Poly(oxy-1,2-ethanediyl),α-hydro-ω-hydroxy- Ethane-1,2-diol, ethoxylated

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test:

Gene mutation toxicity study was performed for the test chemical to evaluate its mutagenic nature. The study was performed as per the preincubation protocol using Salmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system at doses of 0, 100, 333, 1000, 3333 or 10000 µg/plate. Water was used at the vehicle. The plates were incubated for 48 hrs after 20 mins preincubation before the evaluation of the revertant colonies could be made. Concurrent solvent and negative control chemicals were also included in the study. The test chemical did notinduce mutation in theSalmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

In vitro mammalian chromosome aberration study:

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using CHEL cellsin the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 0, 2, 3, 4.5, 6 or 8 mM. Concurrent solvent, untreated and positive control chemicals were also included in the study. 80000 CHEL cells were seeded in 25 cm2 flasks 9-20 hrs before treatment under standard culture conditions. The test chemical treatment was performed continuously for 16 or 24 hrs until harvesting. During the last 2 hrs of culture, colcemid was added. Cells were then collected by trypsin/EDTA treatment. Treated with hypotonic solution of tri-sodium citrate and washed twice with fresh fixative, dropped onto glass slides and air dried. Slides were stained with aqueous solution of 1% Giemsa and coded for analysis. Abpout 100-200 well spread metaphase cells were scored per test point. Based on the observations made, the test chemical did not induce chromosome aberrations in CHEL cell line at dose level of 2 mM. It however induced gene mutation in CHEL cells in the presence and absence of S9 metabolic activation system above 2 mM.

In vitro mammalian cell gene mutation assay:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the test chemical when administered to Chinese Hamster Ovary (CHO) cells. In the genotoxicity test, the test chemical was administered to CHO cells for 3 hrs at the dose levels of 0.5, 1.0, 2.5 or 5.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such asN-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. The positive control ENU showed indication of gene mutations occurring while no other treatment gave rise to gene toxicity. When the mutation frequency was determined, a frequency of 3.79 x 10^-4was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction. Since no tested concentration of Polyethylene glycol, in the absence or presence of S9 liver microsomal fraction, resulted in colonies, we conclude that Polyethylene glycol does not give rise to gene mutations when CHO cells are exposedin vitroto the test chemical at 0, 0.5, 1.0, 2.5 or 5.0 mM for 3 hrs.Based on the results of the current study, it is concluded that the test chemical does not give rise to gene mutations when CHO cells are exposed to the test chemicalin vitroat 0, 0.5, 1.0, 2.5 or 5.0 mM for 3 hrs, in the presence or abscence of metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Gene mutation toxicity study was performed for the test chemical to evaluate its mutagenic nature

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Male Sprague-Dawley rats and male Syrian hamsters were routinely used for the S9 preparation of the liver fractions

Test concentrations with justification for top dose:

0, 100, 333, 1000, 3333 or 10000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: The test chemical was soluble in water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

For strains tested with S9

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

For strains TA100 and TA1535 tested in the absence of S9

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

For strain TA1537 tested in the absence of S9

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

For strain TA98 tested in the absence of S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hr
- Expression time (cells in growth medium): 48 hr
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: At least five dose levels of the chemicals were tested, with three plates per dose level.

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

1) mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold;
2) nomutagenic response: when no increase in the number of revertants was elicited by the chemical;
3) questionable response: when there was an absence of a clear-cut dose-related increase in revertants; when the dose-related increases in the number of revertants were not reproducible; or when the response was of insufficient magnitude to support a determination of mutagenicity

Statistics:

Mean and Standard error of mean

Species / strain:

S. typhimurium, other: TA100, TA98, TA1535 and TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: The chemical was initially tested with strain TA100 in the presence and the absence of the metabolic activation systems, over a wide dose range with an upper limit of 10 mg/plate, or less when solubility problems were encountered. Toxicity was evidenced by one or more of the following phenomena: appearance of his+ pinpoint colonies, reduced numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn. Nontoxic chemicals were tested in the initial experiment up to the 10 mg/plate dose level, or to a level determined by their solubility. Toxic chemicals were tested up to a high dose which exhibited some degree of toxicity.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No mutagenic potential

