Isotridecanol, ethoxylated

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Isotridecanol, ethoxylated

EC Number:

500-241-6

EC Name:

Isotridecanol, ethoxylated

Cas Number:

69011-36-5

Molecular formula:

not available, isomeric structures

IUPAC Name:

Isotridecanol, ethoxylated

Method

Target gene:

Not applicable

Species / strain

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.

Controls

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.

Results and discussion

Test results

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'.

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)

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative