Glycerides, tallow sesqui-, hydrogenated, propoxylated

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance has shown no evidence of causing biologically relevant increases in the frequency of structural chromosome aberrations under any treatment conditions employed in a reliable in vitro chromosome aberration test using human lymphocytes in the presence and absence of metabolic activation.

A structurally related substance is considered to be non-mutagenic in a reliable in vitro gene mutation assay (HPRT test) with Chinese hamster V79 cells in the presence and absence of a metabolic activation system.

A structurally related substance was negative in a reliable chromosome aberration test employing cultured human lymphocytes, either in the presence or absence of a metabolic activation system.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 November 2015 to 28 January 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Test concentrations with justification for top dose:

Preliminary toxicity test: 20.16, 33.59, 55.99, 93.31, 155.52, 259.2, 432, 720, 1200 and 2000 µg/mL.

The maximum final concentration tested in the preliminary toxicity test was 2000 µg/mL as this is the standard limit concentration within this test system as recommended in the regulatory guidelines.

Main tests:
-S9 mix (3 hours) 20, 40, 60, 80 and 100 µg/mL
+S9 mix (3 hours) 20, 40, 60, 80 and 100 µg/mL
-S9 mix (21 hours) 20, 40, 60, 80 and 100 µg/mL

The top dose was based on the results of the preliminary test in which precipitate was evident at 100 µg/mL.

Vehicle / solvent:

Ethanol

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Evaluation criteria:

The following criteria were applied for assessment of assay acceptability:
The concurrent vehicle control was considered acceptable for addition to the laboratories historical vehicle control database (lie within or close to the 95%
confidence interval).
Concurrent positive controls induced a response that were compatible with the laboratories historical positive control database and produced statistically significant increases compared with the concurrent vehicle control.

The test was considered to be clearly positive and able to induce chromosome breaks if, in any of the experimental conditions examined:
At least one of the test concentrations exhibited a statistically significant increase compared with the concurrent vehicle control.
The increase was dose-related when evaluated with an appropriate trend test.
Any of the results were outside the distribution of the historical negative control data.

Providing that all of the acceptance criteria have been met, a negative response was claimed if, in all of the experimental conditions examined:
None of the test concentrations exhibited a statistically significant increase compared with the concurrent negative control.
There was no concentration-related increase when evaluated with an appropriate trend test.
All results were inside the distribution of the historical negative control data.

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

It is concluded that the substance has shown no evidence of causing biologically relevant increases in the frequency of structural chromosome aberrations under any treatment conditions employed in this study.

Executive summary:

In an in vitro chromosome aberration test human lymphocyes were exposed to the test substance in the presence and absence of metabolic activation. The substance did not show any evidence of causing biologically relevant increases in the frequency of structural chromosome aberrations under any treatment conditions employed in this study. Statistically significant increases in numerical aberrations in the form of polyploidy were observed in the absence of S9 mix following treatments for 3 and 21 hours in the absence of S9 mix in this in vitro cytogenetic test system, under the experimental conditions described.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Read-across to K1 study therefore K2 is the maximum Klimisch value.

Justification for type of information:

Read-across approach - see read-across justification in section 13.

Reason / purpose for cross-reference:

read-across source

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Induction of chromosomal damage (clastogenicity).

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

human

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenated (S9) with standard co-facotrs

Test concentrations with justification for top dose:

Experiment 1: Final concentration (ug/ml)
Group: 4(20)-hour without S9 - 0*, 106.25, 212.5, 425, 850*, 1700*, 3400*, MMC 0.4*
Group: 4(20)-hour with S9 - 0*, 106.25, 212.5, 425, 850*, 1700*, 3400*, CP 5*

Experiment 2: Final concentration (ug/ml)
Group: 4(20)-hour without S9 - 0*, 106.25, 212.5, 425, 850*, 1700*, 3400*, MMC 0.2*
Group: 4(20)-hour with S9 - 0*, 425, 850*, 1700*, 3400*, CP 5*

* Dose levels selected for metaphase analysis
MMC = Mitomycin C
CP = Cyclophosphamide

Vehicle / solvent:

Minimal Essential Media (MEM)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHODS
Cells
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a
volunteer who had been previously screened for suitability. The volunteer had not been exposed
to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a
viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was
determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second
and third division metaphase cells and so calculate the average generation time (AGT). The
average AGT for the regular donors used in this laboratory has been determined to be
approximately 17 hours under typical experimental exposure conditions.