 Strain: TA100

Dose	No Activation  (Negative)		No Activation  (Negative)		10% HLI  (Negative)		10% HLI  (Negative)		10% RLI  (Negative)		10% RLI  (Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
ug/Plate	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	120	11.5	115	5.8	105	9.3	105	3	154	3.4	107	4.7
100	127	9	153	4.3	150	13.3	135	3.5	128	1.5	128	17.1
333	128	6	155	12.1	146	12	109	3.6	138	6.4	136	14.4
1000	132	3.7	150	8.1	143	6	140	10.1	125	5.5	116	6.4
3333	132	7.7	141	7.4	137	17.7	137	3.3	129	10.2	120	7.5
10000	119	5.3	141	9.5	137	5.4	127	2.2	155	4.8	137	7.6
Positive Control	422	16.8	330	26	735	17.1	1359	38.4	445	37.5	526	25.3

 Strain: TA1535

Dose	No Activation  (Negative)		No Activation  (Negative)		10% HLI  (Negative)		10% HLI  (Negative)		10% RLI  (Negative)		10% RLI  (Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
ug/Plate	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	33	3.9	30	1.2	11	3.5	9	1.5	12	0.3	14	3.5
100	32	3.7	48	4.7	11	2.1	18	1.8	15	2.8	17	1.2
333	29	2	48	4.7	8	0.6	15	0.7	14	1.7	11	2
1000	29	7.2	47	1.3	16	1.2	17	0.7	13	3.3	13	2.2
3333	33	2.7	41	7.5	12	3.5	17	2.6	11	3.4	13	2.6
10000	32	2.6	54	7.7	12	2.6	17	0.7	14	2.3	6	1.9
Positive Control	452	44.6	292	10	379	23.6	447	18.5	157	13	188	17.4

Conclusions:

The test chemical did not induce mutation in the Salmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Executive summary:

Gene mutation toxicity study was performed for the test chemical to evaluate its mutagenic nature. The study was performed as per the preincubation protocol using Salmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system at doses of 0, 100, 333, 1000, 3333 or 10000 µg/plate. Water was used at the vehicle. The plates were incubated for 48 hrs after 20 mins preincubation before the evaluation of the revertant colonies could be made. Concurrent solvent and negative control chemicals were also included in the study. The test chemical did notinduce mutation in theSalmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical

GLP compliance:

not specified

Type of assay:

other: In vitro mammalian chromosome aberration study

Target gene:

No data

Species / strain / cell type:

mammalian cell line, other: CHEL cells

Details on mammalian cell type (if applicable):

- Type and identity of media: William’s medium supplemented with 10% fetal calf serum and antibiotics
- Properly maintained: No data
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: The cell line has a stable aneuploidy karyotype with modal no. of chromosome of 24 and 21
- Periodically "cleansed" against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction was isolated from young male rats following mixed with β-naphthoflavone and phenobarbital

Test concentrations with justification for top dose:

0, 2, 3, 4.5, 6 or 8 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Untreated negative controls:

yes

Remarks:

Untreated cells

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
Cell at the start of experiment: 80000

DURATION
- Preincubation period:
- Exposure duration: Continuous treatment: 16 hrs
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS:

NUMBER OF CELLS EVALUATED: 100-200 well spread metaphase per test point. Only metaphases with chromosome numbers that differed from the modal numbers by no more than two were considered.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Yes, cytotoxicity was assessed as a reduction in mitotic indices and the confluence status of the cultures

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

The cell line was observed for chromosome breakages, gaps and exchanges

Statistics:

Statistical analysis of data was performed with Fischer’s exact test, accepting P<0.05 as significant

Species / strain:

mammalian cell line, other: CHEL cells

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

above 2 mM

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

13.3% at 8mM

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

mammalian cell line, other: CHEL cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

at 2 mM

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No data

Remarks on result:

other: No mutagenic potential

Table: Mutagenic effects of the test chemical

Dose (mM)	n	Aberrations/100 cells		Aberrant cells	Relative MI (%)
		Chromatid	Chromosome
0	200	2.0	0.5	2.5	100
2.0	150	3.3	2.0	4.7	92
3.0	250	5.5	4.5	8.0b	83
4.5	150	6.7	2.7	8.0a	84
6.0	150	9.3	4.2	11.3a	76
8.0	150	13.3	4.7	13.3a	67
DMBA	100	19.0	6.5	19.0a	42
DMSO	200	3.0	1.0	4.0	102

a Statistically significant at P<0.001 (Fischer’s exact test)

b Statistically significant at P<0.01 (Fischer’s exact test)

Conclusions:

The test chemical did not induce chromosome aberrations in CHEL cell line at dose level of 2 mM. It however induced gene mutation in CHEL cells in the presence and absence of S9 metabolic activation system above 2 mM.

