1,3-Propanediol, 2,2-bis(hydroxymethyl)-, allyl ether

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A bacterial mutation test, chromosome aberration assay and mammalian mutation assay are available for the submission substance. The data from the mouse lymphoma assay an increase in the proportion of small colonies, consistent with chromosomal effects; this positive response was seen in the presence of metabolic activation. Although the clastogenicity assay reports a negative result, it is notable that there is some indication of activity at the highest concentration in the presence of metabolic activation (not considered by the Study Director to be of biological significance). The two studies therefore indicate clastogenic activity for 1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether which should be investigated in a suitable study in vivo.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

16 June 1993 to 21 October 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA (TSCA)

Version / remarks:

Guideline number not specified

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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:

Aroclor 1254 induced S-9

Test concentrations with justification for top dose:

312.5, 625, 1250, 2500 and 5000 µg/plate in the preliminary test
8, 40, 200, 1000 and 5000 µg/plate in the first main experiment
312.5, 625, 1250, 2500 and 5000 µg/plate in the second main experiment
The highest concentration was the maximum concentration in the OECD guideline.

Vehicle / solvent:

Dimethyl sulphoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

A preliminary study was conducted in strains TA100 and WP2 uvrA in the absence of S-9. The two main experiments were conducted with all tester strains in the absence and in the presence of S-9. The bacteria were grown overnight for 10 hours at 37 °C. Aliquots of the bacterial culture (0.1 mL) were added to 0.1 test solution (vehicle control, positive control or test article), 0.5 mL of buffer (treatments in the absence of S-9) or S-9 (for treatments in the presence of S-9) and 2 mL of agar (containing trace amounts of histidine for salmonella strains or tryptophan for the E. coli strain), mixed and poured onto Vogel Bonner plates. The plates were incubated at 38 °C for 48 hours. The plates were then scored for revertant colonies. The preliminary test was plated in duplicate; the main experiments were plated in triplicate.

Evaluation criteria:

For a test substance to be considered positive, a concentration-related and statistically significant increase in the the number of revertants of at least twice the concurrent control should have been observed in at least one strain in either the absence or presence of S-9 in both experiments. Where the results of experiments gave conflicting results, an additional test would be required.

Statistics:

Not stated

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:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

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:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

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:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

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:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

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:

not examined

Positive controls validity:

valid

Additional information on results:

None

Results: Experiment 1 in the absence of S-9

Concentration of test material	Mean number of revertants
	TA98	TA00	TA1535	TA1537	WP2 uvrA
0	22.7	143.3	17.7	4.7	29.0
8	20.7	129.3	23.3	4.3	25.7
40	29.3	118.0	22.3	5.3	23.3
200	26.0	136.0	17.7	4.7	21.3
1000	37.1	125.0	19.0	4.7	220
5000	31.0	154.3	14.3	4.7	20.0
Positive control	161.3	523.7	249.7	347.7	280.0

 

Results: Experiment 1 in the presence of S-9

Concentration of test material	Mean number of revertants
	TA98	TA00	TA1535	TA1537	WP2 uvrA
0	25.7	139.0	25.3	12.7	26.3
312.5	23.7	130.7	22.7	10.7	23.3
625	24.0	146.0	25.3	8.0	20.0
1250	15.0	142.7	19.3	10.7	23.7
2500	27.3	142.0	18.0	9.3	24.3
5000	24.3	133.0	13.7	7.7	23.0
Positive control	165.3	407.7	161.0	108.0	242.0

 

Results: Experiment 2 in the absence of S-9

Concentration of test material	Mean number of revertants
	TA98	TA00	TA1535	TA1537	WP2 uvrA
0	20.3	110.0	17.0	14.0	28.7
312.5	15.3	118.3	16.0	11.3	22.0
625	15.7	103.7	15.3	10.0	23.0
1250	20.7	99.7	17.3	9.7	19.0
2500	17.3	114.7	16.7	10.3	23.3
5000	14.7	101.3	22.3	8.7	20.0
Positive control	143.0	533.3	152.3	591.0	321.7

 

Results: Experiment 2 in the presence of S-9

Concentration of test material	Mean number of revertants
	TA98	TA00	TA1535	TA1537	WP2 uvrA
0	26.0	148.0	28.7	9.0	27.7
8	23.3	111.7	26.0	10.0	27.3
40	24.7	95.3	22.3	7.7	22.3
200	20.0	94.3	27.3	10.0	24.0
1000	19.0	100.3	22.3	8.3	25.7
5000	23.7	93.7	18.3	9.7	27.0
Positive control	154.7	407.7	169.7	134.0	259.3

 

 

Conclusions:

1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether showed no evidence of mutagenicity in the absence and in the presence of metabolic activation when tested up to 5000 µg/plate in strains TA98, TA100, TA535, TA1537 and WP2 uvrA.

Executive summary:

An Ames test was conducted with 1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether according to OECD guideline 471. Testing was conducted in Salmonella typhmurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2 uvrA in the absence and presence of metabolic activation. Two independent tests were conducted up to 5000 µg/plate. There was no evidence of toxicity, precipitation or increases in the number of revertants which were 2 -fold the concurrent controls. The overall conclusion of the study is that the test material has shown no evidence of mutagenicity when tested up to the maximum concentration of 5000 µg/plate.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

16 September 1994 to 01 December 1995

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

RCC (Number of cells in treated cultures/ Number of cells in control cultures) was used as cytotoxicity parameter. Concentrations scored for aberrations were not sufficient cytotoxic ( RCC based) for treatment group in the absence of S-9. In the presence of S-9, equivocal increases in aberrations seen, whch were not statistically significant, were not checked for reproducibility. Only 200 metaphases were scored per concentration.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

RCC was used as cytotoxicity parameter. Concentrations were not sufficient cytotoxic in the absence of S-9. In the presence of S-9, equivocal increases in aberrations seen were not checked for reproducibility. 200 metaphases were scored per concentration.

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Chromosome aberration test

Species / strain / cell type:

mammalian cell line, other: Chinese Hamster Lung

Details on mammalian cell type (if applicable):

Isolated by Koyama et al, 1970
Cloned by Ishidate and Sofuni (1985)
Supplier: Japanese Cancer Research Resouces Bank-Cell Bank
Media: Eagles Minimal Essential medium with Earle's salts, supplemented with 10% foetal calf serum and antibiotics
Culture conditions: 37°C; 5% Carbon dixoide in air

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Preliminary test:
19.5, 39, 78.1, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL for 6 hours with 18 hours recovery in the absence and in the presence of S-9, 24 hours and 48 hours in the absenceof S-9 with no recovery.