Cell Culture
Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented
"in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 15% foetal calf serum,
at 37°C with 5% CO2 in air. The lymphocytes of fresh heparinised whole blood were stimulated
to divide by the addition of phytohaemagglutinin (PHA) at 90 ug/ml final concentration.

Preparation of Test and Control Materials
The test material was accurately weighed, dissolved in Minimal Essential Media (MEM) and
serial dilutions prepared. The molecular weight of the test material was 340 therefore the
maximum dose level was 3400 ug/ml, which was equivalent to 10 mM. The purity of the test
material was 100%. There was no significant change in pH when the test material was dosed into
media and the osmolality did not increase by more than 50 mOsm. Chemical analysis of the test
material formulations was not performed because it is not a requirement of the test method.
Vehicle and positive controls were used in parallel with the test material. The positive control
materials were as follows:

In the absence of S9, mitomycin C (MMC) (Sigma, Batch No. 093K0460) was used at 0.4 and
0.2 ug/ml for cultures in Experiment 1 and 2 respectively. It was dissolved in Minimal Essential
Medium.

In the presence of S9, cyclophosphamide (CP) (Sigma, Batch No.084K1328) was used at
5.0 ug/ml in both experiments. It was dissolved in dimethyl sulphoxide.

Microsomal Enzyme Fraction
Microsomal fraction was prepared in-house at Safepharm Laboratories from the livers of
male Sprague-Dawley rats weighing ~250g. These had received three daily oral doses of a
mixture of phenobarbitone (80 mg/kg) and p-naphthoflavone (l00 mg/kg), prior to S9 preparation
on the fourth day. The S9 was stored at -196°C in a liquid nitrogen freezer.

Culture Conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the
following components, giving, when dispensed into sterile plastic flasks for each culture:
8.05-9.05 ml MEM, 15% (FCS)
0.1 ml Li-heparin
0.1 ml phytohaemagglutinin
0.75 ml heparinised whole blood

With Metabolic Activation (S9) Treatment
After approximately 48 hours incubation at 37°C, 5% CO2 in humidified air, the cultures were
transferred to tubes and centrifuged. Approximately 9 ml of the culture medium was removed,
reserved, and replaced with the required volume of MEM (including serum) and 1.0 ml of the
appropriate solution of vehicle control or test material was added to each culture. For the positive
control, 0.1 ml of the appropriate solution was added to the cultures. 1 ml of 20% S9-mix (ie 2%
final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary
Toxicity Test and of Experiment 1.

In Experiment 2, 1 ml of 10% S9-mix (ie 1% final concentration of S9 in standard co-factors),
was added. All cultures were then returned to the incubator. The nominal final volume of each
culture was 10 ml. After 4 hours at 37°C, the cultures were centrifuged, the treatment medium removed by suction
and replaced with an 8 ml wash ofMEM culture medium. After a further centrifugation the wash
medium was removed by suction and replaced with the original culture medium. The cells were
then re-incubated for a further 20 hours at 37°C in 5% C02 in humidified air.

Without Metabolic Activation (S9) Treatment
In Experiment 1, after approximately 48 hours incubation at 37°C with 5% CO2 in humidified air
the cultures were decanted into tubes and centrifuged. Approximately 9 ml ofthe culture medium
was removed and reserved. The cells were then re-suspended in the required volume of fresh
MEM (including serum) and dosed with 1.0 ml of the appropriate vehicle control, test material
solution or 0.1 ml of positive control solution. The total volume for each culture was a nominal
10 ml. After 4 hours at 37°C, the cultures were centrifuged the treatment medium was removed by
suction and replaced with an 8 ml wash of MEM culture medium. After a further centrifugation
the wash medium was removed by suction and replaced with the reserved original culture
medium. The cells were then returned to the incubator for a further 20 hours.
In Experiment 2, in the absence of metabolic activation, the exposure was continuous for 24
hours. Therefore, when the cultures were established the culture volume was a nominal 9.0 ml.
After approximately 48 hours incubation the cultures were removed from the incubator and dosed
with 1.0 ml of vehicle control, test material dose solution or 0.1 rnl positive control solution. The
nominal final volume of each culture was 10 ml. The cultures were then incubated at 37°C for 24
hours.

Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures using a 4-hour exposure time with and
without metabolic activation followed by a 20-hour recovery period, and a continuous exposure of
24 hours without metabolic activation. The dose range of test material used was
13.28 to 3400 ug/ml. Parallel flasks, containing culture medium without whole blood, were
established for the three exposure conditions so that test material precipitate observations could be
made. Precipitate observations were recorded at the beginning and end ofthe exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each
treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic
index data was used to estimate test material toxicity and for selection of the dose levels for the
main study.