Executive summary:

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using CHEL cellsin the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 0, 2, 3, 4.5, 6 or 8 mM. Concurrent solvent, untreated and positive control chemicals were also included in the study. 80000 CHEL cells were seeded in 25 cm2 flasks 9-20 hrs before treatment under standard culture conditions. The test chemical treatment was performed continuously for 16 or 24 hrs until harvesting. During the last 2 hrs of culture, colcemid was added. Cells were then collected by trypsin/EDTA treatment. Treated with hypotonic solution of tri-sodium citrate and washed twice with fresh fixative, dropped onto glass slides and air dried. Slides were stained with aqueous solution of 1% Giemsa and coded for analysis. Abpout 100-200 well spread metaphase cells were scored per test point. Based on the observations made, the test chemical did not induce chromosome aberrations in CHEL cell line at dose level of 2 mM. It however induced gene mutation in CHEL cells in the presence and absence of S9 metabolic activation system above 2 mM.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

The purpose of this study was to assess toxic and genotoxic effects of the test chemical on Chinese Hamster Ovary (CHO) cells by using several different in vitro-based assays, including genotoxicity tests based on the OECD Guideline No. 476 “In Vitro Mammalian Cell Gene Mutation Test”.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot.

This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.
The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.
HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.

Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable

Additional strain / cell type characteristics:

other: Hypodiploid, modal No. 20

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

0, 0.5, 1.0, 2.5 or 5.0 mM

Vehicle / solvent:

Vehicle(s)/solvent(s) used: Water
Justification for choice of solvent/ vehicle: Polyethylene glycol was easily dissolved in water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

N-ethyl-N-nitrosourea (ENU) was the positive control substance in the tests done without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium with pre-incubation

DURATION
- Pre-incubation: One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.
- Exposure duration: 3 hours
- Expression time: 7 days
- Selection time: 14 days
- Fixation time: 7 days (harvest of cells)

SELECTION AGENT (mutation assays): 6-thioguanine (TG)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Crystal violet

NUMBER OF REPLICATIONS: A minimum of 2 replicates per dose concentration including negative and positive control.

NUMBER OF CELLS EVALUATED: 5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated.

DETERMINATION OF CYTOTOXICITY
- Method: After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.

OTHER EXAMINATIONS: Not applicable
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:

Rationale for test conditions:

No data

Evaluation criteria:

The cell line was observed for gene mutations

Statistics:

No data

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No data

Remarks on result:

other: No mutagenic potential

Table 1A. Effects of the test chemical exposure on gene toxicity in CHO cells. After being exposed to the test chemical for 3 hrs, cells was washed with sterile PBS and then incubated for 7 days at 37°C, 5% CO₂. After 7 days, cells were re-seeded in new 6-well plates in the absence or presence of 10mM TG as a selection agent and returned to the incubator for 14 days at 37°C, 5% CO₂. On day 15, all 6-well plates were stained with crystal violet and the number of colonies were counted manually. The results are presented as the total number of colonies found in the number of independent wells analyzed (e.g. 0 colonies in 4 wells will give 0/4) (n = 2 samples from 2 independent cultures).

 

	With S9		Without S9
	with TG	without TG	with TG	without TG
Neg. control	0/4	593/4	0/4	667/4
Pos. control	0/4	688/4	26/4	557/4
0.5 mM	0/4	543/4	0/4	580/4
1.0 mM	0/4	605/4	0/4	549/4
2.5 mM	0/4	696/4	0/4	600/4
5.0 mM	0/4	570/4	0/4	506/4

 

 

 

 

Table 1B. Mutation frequency in CHO cells after 3 hrs of exposure to test chemical in the absence or presence of 4% S9 liver microsomal fraction. N/A, no colonies present in the samples selected with TG, i.e. no mutation frequency could be determined.