Main test:
39, 78.1, 156.25, 312.5 and 468.75 µg/mL for 24 hours in the absence of S-9 with no recovery
39, 78.1, 156.25, 234.38 and 312.5 µg/mL for 48 hours in the absence of S-9 with no recovery
78.1, 156.25, 312.5, 468.75 µg/mL for 6 hours in the absence of S-9 with 18 hours recovery - not scored for aberrations due to toxicity; treatment repeated with adjusted concentrations
9.75, 19.5, 39 and 58.6 µg/mL for 6 hours in the presence of S-9 with 18 hours recovery - not scored for aberrations due to toxicity; treatment repeated with adjusted concentrations

Repeat main test
40, 80, 160 and 240 µg/mL for 6 hours in the absence of S-9 with 18 hours recovery
5, 10, 20 and 30 µg/mL for 6 hours in the presence of S-9 with 18 hours recovery

Vehicle / solvent:

Dimethyl sulphoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Cyclophosphamide was used as the positive control for the 6 hour treatments in both the absence and presence of S-9, but it requires metabolic activation.

Details on test system and experimental conditions:

CHL cultures were established 24 hours prior to teratment at concentrations of 0.4 x 10^6 cells (6 hour and 24 hour exposure) or 0.15 x 10^6 cells (48 hour exposure) per flask. The cells were exposured to five concentrations of the test material, or vehicle or positive control in the absence and in the presence of metabolic activation. Treatments were performed in duplicate. The treatment regimes were as follows:
6 hours in the absence and presence of S-9 with 18 hours recovery
24 hours continuous treatment in the absence of S-9
48 hours continuous treatment in the absence of S-9
Two hours prior to harvest, mitosis was arrested by the addition of colcemd (0.1 µg/mL). The cells were removed from the flask with trypsin and the suspended in culture medium. A sample of cell suspension was used to determine growth inhibition. The cells were centrifuged and the supernatant removed. The cells were resuspended in 0.075M potassium chloride. After 15 minutes (including 5 minutes centrifugation), the potassium chrloride was removed and the cells were resuspended and then fixed with 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 4 hours prior to slide preparation.
Slides were prepared by resuspending the cells in fresh fixatine and then dropping the cell suspension on to microscope slides and allowed to air dry. The slides were stained with 2% Giemsa for 5 minutes, rinsed, dried and coverslipped.
The first 100 metaphases frome each cutlure were scored for aberrations (gaps, breaks and exchanges based on the scoring system of Savage, 1976, as recommended by UKEMS guidelines). Scoreable metaphases were well spread and having 23 - 27 chromosomes. The incidence of aneuploid cells (27-31 chromosomes) and polyploid cells (>31 chromosomes) were also reported.

Rationale for test conditions:

Study designed based on OECD guideline 473.

Evaluation criteria:

Aberration frequencies for control cultures are commonly in the range of 0-3% cells with aberrations (Ishidate, 1987).
A positive response was considered if the percentage of aberrations (gaps included) was ≥10%; an equivocal response was considered if the percentage of aberrations was ≥5 and <10%; a negative reponse was considered if the percentage of aberrations was <5%.
For polyploid cells, an incidence of >10% was positve.
When evaulating the data, consideration was given to the frequency of chromosome exchange events which are relatively rare in control cultures and designation was based on experience and scientific judgement.

Statistics:

Statistics used, but method not reported.

Species / strain:

mammalian cell line, other: Chinese Hamster Lung cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other:

Remarks:

Highest tested concentration induced no scorable metaphases. Highest scorable concertation did not induce sufficient cytotoxicity ( RCC based).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

Cyclophosphamide was used as the positive control for the 6 hour treatments in both the absence and presence of S-9, but it requires metabolic activation. MMC was used for 24 and 48 h in the absence of S-9.

Species / strain:

mammalian cell line, other: Chinese Hamster Lung Cells

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

not determinable because of methodological limitations

Results: 24 hours in the absence of S-9 with no recovery

Concentration (µg/mL)	Replicate	Toxicity (%)	N	Number of aberrations						Total number of aberrations		Frequency of aberrations (%)		Number of polyploid cells (Percentage)
				g	Ctb	Cte	Csb	Cse	Others	Without gaps	With gaps	Without gaps	With gaps
0 (vehicle control)	A	100	100	2	0	0	0	0	0	0	2	-	-	1
	B		100	0	0	0	1	0	0	1	1	-	-	0
	Total		200	2	0	0	1	0	0	1	3	0.5	1.5	1 (0.5)
39	A	111	100	0	0	0	0	0	0	0	0	-	-	1
	B		100	2	1	0	0	0	0	1	3	-	-	2
	Total		200	2	1	0	0	0	0	1	3	0.5	1.5	3(1.5)
78.1	A	111	100	0	0	0	0	1	0	1	1	-	-	1
	B		100	1	0	0	0	0	0	0	1	-	-	1
	Total		200	1	0	0	0	1	0	1	2	0.5	1.0	2 (1.0)
156.25	A	85	100	1	0	0	1	0	0	1	2	-	-	0
	B		100	0	0	0	0	0	0	0	0	-	-	0
	Total		200	1	0	0	1	0	0	1	2	0.5	1.0	0 (0.0)
Positive control	A	103	100	2	8	19	1	2	0	24	26	-	-	1
	B		50	6	12	17	2	0	0	26	27	-	-	0
	Total		150	8	20	36	3	2	0	50	53	33.33 ***	35.3 ***	1 (0.7)

N:          Number of cells scored
g:           Gaps
Ctb:       Chromatid breaks
Cte:       Chromatid exchange
Csb:      Chromosome break
Cse:      Chromosome exchange
***       Statistically significant (p>0.001)

Results: 48 hours in the absence of S-9 with no recovery

Concentration (µg/mL)	Replicate	Toxicity (%)	N	Number of aberrations						Total number of aberrations		Frequency of aberrations (%)		Number of polyploid cells (percentage)
				g	Ctb	Cte	Csb	Cse	Others	Without gaps	With gaps	Without gaps	With gaps
0 (vehicle control)	A	100	100	0	1	0	0	0	0	1	1	-	-	0
	B		100	0	0	0	0	0	0	0	0	-	-	0
	Total		200	0	1	0	0	0	0	1	1	0.5	0.5	0 (0.0)
39	A	92	100	0	0	1	0	1	0	2	2	-	-	0
	B		100	1	0	0	0	0	0	0	1	-	-	1
	Total		200	1	0	1	0	1	0	2	3	1.0	1.5	1 (0.5)
78.1	A	90	100	0	0	0	0	0	0	0	0	-	-	0
	B		100	1	0	0	0	0	0	0	1	-	-	0
	Total		200	1	0	0	0	0	0	0	1	0.0	0.5	0 (0.0)
156.25	A	61	100	0	0	0	0	0	0	0	0	-	-	0
	B		100	1	2	0	1	0	0	3	4	-	-	1
	Total		200	1	2	0	1	0	0	3	4	1.5	2.0	1 (0.5)
Positive control	A	85	50	5	17	24	3	2	3	32	34	-	-	0
	B		50	7	15	19	8	9	2	35	37	-	-	1
	Total		100	12	32	43	11	11	5	67	71	67.0 ***	71.0 ***	1 (1.0)