Experiment 1
i) 4-hour exposure to the test material without S9-mix followed by 20-hour culture in treatmentfree
media prior to cell harvest.
ii) 4-hour exposure to the test material with S9-mix followed by 20-hour culture in treatmentfree
media prior to cell harvest.

Experiment 2
i) 24-hour continuous exposure to the test material without S9-mix prior to cell harvest.
ii) 4-hour exposure to the test material with S9-mix followed by 20-hour culture in treatmentfree
media prior to cell harvest.

Cell Harvest
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 ug/ml) two hours before the
required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture
medium was drawn off and discarded, and the cells resuspended in 0.075M hypotonic KCl. After
approximately fourteen minutes (including centrifugation), most of the hypotonic solution was
drawn off and discarded. The cells were resuspended and then fixed by dropping the KCI cell
suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least
three times and the cells stored at 4°C for at least four hours to ensure complete fixation.

Preparation of Metaphase Spreads
The lymphocytes were resuspended in several ml of fresh fixative before centrifugation and
resuspension in a small amount of fixative. Several drops of this suspension were dropped onto
clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the
appropriate identification data.

Staining
When the slides were dry they were stained in 5% Gurrs Giemsa for 5 minutes, rinsed, dried and
coverslipped using mounting medium.

Evaluation criteria:

The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test material. These observations were used to select the dose levels for mitotic index evaluation.

The slides were coded using a computerised random number generator. A total of 2000 lymphocyte cell nuclei were counted where possible and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing (Appendix 1). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Non-toxic in +S9 exposure groups but toxic in the 24 h continuous exposure group.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1: Mitotic Index - Preliminary Toxicity Test

4-HOUR TREATMENT, 20-HOUR RECOVERY -S9

4-HOUR TREATMENT, 20-HOUR RECOVERY +S9

24-HOUR TREATMENT -S9

Concentration (ug/ml)	4(20) hours without S9				4(20)h with S9			24h without S9,
µg/ml	Mitotic Index			% of Control	Mitotic Index	% of Control.		Mitotic Index	% of Control
0	5.05			100	4.65	100		6.05	100
13.28	-			-	-	-		-	-
26.56	-			-	-	-		-	-
53.13	-			-	-	-		-	-
106.25	-			-	-	-		-	-
212.5	-			-	-	-		-	-
425	-			-	-	-		5.95	98
850	5.00			99	4.45	96		3.10	51
1700	7.25			144	5.25	113		2.19	36
3400	4.80			95	5.05	109		NM	0

- = Not assessed for mitotic index

NM = No scorable metaphases

.

Table 2: Mitotic Index - Experiment 1:

Dose level	4 hours treatment without S9				4 hours treatment with S9
µg/ml	A	B	mean	% of control	A	B	mean	% of control
0	8.35	6.25	7.30	100	5.20	7.60	6.40	100
425	-	-	-	-	-	-	-	-
850	5.05	5.20	5.13	70	5.55	6.90	6.23	97
1700	5.50	7.25	6.38	87	3.90	3.45	3.68	57
3400	9.25	5.10	7.18	98	4.20	4.50	4.35	68
MMC 0.4	1.35	1.50	1.43	20	NA	NA	NA	NA
CP 5	NA	NA	NA	NA	1.25	1.20	1.23	19

MMC = Mitomycin C

CP = Cyclophosphamide

NA = Not applicable

- = Not assessed for mitotic index

.

Table 3: Mitotic Index - Experiment 2:

Dose level	4 hours treatment without S9				4 hours treatment with S9
µg/ml	A	B	mean	% of control	A	B	mean	% of control
0	4.45	9.50	6.98	100	4.75	5.25	5.00	100
425	-	-	-	-	-	-	-	-
850	3.80	5.40	4.60	66	3.85	5.95	4.90	98
1700	3.40	2.90	3.15	45	4.05	4.90	4.48	90
3400	0.75	0.95	0.85	12	4.80	5.15	4.98	100
MMC 0.2	1.45	1.50	1.48	21	NA	NA	NA	NA
CP 5	NA	NA	NA	NA	1.65	1.45	1.55	31

CP = Cyclophosphamide

MMC Mitomycin C

NA = Not applicable

- = Not assessed for mitotic index

.