 

	With S9	Without S9
Neg. control	N/A	N/A
Pos. control	N/A	3.79x10^-4
0.5 mM	N/A	N/A
1.0 mM	N/A	N/A
2.5 mM	N/A	N/A
5.0 mM	N/A	N/A

Conclusions:

The test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence or abscence of metabolic activation.

Executive summary:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the test chemical when administered to Chinese Hamster Ovary (CHO) cells.

In the genotoxicity test, the test chemical was administered to CHO cells for 3 hrs at the dose levels of 0.5, 1.0, 2.5 or 5.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test.

The positive control ENU showed indication of gene mutations occurring while no other treatment gave rise to gene toxicity.

When the mutation frequency was determined, a frequency of 3.79 x 10^-4was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction. Since no tested concentration of Polyethylene glycol, in the absence or presence of S9 liver microsomal fraction, resulted in colonies, we conclude that Polyethylene glycol does not give rise to gene mutations when CHO cells are exposed in vitro to the test chemical at 0, 0.5, 1.0, 2.5 or 5.0 mM for 3 hrs.

Conclusion

Based on the results of the current study, it is concluded that the test chemical does not give rise to gene mutations when CHO cells are exposed to the test chemical in vitro at 0, 0.5, 1.0, 2.5 or 5.0 mM for 3 hrs, in the presence or abscence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Data available for the test chemical was reviewed to determine its mutagenic nature. The studies are as mentioned below:

Ames test:

Gene mutation toxicity study was performed for the test chemical to evaluate its mutagenic nature. The study was performed as per the preincubation protocol using Salmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system at doses of 0, 100, 333, 1000, 3333 or 10000 µg/plate. Water was used at the vehicle. The plates were incubated for 48 hrs after 20 mins preincubation before the evaluation of the revertant colonies could be made. Concurrent solvent and negative control chemicals were also included in the study. The test chemical did notinduce mutation in theSalmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

In vitro mammalian chromosome aberration study:

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using CHEL cellsin the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 0, 2, 3, 4.5, 6 or 8 mM. Concurrent solvent, untreated and positive control chemicals were also included in the study. 80000 CHEL cells were seeded in 25 cm2 flasks 9-20 hrs before treatment under standard culture conditions. The test chemical treatment was performed continuously for 16 or 24 hrs until harvesting. During the last 2 hrs of culture, colcemid was added. Cells were then collected by trypsin/EDTA treatment. Treated with hypotonic solution of tri-sodium citrate and washed twice with fresh fixative, dropped onto glass slides and air dried. Slides were stained with aqueous solution of 1% Giemsa and coded for analysis. Abpout 100-200 well spread metaphase cells were scored per test point. Based on the observations made, the test chemical did not induce chromosome aberrations in CHEL cell line at dose level of 2 mM. It however induced gene mutation in CHEL cells in the presence and absence of S9 metabolic activation system above 2 mM.

In vitro mammalian cell gene mutation assay:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the test chemical when administered to Chinese Hamster Ovary (CHO) cells. In the genotoxicity test, the test chemical was administered to CHO cells for 3 hrs at the dose levels of 0.5, 1.0, 2.5 or 5.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such asN-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. The positive control ENU showed indication of gene mutations occurring while no other treatment gave rise to gene toxicity. When the mutation frequency was determined, a frequency of 3.79 x 10^-4was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction. Since no tested concentration of Polyethylene glycol, in the absence or presence of S9 liver microsomal fraction, resulted in colonies, we conclude that Polyethylene glycol does not give rise to gene mutations when CHO cells are exposed in vitro to the test chemical at 0, 0.5, 1.0, 2.5 or 5.0 mM for 3 hrs.Based on the results of the current study, it is concluded that the test chemical does not give rise to gene mutations when CHO cells are exposed to the test chemical in vitro at 0, 0.5, 1.0, 2.5 or 5.0 mM for 3 hrs, in the presence or abscence of metabolic activation.

Based on the data available for the target chemical, the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to be genetoxic in vitro as per the criteria mentoned in CLP regulation.

Justification for classification or non-classification

Based on the data available for the target chemical, the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to be genetoxic in vitro as per the criteria mentoned in CLP regulation.