lawn
***: Severe thinning of the background lawn
Strain: WP2 uvrA
Dose-level [µL/plate]	Without metabolic activation			Mean	S. E.	With metabolic activation			Mean	S. E.
	Plate counts					Plate counts
Untreated	30	31	32	31	0.6	37	35	38	37	0.9
0.00	29	29	29	29	0.0	35	33	39	36	1.8
0.313	30	30	30	30	0.0	32	41	41	38	3.0
0.625	28	28	26	27*	0.7	33	39	40	37	2.2
1.25	29	22	22	24**	2.3	39	35	32	35	2.0
2.50	20	25	24	23***	1.5	35	32	37	35	1.5
5.00	25	29	25	26***	1.3	28	26	26	27	0.7
Controls		S9	Plate counts			Mean	S. E.
MMS	500 µg/plate	-	112	114	117	23.4	1.5
DMSO	50 µg/plate	+	35	33	39	5.2	1.8
2-Aminoanthracene	20 µg/plate	+	135	150	167	30.5	9.2
*: Slight thinning of the background lawn
** : Moderate thinning of the background lawn
***: Severe thinning of the background lawn
Strain: TA98
Dose-level [µL/plate]	Without metabolic activation			Mean	S. E.	With metabolic activation			Mean	S. E.
	Plate counts					Plate counts
Untreated	27	31	32	30	1.5	35	35	36	35	0.3
0.00	33	34	29	32	1.5	34	39	39	37	1.7
0.313	26	32	25	28	2.2	29	31	30	30	0.6
0.625	32	31	25	29	2.2	28	30	34	31	1.8
1.25	29	29	29	29	0.0	30	30	29	30	0.3
2.50	24	25	25	25	0.3	27	25	28	27	0.9
5.00	27	25	29	27	1.2	30	35	28	31	2.1
Controls		S9	Plate counts			Mean	S. E.
DMSO	50 µl/plate	-	33	34	29	1.2	1.5
2-Nitrofluorene	2 µl/plate	-	125	123	127	4.5	1.2
DMSO	50 µl/plate	+	34	39	39	6.2	1.7
2-Aminoanthracene	2 µl/plate	+	507	681	643	1.9	52.8
Strain: TA100
Dose-level [µL/plate]	Without metabolic activation			Mean	S. E.	With metabolic activation			Mean	S. E.
	Plate counts					Plate counts
Untreated	125	118	128	124	3.0	156	158	147	154	3.4
0.00	124	132	122	126	3.1	147	148	159	151	3.8
0.00244	128	123	132	128	2.6	NT
0.00488	120	122	120	121	0.7	NT
0.00977	78	79	78	78*	0.3	123	120	128	124	2.3
0.0195	98	97	91	95**	2.2	121	116	118	118	1.5
0.0391	89	86	85	87***	1.2	115	128	124	122	3.8
0.0781	82	89	80	84***	2.7	129	127	121	126	2.4
0.156	NT					137	134	126	132	3.3
0.313	NT					78	77	68	74*	3.2
Controls		S9	Plate counts			Mean	S. E.
Sodium Azide	1 µg/pl	-	680	542	623	615	40.0
DMSO	50 µL/pl	+	147	148	159	151	3.8
2-Aminoanthracene	2 µg/pl	+	1316	1086	1218	1207	66.6
*: Slight thinning of the background lawn
** : Moderate thinning of the background lawn
***: Severe thinning of the background lawn
NT: Not tested