Table 4: Results of the Chromosome Aberration Test - Experiment 1 without metabolic Activation (S9)

Treatment group [µg/ml]	Replicate	Mitotic Index	Number of cells Scored	Number of aberrations						Total Number of Aberrations		Frequency of Aberrant Cells [%]
				Gaps	Chromatid		Chromosome
					Breaks	Exchanges	Breaks	Exchanges	Others X	+Gaps	-Gaps	+Gaps	-Gaps
Vehicle control	A	8.35	100	1	0	1	0	0	0	2	1	2	1
	B	6.25	100	3	0	0	0	0	0	3	0	3	0
	total		200	4	0	0	1	0	0	5	1	5	1
		(100)										(2.5)	(0.5)
850	A	5.05	100	0	0	0	0	0	0	0	0	0	0
	B	5.20	100	2	0	0	0	0	0	2	0	2	0
	total		200	2	0	0	0	0	0	2	0	2	0
		(70)										(1.0)	(0.0)
1700	A	5.50	100	1	0	0	1	0	0	2	1	2	1
	B	7.25	100	0	0	0	0	0	0	0	0	0	0
	total		200	1	0	0	1	0	0	2	1	2	1
		(87)										(1.0)	(0.5)
3400	A	9.25	100	0	0	0	0	0	0	0	0	0	0
	B	5.10	100	0	0	0	0	0	0	0	0	0	0
	total		200	0	0	0	0	0	0	0	0	0	0
		(98)										(0.0)	(0.0)
positive control MMC 0.4	A	1.35	50a	8	40	39	5	0	0	92	84	36	36
	B	1.50	50a	11	34	42	2	0	1	90	79	37	34
	total		100	19	74	81	7	0	1	182	163	73	70***
		(20)										73.0)	(70.0)

MMC = Mitomycin C

a = Slide evaluation terminated at 50 cells because approximately 50% cells with aberrations had been observed

*** = p < 0.001

.

Table 5: Results of Chromosome Abberation test Experiment 1 with metabolic activation (S9)

Treatment group [µg/ml]	Replicate	Mitotic Index	Number of cells Scored	Number of aberrations						Total Number of Aberrations		Frequency of Aberrant Cells [%]
				Gaps	Chromatid		Chromosome
					Breaks	Exchanges	Breaks	Exchanges	Others X	+Gaps	-Gaps	+Gaps	-Gaps
Vehicle control	A	5.20	100	0	0	0	0	0	0	0	0	0	0
	B	7.60	100	1	0	0	0	0	0	1	0	1	0
	total		200	1	0	0	0	0	0	1	0	1	0
		(100)										(0.5)	(0.0)
850	A	5.55	100	1	0	0	4	0	0	5	4	2	1
	B	6.90	100	3	0	0	2	0	0	5	2	4	1
	total		200	4	0	0	6	0	0	10	6	6	2
		(97)										(3.0)	(1.0)
1700	A	3.90	100	0	0	0	0	0	0	0	0	0	0
	B	3.45	100	0	1	0	1	0	0	2	2	2	2
	total		200	0	1	0	1	0	0	2	2	2	2
		(57)										(1.0)	(1.0)
3400	A	4.20	100	0	0	0	0	0	0	0	0	0	0
	B	4.50	100	1	0	0	0	0	0	1	0	1	0
	total		200	1	0	0	0	0	0	1	0	1	0
		(68)										(0.5)	(0.0)
positive control CP 5	A	1.25	50a	10	19	21	1	0	0	51	41	26	22
	B	1.20	500a	10	24	9	2	0	0	45	35	22	20
	total		100	20	43	30	3	0	0	96	76	48	42***
		(19)										(48.0)	(42.0)

CP = Cyclophosphamide

a = Slide evaluation terminated at 50 cells because approximately 50% cells with aberrations had been observed

*** = P < 0.001

.

Table 6:Results of the Chromosome Aberration Test - Experiment 2 without metabolic Activation

Treatment group [µg/ml]	Replicate	Mitotic Index	Number of cells Scored	Number of aberrations						Total Number of Aberrations		Frequency of Aberrant Cells [%]
				Gaps	Chromatid		Chromosome
					Breaks	Exchanges	Breaks	Exchanges	Others X	+Gaps	-Gaps	+Gaps	-Gaps
Vehicle control	A	4.45	100	1	0	0	0	0	0	1	0	1	0
	B	9.50	100	0	0	0	0	0	0	0	0	0	0
	total		200	1	0	0	0	0	0	1	0	1	0
		(100)										(0.5)	(0.0)
850	A	3.80	100	0	0	0	0	0	0	0	0	0	0
	B	5.40	100	1	0	0	0	1	0	2	1	2	1
	total		200	1	0	0	0	1	0	2	1	2	1
		(66)										(1.0)	(0.5)
1700	A	3.40	100	4	0	0	0	1	0	5	1	5	1
	B	2.90	100	3	1	0	0	0	0	4	1	4	1
	total		200	7	1	0	0	1	0	9	2	9	2
		(45)										(4.5)	(1.0)
3400	A	0.75	91b	0	1	0	0	0	0	1	1	1	1
	B	0.95	100	0	1	0	0	0	0	1	1	1	1
	total		191	0	2	0	0	0	0	2	2	2	2
		(12)										(1.0)	(1.0)
positive control MMC 0.2	A	1.45	50a	20	15	10	2	0	0	47	27	29	19
	B	1.50	50a	10	21	10	0	0	0	41	31	24	22
	total		100	30	36	20	2	0	0	88	58	53	41***
		(21)										(53.0)	(41.0)

MMC = Mitomycin C

a = Slide evaluation terminated at 50 cells because approximately 50% cells with aberrations had been observed

b = insufficient metaphases to finish

*** = P < 0.001

.

Table 7:Results of the Chromosome Aberration Test - Experiment 2 with metabolic Activation

Treatment group [µg/ml]	Replicate	Mitotic Index	Number of cells Scored	Number of aberrations						Total Number of Aberrations		Frequency of Aberrant Cells [%]
				Gaps	Chromatid		Chromosome
					Breaks	Exchanges	Breaks	Exchanges	Others X	+Gaps	-Gaps	+Gaps	-Gaps
Vehicle control	A	4.75	100	3	1	0	0	0	0	4	1	4	1
	B	5.25	100	3	0	0	2	0	0	5	2	5	2
	total		200	6	1	0	2	0	0	9	3	9	3
		(100)										(4.5)	(1.5)
850	A	3.85	100	0	0	0	0	0	0	0	0	0	0
	B	5.95	100	2	2	1	1	0	0	6	4	5	3
	total		200	2	2	1	1	0	0	6	4	5	3
		(98)										(2.5)	(1.5)
1700	A	4.05	100	0	0	0	0	0	0	0	0	0	0
	B	4.90	100	0	0	0	0	0	0	0	0	0	0
	total		200	0	0	0	0	0	0	0	0	0	0
		(90)										(0.0)	(0.0)
3400	A	4.80	100	0	0	0	0	0	0	0	0	0	0
	B	5.15	100	2	0	0	0	0	0	2	0	2	0
	total		200	2	0	0	0	0	0	2	0	2	0
		(100)										(1.0)	(0.0)
positive control CP 5	A	1.65	100	16	13	4	2	0	0	35	19	30	18
	B	1.45	100	9	9	4	3	0	0	25	16	21	15
	total		200	25	22	8	5	0	0	60	35	51	33***
		(31)										(25.5)	(16.5)

CP = Cyclophosphamide

*** =P<0.00I

.

Table 8: Mean Frequency of Polyploid Cells - Experiment 1:

Harvest time (24h)

Dose [µg/ml]	with S9 (4 hrs)	without S) (4 hrs)
0	0.0	0.0
850	0.0	0.0
1700	0.0	0.0
3400	0.0	0.0
MMC 0.4	0.0	NA
CP 5	NA	0.0

MMC = Mitomycin C

CP = Cyclophosphamide

NA = Not applicable

.

Table 8: Mean Frequency of Polyploid Cells - Experiment 2:

Harvest time (24h)

Dose [µg/ml]	with S9 (4 hrs)	without S) (4 hrs)
0	0.0	0.0
850	1.0	0.0
1700	0.0	0.0
3400	0.5	0.0
MMC 0.2	0.0	NA
CP 5	NA	0.0

MMC = Mitomycin C

CP = Cyclophosphamide

NA = Not applicable

Conclusions:

The substance was negative in the chromosome aberration test employing cultured human lymphocytes, either in the presence or absence of a metabolic activation system (rat liver S9).

Executive summary:

The results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells are described. Duplicate cultures of human lymphocytes, treated with the substance, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, ie. in Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The substance was non-toxic in the pulse exposure groups but was toxic in the 24 hours continuous exposure group and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that was the 10mM maximum recommended dose level, 3400 ug/ml.

In conclusion, the substance was considered to be non-clastogenic to human lymphocytes in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Read-across to K1 study therefore K2 is the maximum Klimisch value.

Justification for type of information:

Read-across approach - see read-across justification in section 13.

Reason / purpose for cross-reference:

read-across source

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Type and identity of media: PAA Ready Mix (2 % FBS)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced male rat liver S9 mix

Test concentrations with justification for top dose:

initial assay and independent repeat assay: 400, 800, 1200, 1600, 2000, 2400, 2800 µg/ml (-S9) and 42, 84, 168, 336, 672, 1344, 2688 µg/ml (+S9)

Vehicle / solvent:

Vehicle(s)/solvent(s) used: ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethylmethanesulfonate (EMS, 900 µg/ml, -S9); dimethylbenzanthracene (DMBA, 20 µg/ml, +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 hours
- Expression time (cells in growth medium): 6-8 days

NUMBER OF REPLICATIONS: at least 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

- Mutant frequencies will only be used for assessment, if at least 5 dishes per culture were available and relative survival to treatment, relative population growth and absolute cloning efficiency were 10% or greater.
- A trial will be considered positive if a concentration-related and in parallel cultures reproducible increase in mutant frequencies is observed. To be relevant, the increase in mutant frequencies should be at least two to three times that of the highest negative or negative control value observed in the respective trial. If this result can be reproduced in a second trial, the test substance is considered to be mutagenic.
- Despite these criteria, a positive result will only be considered relevant, if no significant change in osmolality compared to the negative control can be observed. Otherwise, unphysiological culture conditions may be the reason for the positive result (Scott et al, 1991).
- A test substance will be judged as equivocal if there is no strictly concentration related increase in mutation frequencies but if one or more concentrations induce a reproducible and biologically relevant increase in mutant frequencies in all trials.
- An assay will be considered negative if no reproducible and relevant increases of mutant frequencies were observed.

Statistics:

All acceptable groups are included in the weighted analysis of variance followed by pairwise comparisons to the negative control on a nominal significance level of a =0.05 using the Dunnett test (Dunnett, 1955). The regression analysis part is performed on the basis of the actual concentrations thereby omitting the positive, untreated and negative controls. If there is a significant concentration related increase of the mutant frequency (a = 0.05) in the main analysis the highest concentration will be dropped and the analysis will be repeated. This procedure will be repeated until p > 0.05.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

2,2',2''-Nitrilotriethanol, propoxylated was evaluated for point mutagenic effects at the hypoxanthine-guanine phosphoribosyl transferase locus (forward mutation assay) in V79 cell cultures. Without S9 mix concentrations of up to and including 2800 µg/ml were employed. With S9 mix concentrations up to and including 2688 µg/ml were used. Without and with S9 mix 2,2',2''-Nitrilotriethanol, propoxylated induced no decreases in survival to treatment or in relative population growth. Precipitation of 2,2',2''-Nitrilotriethanol, propoxylated in the culture medium was not observed. However, 2,2',2''-Nitrilotriethanol, propoxylated was tested up to at least the limit concentration of 10 mM which is equal to approximately 2650 µg/ml 2,2',2''-Nitrilotriethanol, propoxylated. Without and with S9 mix there was no biologically relevant increase in mutant frequency above that of the negative controls. Ethylmethanesulfonate and dimethylbenzanthracene induced clear mutagenic effects and demonstrated the sensitivity of the test system and the activity of the S9 mix. Based on these results, 2,2',2''-Nitrilotriethanol, propoxylated is considered to be non-mutagenic in the V79/HPRT forward mutation assay, both with and without metabolic activation.

Conclusions:

The substance is considered to be non-mutagenic in the in vitro gene mutation assay (HPRT test) with Chinese hamster V79 cells in the presence and absence of a metabolic activation system.

Executive summary:

Point mutagenic effects at the hypoxanthine-guanine phosphoribosyl transferase locus (forward mutation assay) in V79 cell cultures of the substance was evaluated in the presence and absence of metabolic activation system (S9 fraction). The substance did not induce decreases in survival both in the presence and absence of a metabolic activation system at a concentrations up to 2800 µg/L (with metabolic activation) and 2688 µg/L (without metabolic activation). The substance is considered to be non-mutagenic in the in vitro gene mutation assay (HPRT test) with Chinese hamster V79 cells in the presence and absence of a metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the findings of a reliable in vitro chromosome aberration test conducted on the substance and two relaible in vitro studies (gene mutation assay (HPRT test) and chromosome aberration test) conducted on a structurally similar substance, classification of the substance is not justified.