Table 3: Detailed results of Main Assay III
MAIN ASSAY III	Pre-incubation method
Strain: TA1535
Dose-level [µL/plate]	Without metabolic activation			Mean	S. E.
	Plate counts
Untreated	14	14	14	14	0.0
0.00	15	15	14	15	0.3
0.00977	18	21	21	20	1.0
0.0195	15	14	15	15	0.3
0.0391	15	17	15	16	0.7
0.0781	16	13	13	14	1.0
0.156	12	12	13	12*	0.3
0.313	13	13	12	13**	0.3
Controls		S9	Plate counts			Mean	S. E.
Sodium Azide	1 µg/plate	-	412	463	394	423	20.7
* : Slight thinning of the background lawn
**: Moderate thinning of the background lawn
Strain: TA1537
Dose-level [µL/plate]	Without metabolic activation			Mean	S. E.
	Plate counts
Untreated	13	15	14	14	0.6
0.00	14	17	16	16	0.9
0.000610	19	20	16	18	1.2
0.00122	15	16	13	15	0.9
0.00244	18	19	20	19	0.6
0.00488	17	15	16	16	0.6
0.00977	10	10	12	11**	0.7
Controls		S9	Plate counts			Mean	S. E.
DMSO	50 µL/pl	-	14	17	16	16	0.9
9-Aminoacridine	50 µg/pl	-	89	103	127	106	11.1
**: Moderate thinning of the background lawn
Strain: WP2 uvrA
Dose-level [µL/plate]	Without metabolic activation			Mean	S. E.
	Plate counts
Untreated	30	26	27	28	1.2
0.00	34	31	27	31	2.0
0.0391	29	26	32	29	1.7
0.0781	25	30	29	28	1.5
0.156	27	26	34	29	2.5
0.313	27	29	30	29	0.9
0.625	26	22	22	23*	1.3
1.25	21	23	24	23**	0.9
Controls		S9	Plate counts			Mean	S. E.
MMS	500 µg/pl	-	156	161	179	165	7.0
*:  Slight thinning of the background lawn
**: Moderate thinning of the background lawn
Strain: TA100
Dose-level [µL/plate]	Without metabolic activation			Mean	S. E.
	Plate counts
Untreated	154	142	141	146	4.2
0.00	120	136	131	129	4.7
0.000305	131	134	139	135	2.3
0.000610	141	121	121	128	5.7
0.00122	156	138	159	151	6.6
0.00244	121	129	128	126	2.5
0.00488	92	100	90	94*	3.1
0.00977	88	76	78	81**	3.7
Controls		S9	Plate counts			Mean	S. E.
Sodium Azide	1 µg/plate	-	715	883	779	792	49.0
*  : Slight thinning of the background lawn
**: Moderate thinning of the background lawn

Table 4: Historical control data
TA 1535 Plate incorporation					TA 1535 Pre-incubation
	Negative Control		Positive control			Negative Control		Positive control
	-S9	+S9	-S9	+S9		-S9	+S9	-S9	+S9
Mean	18	17	518	136	Mean	18	17	528	98
SD	2.6	2.2	81.7	27.9	SD	2.5	2.2	78.4	14.7
UCL	23.2	21.3	681.3	191.6	UCL	23.0	21.5	684.5	127.2
LCL	12.8	12.7	354.6	79.9	LCL	12.8	12.7	370.7	68.5
n	491	492	491	492	n	393	379	393	378
min	13	12	299	81	min	12	12	374	54
max	16	22	750	241	max	25	23	774	179
TA 1537 Plate incorporation					TA 1537 Pre-incubation
	Negative Control		Positive control			Negative Control		Positive control
	-S9	+S9	-S9	+S9		-S9	+S9	-S9	+S9
Mean	17	21	177	114	Mean	18	21	169	98
SD	2.3	2.9	40	21	SD	2.3	2.4	46.4	12.4
UCL	21.7	26.7	257.2	156.7	UCL	22.1	25.7	261.8	122.3
LCL	12.6	15.2	97.0	72	LCL	13.0	16.1	76.3	72.8
n	495	494	495	494	n	386	371	386	370
min	11	15	92	75	min	12	15	72	69
max	27	33	407	181	max	27	27	412	142
TA 98 Plate incorporation					TA 98 Pre-incubation
	Negative Control		Positive control			Negative Control		Positive control
	-S9	+S9	-S9	+S9		-S9	+S9	-S9	+S9
Mean	31	38	162	508	Mean	31	39	163	628
SD	3.3	3.9	29.3	157.8	SD	3.1	3.2	26.7	180
UCL	37.7	46.0	220.3	823.5	UCL	37.4	45.8	216.5	987.7
LCL	24.3	30.5	103.0	192.2	LCL	25.1	33.1	109.8	267.6
n	497	497	497	497	n	377	353	377	353
min	23	29	109	203	min	24	33	115	188
max	43	53	259	994	max	43	50	256	1377
TA 100 Plate incorporation					TA 100 Pre-incubation
	Negative Control		Positive control			Negative Control		Positive control
	-S9	+S9	-S9	+S9		-S9	+S9	-S9	+S9
Mean	141	151	633	1192	Mean	143	150	676	1118
SD	17.7	18.3	157.0	230.2	SD	16.7	15.5	154.3	201.8
UCL	176.1	188.0	947.3	1652.1	UCL	176.6	181.2	984.8	1521.5
LCL	105.4	114.3	319.3	731.4	LCL	109.7	119.2	367.5	714.2
n	499	498	499	498	n	386	366	386	365
min	109	86	144	607	min	112	119	325	745
max	182	196	1081	2257	max	192	197	1081	1701
WP2 uvrA Plate incorporation					WP2 uvrA Pre-incubation
	Negative Control		Positive control			Negative Control		Positive control
	-S9	+S9	-S9	+S9		-S9	+S9	-S9	+S9
Mean	28	34	178	194	Mean	28	33	189	198
SD	2.9	3.5	20.1	36.9	SD	3.0	3.2	41.6	33.8
UCL	34.0	40.5	218.7	268,3	UCL	33.7	39.5	272.0	265.5
LCL	22.4	26.7	138.1	120,6	LCL	21.7	26.5	105.5	130.3
n	430	430	430	430	n	315	302	315	301
min	21	26	127	117	min	20	24	113	129
max	38	47	240	333	max	38	44	351	322
SD: Standard Deviation
UCL: Upper Confidence Limit (mean value + 2 SD)
LCL: Lower Confidence Limit (mean value - 2 SD)
n: number of experiments

Conclusions:

Interpretation of results: negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse mutation assay

Mutagenicity in bacteria was assessed in a study performed with the registered substance isotridecanol, ethoxylated, < 2.5 EO (CAS No. 69011-36-5, EC No. 500-241-6) according to OECD guideline 471 (adopted 1997) under GLP conditions (Sasol/BASF, 2019a). Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strain WP2 uvrA were treated using the plate incorporation method and the preincubation method, both with and without the addition of a metabolic activation system (rat liver S9 mix). The dose levels selected for the different strains in the plate incorporation experiment (first assay) were the following: TA1535, WP2 uvrA, TA98 (±S9): 0.313, 0.625, 1.25, 2.50 and 5.00 µg/plate; TA1537 (±S9) and TA100 (+S9): 0.00977, 0.0195, 0.0391, 0.0781, 0.156 and 0.313 µg/plate; TA100 (-S9): 0.00244, 0.00488, 0.00977, 0.0195, 0.0391 and 0.0781 µg/plate. Cytotoxicity was observed with TA1537 and TA100 tester strains at the highest dose levels, both in the absence and presence of S9 mix. No cytotoxicity was observed with the remaining tester strains and, therefore, the same dose levels were applied in the pre-incubation assay (second assay). However, in this assay, severe cytotoxicity was observed with most tester strains in the absence of metabolic activation and a third experiment (pre-incubation method) was necessary in order to have a sufficiently high number of analysable concentrations. In this third assay the following dose levels were used: TA1535 (-S9): 0.00977, 0.0195, 0.0391, 0.0781, 0.156 and 0.313 µg/plate; TA1537 (-S9): 0.000610, 0.00122, 0.00244, 0.00488, 0.00977 and 0.0195 µg/plate; WP2 uvrA (-S9): 0.0391, 0.0781, 0.156, 0.313, 0.625 and 1.25 µg/plate; TA100 (-S9): 0.000305, 0.000610, 0.00122, 0.00244, 0.00488 and 0.00977 µg/plate. All tests were carried out in triplicates. The vehicle (dimethyl sulfoxide, DMSO) control and untreated control produced counts of revertant colonies within an acceptable range. All positive controls used induced significant increases in the frequency of revertant colonies, both with and without the metabolising system. No mutagenic activity of the test substance to any of the tester strains was observed with and without metabolic activation at any dose level. Thus, under the conditions of this test, the test substance can be regarded as not mutagenic in bacteria.

In vitro mammalian cell micronucleus test

The registered substance isotridecanol, ethoxylated, < 2.5 EO (CAS No. 69011-36-5, EC No. 500-241-6) was assayed for its ability to induce micronuclei in human lymphocytes, following in vitro treatment in the presence and absence of an S9 metabolic activation system (rat liver S9 mix). Experiments were conducted according to OECD guideline 487 (adopted 2016) and observing GLP conditions (Sasol/BASF, 2020). A total of three experiments were performed: a short-term treatment, in which cells were treated for 3 h both in the absence and presence of S9 mix and harvested at 32 h, and a long-term (continuous) treatment where cells were treated in the absence of S9 mix, until harvest at 31 h. The dose levels applied were 0.195, 0.293, 0.439, 0.658, 0.988, 1.48, 2.22, 3.33 and 5.00 µL/mL. An additional dose of 0.130 µL/mL was included for the continuous treatment. Dimethyl sulfoxide (DMSO) served as vehicle. Since severe cytotoxicity was observed at all dose levels in all experiments, a second assay needed to be performed. The dose levels applied in the second assay were: ±S9 (3 h treatment, 31 h harvest): 0.00780, 0.0117, 0.0176, 0.0263, 0.0395, 0.0593, 0.0889, 0.133 and 0.200 µL/mL; -S9 (32 h treatment, 32 h harvest): 0.000520, 0.000780, 0.00117, 0.00176, 0.00263, 0.00395, 0.00593, 0.00889, 0.0133 and 0.0200 µL/mL. In the presence of S9 metabolic activation an adequate level of cytotoxicity to select dose levels for scoring was not achieved. Therefore, this treatment series was repeated by using a very narrowly spaced concentration interval. The following dose levels were selected: 0.0654, 0.0752, 0.0865, 0.0994, 0.114, 0.132, 0.151, 0.174 and 0.200 µL/mL. Each experiment included appropriate negative and positive controls. Two replicate cell cultures were prepared at each test point. The actin polymerisation inhibitor Cytochalasin B was added after 3 h treatment to allow the selective analysis of micronucleus frequency in binucleated cells. The cytokinesis block proliferation index (CBPI) was calculated in order to evaluate cytotoxicity. 2000 binucleated cells per culture were scored to assess the frequency of micronucleated cells as well as the number of micronuclei per cell, while 1000 cells in total per concentration were assessed for cytotoxicity. Adequate cell proliferation was observed in negative control cultures and an appropriate number of doses and cells was analysed. The negative and positive control treatments yielded the expected results. No statistically significant increase in the incidence of micronucleated cells over the concurrent solvent control value was observed at any dose level at any treatment condition for the test substance. It can therefore be concluded that the test substance does not induce micronuclei in human lymphocytes after in vitro treatment under the reported experimental conditions.

In vitro mammalian cell gene mutation test

The mutagenic potential of the registered substance isotridecanol, ethoxylated, < 2.5 EO (CAS No. 69011-36-5, EC No. 500-241-6) in mammalian cells was assessed in a HPRT-assay according to OECD guideline 476 (adopted 2016) under GLP conditions (Sasol/BASF, 2019b). Following pre-tests with concentrations ranging from 0.00328 - 5.00 µL/mL, Chinese hamster V79 cells were exposed for 3 h to test substance concentrations of 0.00205, 0.00410, 0.00819, 0.0102, 0.0128, 0.0160 and 0.0200 µg/mL in the absence of metabolic activation, and at concentrations of 0.0307, 0.0614, 0.123, 0.154, 0.192, 0.240 and 0.300 µL/mL in the presence of metabolic activation by rat liver S9 mix. In both treatment series cytotoxic effects  were observed at one or several concentrations. No relevant increase in mutant numbers or mutant frequency was observed following treatment with the test substance at any dose level, in the absence or presence of S9 mix. Negative and positive control treatments were included in each mutation experiment. Significant increases in mutant numbers or mutant frequency were obtained with the positive control treatments, indicating the correct functioning of the assay system. It is concluded that the test substance does not induce gene mutation in Chinese hamster V79 cells after in vitro treatment in the absence or presence of S9 metabolic activation under the reported experimental conditions.

Justification for classification or non-classification

The available data on genetic toxicity obtained with isotridecanol, ethoxylated, < 2.5 EO (CAS No. 69011-36-5, EC No. 500-241-6) do not meet the criteria for classification according to the CLP Regulation (EC) No. 1272/2008 and are therefore conclusive but not sufficient for classification.