N:          Number of cells scored
g:           Gaps
Ctb:       Chromatid breaks
Cte:       Chromatid exchange
Csb:      Chromosome break
Cse:      Chromosome exchange
***       Statistically significant (p>0.001)

Results: 6 hours in the absence of S-9 with 18 hours recovery

Concentration (µg/mL)	Replicate	Toxicity (%)	N	Number of aberrations						Total number of aberrations		Frequency of aberrations (%)		Number of polyploid cells (percentage)
				g	Ctb	Cte	Csb	Cse	Others	Without gaps	With gaps	Without gaps	With gaps
0 (vehicle control)	A	100	100	1	0	0	0	0	0	0	1	-	-	0
	B		100	1	0	0	0	0	0	0	1	-	-	0
	Total		200	2	0	0	0	0	0	0	2	0.0	1.0	0 (0.0)
80	A	94	100	0	2	2	0	1	0	4	4	-	-	2
	B		100	0	0	0	0	0	0	0	0	-	-	1
	Total		200	0	2	2	0	1	0	4	4	2.0	2.0	3 (1.5)
160	A	93	100	0	0	0	0	1	0	1	1	-	-	0
	B		100	2	0	0	0	0	0	0	2	-	-	0
	Total		200	2	0	0	0	1	0	1	3	0.5	1.5	0 (0.0)
240	A	85	100	1	0	0	0	0	0	0	1	-	-	0
	B		100	2	0	0	0	0	0	0	2	-	-	1
	Total		200	3	0	0	0	0	0	0	3	0.0	1.5	1 (0.5)
CP	A	104	100	0	0	0	0	1	0	1	1	-	-	1
	B		100	1	0	0	0	0	0	0	0	-	-	1
	Total		200	1	0	0	0	1	0	1	1	0.5	0.5	2 (1.0)

CP:        Cyclophosphamide
N:          Number of cells scored
g:           Gaps
Ctb:       Chromatid breaks
Cte:       Chromatid exchange
Csb:      Chromosome break
Cse:      Chromosome exchange

Results: 6 hours in the presence of S-9 with 18 hours recovery

Concentration (µg/mL)	Replicate	Toxicity (%)	N	Number of aberrations						Total number of aberrations		Frequency of aberrations (%)		Number of polyploid cells (percentage)
				g	Ctb	Cte	Csb	Cse	Others	Without gaps	With gaps	Without gaps	With gaps
0 (vehicle control)	A	100	100	0	1	2	2	1	0	6	6	-	-	2
	B		100	0	0	0	0	0	0	0	0	-	-	2
	Total		200	0	1	2	2	1	0	6	6	3.0	3.0	4 (2.0)
10	A	109	100	1	0	0	0	0	0	0	1	-	-	0
	B		100	0	0	0	0	1	0	1	1	-	-	0
	Total		200	1	0	0	0	1	0	1	2	0.5	1.0	0 (0.0)
20	A	112	100	2	0	1	0	0	0	1	3	-	-	0
	B		100	4	1	0	2	2	0	4	8	-	-	2
	Total		200	6	1	1	2	2	0	5	11	2.5	5.5	2 (1.0)
30	A	46	100	0	5	1	1	2	0	9	9	-	-	3
	B		100	0	1	3	1	0	0	4	4	-	-	1
	Total		200	0	6	4	2	2	0	13	13	6.5	6.5	4 (2.0)
CP	A	73	50	9	14	30	6	3	5	41	43	-	-	1
	B		50	15	12	26	4	0	1	34	38	-	-	0
	Total		100	24	26	56	10	3	6	75	81	75.0 ***	81.0 ***	1 (1.0)

CP:        Cyclophosphamide
N:          Number of cells scored
g:           Gaps
Ctb:       Chromatid breaks
Cte:       Chromatid exchange
Csb:      Chromosome break
Cse:      Chromosome exchange
***       Statistically significant (p>0.001)

Conclusions:

The test material showed some equivocal evidence of clastogenicity in the presence of metabolic activation which was considered by the Study Director to be of no biological relevance, due to increased aberration frequency noticed in one of vehicle control cultures. There was no evidence of clastogenicity in the absence of metabolic activation, but the concentrations which were scored for aberrations did not show adequate levels of toxicity.No conclusion been made due to equivocal resuls.

Executive summary:

A chromosome aberration study was conducted based on OECD guideline 473. Chinese Hamster Lung (CHL) cells were exposed to the substance 1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether for 6-hours in the absence and in the presence of metabolic activation with 18 hours recovery. Exposures of 24-hours and 48-hours were also conducted in the absence of metabolic activation with no recovery. The concentrations selected for the main experiment were based on the results of a preliminary experiment and for the 6-hour treatments on an initial main experiment which was not scored for aberrations due to the levels of toxicity. The test material has shown some equivocal evidence of clastogenicity in the presence of metabolic activation which was considered by the Study Director to be of no biological relevance since the number of aberrations were comparable to a single vehicle control which showed 3% of cells with aberrations (the other replicate vehicle control culture showed no damage). Although the number of aberrations was concentration related (0.5% aberrant cells at 10 µg/mL; 2.5% aberrant cells at 20 µg/mL and 6.5% aberrant cells at 30 µg/mL), the number of aberrations was not statistically significant. The experiment was not repeated to determine reproducibility. Although there was no evidence of clastogenicity in the absence of metabolic activation, the concentrations which were scored for aberrations did not show adequate levels of toxicity.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

August 2017 to October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

OECD 490 adopted July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase (tk) gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection, Rockville, Maryland (ATCC code: CRL 9518)
- Suitability of cells: as specified in the OECD guideline

The generation time and mutation rates (spontaneous and induced) were checked in the test laboratory. The cells were checked at regular intervals for the absence of mycoplasmal contamination. Permanent stocks of the L5178Y TK+/− cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use. Prior to use, cells were cleansed of pre-existing mutants.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 tissue homogenate obtained from the liver of Sprague-Dawley rats induced with phenobarbital - 5,6-benzoflavone (supplied by Trinova Biochem GmbH)

Test concentrations with justification for top dose:

Cytotoxicity assay: 1760, 880, 440, 220, 110, 55.0, 27.5, 13.8 and 6.88 µg/mL; the top dose of 1760 µg/mL corresponds to 10 mM (the upper limit specified in the test guideline)
Mutation assay without S9: 410, 342, 285, 237, 198 and 165 µg/mL
Mutation assay with S9: 41.0, 29.3, 20.9, 14.9, 10.7 and 7.62 µg/mL

Vehicle / solvent:

Dimethylsulphoxide (DMSO); a preliminary solubility trial indicated that the test item was miscible in DMSO at 200 µL/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Details on test system and experimental conditions:

A cell suspension (1×10E6 cells/mL) in complete medium was prepared. A common pool was used for each experiment to prepare the test cultures in appropriately labelled conical screw-cap tissue culture tubes. The cultures were incubated at 37°C for 3 hours in the presence and absence of S9 homogenate, and for 24 hours without S9 homogenate. At the end of the incubation period, the treatment medium was removed and the cultures centrifuged and washed twice with Phosphate Buffered Saline (PBS).

A preliminary cytotoxicity test was performed in order to select appropriate dose levels for the mutation assays. Treatments were performed in the absence and presence of S9 metabolic activation for 3 hours and for 24 hours only in the absence of S9 metabolic activation. A single culture was used at each test point. After washing in Phosphate Buffered Saline (PBS), cells were resuspended in 20 mL of complete medium (10%). Cell concentrations were adjusted to 8 cells/mL using complete medium (20%) and, for each dose level, 0.2mL was plated into 96 microtitre wells. The plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 8-9 days. Wells containing viable clones were identified by eye using background illumination and then counted.

The mutation assay was performed including vehicle and positive controls, in the absence and presence of S9. Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture. In the main experiment, the cells were exposed to the test item for a short treatment time (3 hours). Since positive results were obtained, no further experiment was performed. After washing in Phosphate Buffered Saline (PBS), cells were resuspended in fresh complete medium (10%) and cell densities were determined. The number of cells was adjusted to
give 2×10E5 cells/mL. The cultures were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.

During the expression period (two days after treatment), the cell populations were subcultured in order to maintain them in exponential growth. At the end of this period, the celldensities of each culture were determined and adjusted to give 2×10E5 cells/mL.

Plating for 5-trifluorothymidine resistance: After dilution, the cell suspensions in complete medium B (20%) were supplemented with trifluorothymidine (final concentration 3.0 µg/mL) and an estimated 2×10E3 cells were plated in each well of four 96-well plates. Plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for one to two weeks and wells containing clones were identified by eye using background illumination and counted. In addition, the number of wells containing large colonies as well as the number of those containing small colonies were scored.

Plating for viability: After dilution, in complete medium A (20%), an estimated 1.6 cells/well were plated in each well of two 96-well plates. These plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for one to two weeks and wells containing clones were identified as above and counted.

Survival (viability) calculated based on plating efficiency; cloning efficiences in test and control cultures were compared to give the relative cloning efficiency (RCE). The relative total growth (RTG) was also calculated. This is a product of the relative suspension growth (RSG) and the Day 2 relative cloning efficiency (RCE), expressed as a percentage of the concurrent negative control, for each culture. Mutation frequency was calculated using Poisson statistics.

Rationale for test conditions:

As specified in the OECD test guideline (OECD 490).

Evaluation criteria:

The assay was considered valid if the following criteria were met:
1. The cloning efficiencies at Day 2 in the untreated/solvent control cultures fell within the range of 65-120%.
2. The untreated/solvent control suspension growth over 2 days fell within the range: 8-32 (3 hour treatment), 32-180 (24 hour treatment).
3. The mutant frequencies in the untreated/solvent control cultures fell within the range of 50−170×10E−6 viable cells.

Every assay was also evaluated as to whether the positive control met at least one of the following two acceptance criteria:
1. The positive control induced a clear increase above the spontaneous background (induced mutent frequency = IMF) of at least 300×10E−6. At least 40% of the IMF was reflected in the small colony MF.
2. The positive control induced a clear increase in the small colony IMF of at least 150×10E−6.

For a test item to be considered mutagenic in this assay, it is required that:
1. The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126×10E−6) at one or more doses.
2. There is a significant dose-relationship as indicated by the linear trend analysis.
Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance.

Statistics:

Statistical analysis was performed according to UKEMS guidelines (RobinsonW.D., 1990). Parameters evaluated were consistency between plates, heterogeneity factors for replicate cultures, overall consistency, updated heterogeneity factors, comparison of each treatment with the control, and test for linear trend

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Cytotoxicity test: In the absence of S9, dose-related precipitation of the test item was noted at the end of the 3-hour treatment period, at the three highest concentrations tested. ~In the absence of S9, using the 3-hour treatment time, no cells survived treatment at the three highest dose levels. No relevant toxicity was noted over the remaining concentrations tested. Using the 24 hour treatment time, no cells survived treatment at the three highest concentrations; moderate toxicity was noted at the next two lower concentrations, reducing relative survival (RS) to 25% and 30%; slight toxicity was observed at 55.0 µg/mL, reducing RS to 66%, while no relevant toxicity was noted over the remaining concentrations tested. Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), no cells survived treatment starting from 55.0 µg/mL; test item treatment at 27.5 µg/mL yielded moderate toxicity, reducing RS to 19%; slight reduction of RS (63%) was noted at the next lower concentration of 13.8 µg/mL, while no relevant toxicity was observed at the lowest concentration tested.

Mutation assay:
No precipitate was noted upon addition of the test item to the cultures or by the end of the 3 hour treatment incubation period. Solvent and positive control cultures were included in each treatment series. The mutant frequencies in the solvent control cultures fell within the normal range (50-170×10E-6 viable cells). The cloning efficiencies at Day 2 in the negative control cultures fell within the range of 65 - 120%. The control suspension growth over 2 days fell within the range of 8 - 32. Positive control item Benzo(a)pyrene treatment yielded an absolute increase in total mutation frequency (MF) higher than 300×10E-6, but an increase in the small colony mutant frequency slightly lower than the acceptable one. However, based on the overall evaluation of the results obtained, the responsiveness of the test system was regarded as adequately demonstrated and the study was accepted as valid.

In the absence of S9 metabolic activation, no cells survived treatment at the highest dose level; marked toxicity, reducing Relative Total Growth (RTG) to 8% of the concurrent negative control, was noted at 342 µg/mL; moderate toxicity was observed at 285 µg/mL (RTG = 24%), while dose related, slight to mild, toxicity was noted over the remaining concentrations tested. In the presence of S9 metabolism, marked toxicity was observed at the two highest concentrations, reducing RTG to 4% and 10% over the concurrent negative control, treatment at 20.9 µg/mL yielded moderate toxicity (RTG = 26%), while slight toxicity was noted at the remaining concentrations tested. At low survival levels, the mutation data are prone to a variety of artefacts (selection effects, sampling error, founder effects), therefore mutation data obtained at 342 µg/mL, in the absence of S9 homogenate, and at 41.0 µg/mL in the presence of S9 were excluded from the statistical analyses.

Statistically significant increases in mutant frequency, which did not reach the Global Evaluation Factor (GEF), were observed at the two highest analysable concentrations, in the absence of S9 metabolism. Marked and dose related increases of mutation frequency were observed in the presence of S9 metabolic activation; at the two highest analysable concentrations, the InducedMutation Frequency (IMF) was higher than GEF. A linear trend was also indicated (p<0.001), both in the absence and presence of S9 metabolism. The small and large colony mutant frequencies were estimated for test item concentrations which elicited a positive response and for the negative and positive controls; the proportion of small mutant colonies was calculated. The ratio of small versus large colonies is shifted towards the small colonies at concentrations producing a mutagenic effect indicating that the test item induces mainly gross structural changes at the chromosomal level. Since clear positive results were obtained, no additional experiment (24 hour) was performed.

The pH values and osmolality of the post-treatment media were determined: the addition of the test item solution did not have any obvious effect on the osmolality or pH of the treatment medium.

Negative controls

Assay No.	S9	Suspension Growth	Cloning efficiency (%)
1	-	22	117
1	+	16	107

 

Mutation assay: 3 hour treatment time without S9: Relative Total Growth

Dose level (µg/mL)		10^6cells/mL Post treatment	10^6cells/mL Day 1	10^6cells/mL Day 2	Total wells	Plate counts # Day 2		Cloning efficiency Day 2	TSG	RTG
0.00	A	0.60	0.75	0.86	192	80	80	1.17	22.0	100%
	B	0.67	0.72	1.03	192	82	83
165	A	0.60	0.49	0.67	192	78	78	1.19	11.0	50%
	B	0.58	0.51	0.83	192	82	87
198	A	0.39	0.50	0.51	192	84	86	1.42	6.8	37%
	B	0.43	0.54	0.74	192	87	87
237	A	0.45	0.44	0.81	192	83	80	1.19	8.5	39%
	B	0.34	0.46	1.16	192	80	84
285	A	0.34	0.45	0.70	192	81	78	1.15	5.3	24%
	B	0.34	0.43	0.71	192	79	85
342	A	0.21	0.27	0.81	192	80	75	1.18	1.9	8%
	B	0.20	0.28	0.54	192	87	82
410	A	0.20	0.04	£	0	0	0			0%
	B	0.21	0.02	£	0	0	0
0.00	A		0.00	0.00	0	0	0
10.00

 # = wells with clones/plate – 1.6 cells in each well of two 96-well plates plated for viability

A and B = replicate cultures

£ = insufficient number of cells recovered

Mutation assay: 3 hour treatment time without S9: Mutant Frequency

Dose level (µg/mL)		Total wells	Resistant mutant # after Day 2				Mean mutant frequency §		IMF §
0.00	A	384	12	7	10	13	53.9	-	-
	B	384	15	10	13	11
165	A	384	17	19	14	14	78.5	NS	24.67
	B	384	19	14	13	19
198	A	384	25	19	23	15	70.1	NS	16.19
	B	384	12	11	15	18
237	A	384	19	15	15	20	88.4	*	34.56
	B	384	18	25	19	15
285	A	384	20	21	23	21	113.9	**	60.05
	B	384	17	30	24	21
342	A	384	17	21	21	26	129.3	$	75.39
	B	384	30	29	34	21
0.00	A	0	0	0	0	0		-
10.00
Linear trend								***

# = wells with clones/plate – 2 x 10³cells/well of four 96-well plates platedfor 5-TFT resistance

§ = per 10⁶viable cells

A and B = replicate cultures

^a= Induced mutant frequency (IMF) > global evaluation factor (GEF = 126 x 10^-6)

NS = not statistically significant

* = statistically significant at p < 5%; ** = p < 1%; *** = p < 0.1%

$ = treatment excluded from test statistics due to excessive toxicity

Mutation assay: 3 hour treatment time with S9: Relative Total Growth

Dose level (µg/mL)		10^6cells/mL Post treatment	10^6cells/mL Day 1	10^6cells/mL Day 2	Total wells	Plate counts # Day 2		Cloning efficiency Day 2	TSG	RTG
0.00	A	0.54	0.79	0.78	192	79	80	1.07	16.3	100%
	B	0.52	0.81	0.77	192	78	78
7.62	A	0.43	0.54	0.79	192	80	83	1.17	9.7	65%
	B	0.42	0.50	0.98	192	80	82
10.7	A	0.41	0.57	0.83	192	74	72	1.12	9.0	58%
	B	0.31	0.60	0.91	192	84	86
14.9	A	0.51	0.49	0.91	192	75	73	1.00	9.4	53%
	B	0.41	0.44	0.84	192	79	79
20.9	A	0.33	0.32	0.36	192	90	92	2.01	2.3	26%
	B	0.39	0.30	0.47	192	94	92
29.3	A	0.40	0.28	0.17	192	93	92	1.64	1.1	10%
	B	0.37	0.20	0.38	192	80	84
41.0	A	0.41	0.10	0.13	192	76	78	1.06	0.6	4%
	B	0.41	0.12	0.16	192	80	79
B(a)P 2.00	A	0.40	0.45	0.68	192	72	72	0.87	6.0	30%

 # = wells with clones/plate – 1.6 cells in each well of two 96-well plates plated for viability

A and B = replicate cultures

£ = insufficient number of cells recovered

Mutation assay: 3 hour treatment time with S9: Mutant Frequency

Dose level (µg/mL)		Total wells	Resistant mutant # after Day 2				Mean mutant frequency §		IMF §
0.00	A	384	12	12	13	11	62.9	-	-
	B	384	8	14	13	14
7.62	A	384	20	22	23	17	89.4	NS	26.44
	B	384	16	17	16	14
10.7	A	384	15	16	20	20	108.7	*	45.79
	B	384	29	20	25	16
14.9	A	384	24	17	20	15	139.2	**	76.26
	B	384	28	26	30	26
20.9	A	384	63	65	77	66	283.5	**	22.6^a
	B	384	67	57	65	59
29.3	A	384	51	46	40	41	214.5	**	151.6^a
	B	384	46	46	53	41
41.0	A	384	17	19	22	18	128.2	$	65.26
	B	384	26	26	27	27
B(a)P 2.00	A	384	71	69	76	69	782.2	-	719.3^a
Linear trend								***

 # = wells with clones/plate – 2 x 10³cells/well of four 96-well plates plated for 5-TFT resistance

§ = per 10⁶viable cells

A and B = replicate cultures

^a= Induced mutant frequency (IMF) > global evaluation factor (GEF = 126 x 10^-6)

NS = not statistically significant

* = statistically significant at p < 5%; ** = p < 1%; *** = p < 0.1%

$ = treatment excluded from test statistics due to excessive toxicity

Mutation assay: small and large colony mutant frequencies

Dose level (µg/mL)	S9	Mutant frequency per 10^6viable cells		Proportion small colony mutants
		Small colony	Large colony
0.00	-	14.1	38.4	0.27
0.00	+	11.7	49.9	0.19
20.9	+	103.1	112.1	0.48
29.3	+	86.4	94.3	0.48
B(a)P 2.00	+	118.0	574.1	0.17

Conclusions:

1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether induced mutations at the TK locus of L5178Y mouse lymphoma cells in the presence of S9 metabolic activation. Colony size analysis of the concentrations resulting in a positive response showed an increase in the proportion of small colonies, indicative of gross structural changes at the chromosomal level.

Executive summary:

An in vitro cell gene mutation assay was performed with 1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether using mouse lymphoma L5178Y cells, according to GLP and OECD test guideline 490. The assay examines mutagenic activity by assaying for the induction of 5 -trifluorothymidine resistant mutants in the presence and absence of exogenous metabolic activation (S9 homogenate). Following preliminary solubility and cytotoxicity assays, the following test concentrations were used in the mutation assays: 165, 198, 237, 285, 342 and 410 µg/mL without S9 and 7.62, 10.7, 14.9, 20.9, 29.3 and 41.0 µg/mL with S9. The cells were incubated with the test substance for 3 hours, either in the presence of absence of S9. Sterility, vehicle (DMSO) and positive (benzo(a)pyrene) controls were included. Adequate levels of cytotoxicity, covering a range from the maximum to slight or no toxicity, were observed in both treatment series. Slight increases of mutation frequency, which did not reach the Global Evaluation Factor (GEF), were noted in the absence of S9 metabolism. Marked and dose related increases of mutation frequency were observed in the presence of S9 metabolic activation; at the two highest analysable concentrations, the Induced Mutation Frequency (IMF) was higher than GEF. A statistically significant linear trend was also indicated, both in the absence and presence of S9 metabolism. Since the results were clearly positive, no further experiment was performed. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments, indicating the correct functioning of the assay system. It was concluded that ally pentaerythritol induces mutations at the TK locus of L5178Y mouse lymphoma cells in the presence of S9 metabolic activation. Colony size analysis of the concentrations resulting in a positive response showed an increase in the proportion of small colonies, indicative of gross structural changes at the chromosomal level.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The clastogenic potential of 1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether to induce micronuclei in the mouse bone marrow erythropoietic cells was evaluated in CD-1 mice received a single intraperitoneal dose of 100, 200 and 400 mg/kg and sacrified 24, 48 and 72 hours post-dose. Analysis of the data indicated there was not a statistically significant increase in the frequency of MPCEs

in any group of mice treated with the test item when compared to the concurrent negative control group. Statistically significant increases in the PCE/NCE ratios were detected at the 48 hour sacrifice

time in the 100 and 400 mg/kg group of mice treated with 1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether. The biological significance of this increase (if any) is unknown, since no dose response was observed and such an increase was not noticed at 24 or 72hours ethier. Therefore, 1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether was considered negative (non-clastogenic) in the mouse MNT at all the time intervals evaluated under the criteria and the experimental conditions.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 22, 1992 – November 5, 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian bone marrow chromosome aberration test

Specific details on test material used for the study:

- Storage condition: stored at ambient temperature in the container received from the sponsor
- Stability under test conditions: no apparent change in its physical state during storage
-Test item solutions were prepared fresh prior to dosing.
- No treatment of test material prior to testing.
- No preliminary purification step.
- Aliquots of allyl pentaerythritol was weighed and diluted with corn oil.

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

Historically, mice have been used in the MNT and have been shown to exhibit micronuclei indicative of chromosome breakage or lagging chromosomes (Heddle et al., 1983; Salamone et al., 1980; Schmid, 1975; Von Ledebur and Schmid, 1973). Intraperitoneal injection was chosen to maximize the bioavailability of allyl pentaerythritol to the target cells.

Sex:

male/female

Details on test animals or test system and environmental conditions:

August 4, 1992 - November 5, 1992 TEST ANIMALS
- Source: CD-1® mice from Charles River Laboratories, Wilmington, MA01887
- Age at study initiation: 10 weeks of age
- Weight at study initiation: At the time of dosing, body weights ranged between 32- 43 grams for males and 26-35 grams for females.
- Assigned to test groups randomly: Mice were randomized by body weight and assigned to groups by use of a computer generated random number list and individually identified with an ear tag.
- Fasting period before study: no
- Housing: Mice were housed in stainless steel wire mesh cages with a maximum of five per cage according to sex and dose group. Waste material was removed twice per week.
- Diet (e.g. ad libitum): Harlan Teklad Rodent Diet
- Water (e.g. ad libitum): fresh tap water was available ad libitum
- Acclimation period: All mice were acclimated to laboratory conditions for 26 days prior to initiation of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°c ± 3°C
- Humidity (%): 50% ± 20
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

IN-LIFE DATES: From: To: Aug 4 – Aug 7, 1992

Route of administration:

intraperitoneal

Vehicle:

- Vehicle:corn oil
- Concentration of test material in vehicle: 10, 20 and 40 mg/ml.

Details on exposure:

Single doses of allyl pentaerythritol (100, 200 and 400 mg/kg) was administered by ip injection to 9 groups of mice (5/sex/group).
Concurrently, the negative control, corn oil, was administered to three groups of mice, and a group of these mice was included in each sacrifice time.
The positive control, TEM (Triethylenemelamine) at 0.5 mg/kg, was administered to one group of mice and sacrificed 24 hours post-dose.

Duration of treatment / exposure:

A single intraperitoneal dose of allyl pentaerythritol (100, 200 and 400 mg/kg) and a group of mice from each dose level was sacrificed at 24, 48 and 72 hours post-dose.

Frequency of treatment:

A single intraperitoneal dose.

Post exposure period:

A group of mice from each dose level was sacrificed at 24, 48 and 72 hours post-dose

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Five/Sex/group

Control animals:

yes, concurrent vehicle

Positive control(s):

Triethylenemelamine (TEM);
- Route of administration: by ip injection
- Doses / concentrations: TEM was dissolved in 0.9% Saline, dose level was 0.5 mg/kg

Tissues and cell types examined:

1000 PCEs per mouse were scored for the presence of MPCEs, as well as for the number of MNCEs present in the optical field containing these 1000 PCEs. The data were expressed as the total number of mean percentage of MPCEs for 10,000 PCEs per group (1000 PCEs/mouse, whenever possible). An additional 1000 erythrocytes (PCE and NCE) were also scored per mouse and the proportion was expressed as the PCE/NCE ratio.

Details of tissue and slide preparation:

All mice were sacrificed by cervical dislocation and their femurs removed. One end of each femur (one proximal, one distal to the iliac end) was opened with scissors until a small opening to the marrow canal was visible. A 1 ml tuberculin syringe, fitted with a 5/8” 25 gauge needle containing approximately 0.2 ml fetal bovine serum, was inserted into the marrow cavity and the marrow gently flushed (to assure maximal dispersion) into a 5 ml round bottom culture tube containing 1.0 ml of fetal bovine serum. Each femur was flushed with fetal bovine serum until the bone appeared almost translucent. The cell suspensions were centrifuged for 5 minutes at 1000 rpm. The supernatant was removed leaving a small amount of fetal bovine serum with the cell pellet. With a pasteur pipette, the cell pellet was resuspended into a homogenous cell suspension. A small drop of the cell suspension was spread immediately onto an ethanol pre-cleaned glass slide. The smears were quickly dried on a slide warmer (~56°C), dipped in absolute methanol (~2 seconds) and air-dried. Slides were stained with a Modified Wrights Stain Pak (4481) containing polychrome methylene blue-eosin in an Ames Hema-Tek® 1000 automatic slide stainer. Slides were cleared in xylene,air-dried and coverslipped with Permaslip®.

Evaluation criteria:

Although micronuclei are uniformly round bodies in the cytoplasm of erythrocytes, they may appear almond-, ring-, or teardrop-shaped. Cytoplasmic inclusions which were light reflective, improperly shaped or stained, or which were not in the focal plane of the erythrocyte were considered artifacts and were not scored as micronuclei. Erythrocytes containing one or more than one micronucleus were scored as one micronucleated erythrocyte.

A test article is considered positive if it produced a statistically significant increase in the number of MPCEs as compared to the negative control.

Statistics:

One-tailed-tests were used to make pairwise comparisons between each treatment group and its concurrent negative control for statistically significant increases in the number of MPCEs. The proportion of PCEs per 1000 erythrocytes per mouse was evaluated by pairwise two-tailed-tests after an arcsin transformation was performed. Statistical significance was judged at p ≤ 0.05 and p ≤ 0.01 levels. All comparisons were made for each sacrifice time separately, comparing the treated group versus the concurrent negative control group.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 100, 250, 500, 750 and 1000 mg/kg of body weight
- Solubility: Aliquots of allyl pentaerythritol was weighed and diluted with corn oil. Apparent homogeneous solutions were obtained and maintained on a magnetic stirplate at all dose levels evaluated.
- Clinical signs of toxicity in test animals: Pharmacotoxic signs (endpoints examined including abnormal gait, ataxia, body drop, decreased activity, decreased body tone, loss of righting, tonic convulsions, writhing) were observed at the dosage of 500, 750 and 1000 mg/kg dose groups.
- Rationale for exposure: Intraperitoneal injection was chosen to maximize the bioavailability of allyl pentaerythritol to the target cells.
- Harvest times: 24, 48 and 72 hours post-dose

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): there was not a statistically significant increase in the frequency of MPCEs in any group of mice treated with allyl pentaerythritol when compared to the concurrent negative control group.
- Ratio of PCE/NCE (for Micronucleus assay): Statistically significant increases in the PCE/NCE ratios were detected at the 48 hour sacrifice time in the 100 and 400 mg/kg group of mice treated with allyl pentaerythritol, but not at 24 nor 72 hour sacrifice time. The biological significance of this increase (if any) is unknown. No statistically significant changes were observed in other groups.

Pharmacotoxic Signs in the dose range fining study:

Mice were observed for death and pharmacotoxic signs immediately, 24, 48 and 72 hours post-dose and the surviving mice were sacrificed at the last observation time.

Table 1. Pharmacotoxic signs observed in mice dosed with allyl pentaerythritol at dosages of 100, 250, 500, 750 and 1000 mg/kg of body weight.

	Immediate		24 hrs			48 hrs			72 hrs
Pharmacotoxic Signs 100 mg/kg	M	F	M	F	M		F	M		F
Decreased body tone			0	1	0		1	0		1

 

	Immediate		24 hrs			48 hrs			72 hrs
Pharmacotoxic Signs 250 mg/kg	M	F	M	F	M		F	M		F
Abnormal gait	2	2	1	1
Decreased body tone			1	1	1		1	1		1
Piloerection	2	0	2	0	2		0	1		0
Writhing	2	2

	Immediate		24 hrs			48 hrs			72 hrs
Pharmacotoxic Signs 500 mg/kg	M	F	M	F	M		F	M		F
Abnormal gait	2	2	1
Ataxia	1	1
Decreased activity	1	1
Decreased body tone	2	2
Piloerection			2	2	1		1	1		1
Writhing	2	2

 

	Within 7 minutes		24 hrs			48 hrs			72 hrs
Pharmacotoxic Signs 750 mg/kg	M	F	M	F	M		F	M		F
Abnormal gait	2	2	2	2
Ataxia	2	2
Body drop	2	2
Decreased activity	2	2
Decreased body tone	2	2	2	2	2		1	2		1
Piloerection			2	1	1		1	1		0
Tonic convulsions	2	2
Writhing	2	2

 

	Within 15 minutes		24 hrs		48 hrs		72 hrs
Pharmacotoxic Signs 1000 mg/kg	M	F	M	F	M	F	M	F
Abnormal gait	2	2	2	2
Ataxia	2	2
Body drop	2	2
Decreased activity	2	2
Decreased body tone	2	2	2	2	2	2	2	1
Loss of righting	2	2
Tonic convulsions	2	2
Writhing	2	2

 

 

Pharmacotoxic Signs in the main study:         

Table 2. Pharmacotoxic signs observed in the MNT in mice treated with allyl pentaerythritol:           

A) 100 mg/kg group, B) 200 mg/kg group and C) 400 mg/kg group:

Pharmacotoxic Signs	Immediate		24 hrs		48 hrs		72 hrs
	M (15)	F (15)	M (15)	F (15)	M (15)	F (15)	M (15)	F (15)
A) Piloerection Writhing	2 4	1 2	5	4	2	2	2	1
B) Abnormal gait Decrease body tone Piloerection Writhing	15   10 15	15   3 15	7 3 7	4 0 7	9 2 8	4 0 7	1 1 1	0 0 2
C) Abnormal gait Body drop Decrease body tone Decrease activity Increase activity Piloerection Writhing	15 14 1 15     15	15 12 0 15     15	11   2 1 2 14	12   0 0 1 13	8   2 1 1 9	7   0 0 1 9	1     5	0     5

 

( ) = Number of mice per group in each observation time.

Conclusions:

1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether at dosages of 100, 200 and 400 mg/kg was considered negative (non-clastogenic) in the mouse micronucleus test at all the time intervals (24, 48 and 72 hours) evaluated under the criteria and the experimental conditions.

Executive summary:

The purpose of this study was to evaluate the clastogenic potential of 1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether to induce micronuclei in the mouse bone marrow erythropoietic cells.

Method:

A range-finding test was performed to find suitable dose levels of the test item. Each mouse received a single intraperitoneal dose of 1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether (100,250,500,750and1000mg/kg of bodyweight). Based on the severe pharmacotoxic signs observed at the dosages of 500, 750 and 1000 mg/kg dose groups, 100, 200 and 400 mg/kg were selected as the doses for the main study. Thirteen groups of CD-1® mice (10 weeks of age) were used in the main study. Each group of mice was comprised of ten animals (five/sex). Each 3 groups mice received a single intraperitoneal dose of 1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether (100, 200 and 400 mg/kg) and a group of mice from each dose level was sacrificed at 24, 48 and 72 hours post-dose. Concurrently, the negative control, corn oil, was administered to three groups of mice, and a group of these mice was included in each sacrifice time. The positive control, TEM at 0.5 mg/kg, was administered to one group of mice and sacrificed 24 hours post-dose. Bone marrow extracted and smear preparation made and stained. One thousand PCEs per mouse were scored for the presence of MPCEs,as well as for the number of MNCEs present in the optical fields containing these 1000 PCEs. The data were expressed as the total number of mean percentage of MPCEs for 10,000 PCEs per group (1000 PCEs/mouse,wheneverpossible). An additional 1000 erythrocytes (PCE and NCE) were also scored per mouse and the proportion was expressed as the PCE/NCE ratio.The cytotoxicity is determined from the PCE/NCE ratio which shifts in favor of NCEs after treatment with a cytotoxic compound. Statistical analyses were performed and a test article is considered positive if it produced a statistically significant increase in the number of MPCEs as compared to the negative control.

Results:

There was no premature death seen in any of the dose groups, the following pharmacotoxic signs were observed: piloerection, writhing, abnormal gait, decreased body tone, activity changes, body drop. No

pharmacotoxic signs were observed in mice administered the negative or positive controls. Analysis of the data indicated there was not a statistically significant increase in the frequency of MPCEs in any group of mice treated with 1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether when compared to the concurrent negative control group. Statistically significant increases in the PCE/NCE ratios were detected at the 48 hour sacrifice time in the 100 and 400 mg/kg group of mice. The biological significance of this increase (if any) is unknown, since there is no dose response and not obsered at 24 nor 72h groups. Mice treated with the positive control produced a statistically significant increase (p ≤ 0.01) in the frequency of MPCE and a statistically significant depression (p ≤0.01) in the PCE/NCE ratio. In the MNT for 1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether both the negative and positive controls are within the range of this historical data.

Conclusion

The test item, 1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether at dosages of 100, 200 and 400 mg/kg was considered negative (non-clastogenic) in the mouse MNT at all the time intervals evaluated under the criteria and the experimental conditions of the test protocol.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames Test

An Ames test was conducted with 1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether according to OECD guideline 471. Testing was conducted in Salmonella typhmurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2 uvrA in the absence and presence of metabolic activation. Two independent tests were conducted up to 5000 µg/plate. There was no evidence of toxicity, precipitation or increases in the number of revertants which were 2 -fold the concurrent controls. The overall conclusion of the study is that the test material has shown no evidence of mutagenicity when tested up to the maximum concentration of 5000 µg/plate.

Clastogenicity Assay

A chromosome aberration study was conducted based on OECD guideline 473. Chinese Hamster Lung (CHL) cells were exposed to the test article for 6-hours in the absence and in the presence of metabolic activation with 18 hours recovery. Exposures of 24-hours and 48-hours were also conducted in the absence of metabolic activation with no recovery. The test material has shown some equivocal evidence of clastogenicity in the presence of metabolic activation which was considered by the Study Director to be of no biological relevance since the number of aberrations were comparable to a single vehicle control which showed 3% of cells with aberrations (the replicate vehicle showed no damage). Although the number of aberrations was concentration related (0.5% aberrant cells at 10 µg/mL; 2.5% aberrant cells at 20 µg/mL and 6.5% aberrant cells at 30 µg/mL), the number of aberrations was not statistically significant. The experiment was not repeated to determine reproducibility. Although there was no evidence of clastogenicity in the absence of metabolic activation, the concentrations which were scored for aberrations did not show adequate levels of toxicity.

Mouse lymphoma Assay

An in vitro gene mutation assay was performed with 1,3 -propanediol, 2,2 - bis(hydroxymethyl) -, allyl ether using mouse lymphoma L5178Y cells, according to GLP and OECD test guideline 490. The assay examines mutagenic activity by assaying for the induction of 5 -trifluorothymidine resistant mutants in the presence and absence of exogenous metabolic activation (S9 homogenate). Following preliminary solubility and cytotoxicity assays, the following test concentrations were used in the mutation assays: 165, 198, 237, 285, 342 and 410 µg/mL without S9 and 7.62, 10.7, 14.9, 20.9, 29.3 and 41.0 µg/mL with S9. The cells were incubated with the test substance for 3 hours, either in the presence of absence of S9. Sterility, vehicle (DMSO) and positive (benzo(a)pyrene) controls were included. Adequate levels of cytotoxicity, covering a range from the maximum to slight or no toxicity, were observed in both treatment series. Slight increases of mutation frequency, which did not reach the Global Evaluation Factor (GEF), were noted in the absence of S9 metabolism. Marked and dose related increases of mutation frequency were observed in the presence of S9 metabolic activation; at the two highest analysable concentrations, the Induced Mutation Frequency (IMF) was higher than GEF. A statistically significant linear trend was also indicated, both in the absence and presence of S9 metabolism. Since the results were clearly positive, no further experiment was performed. The mutant frequencies in the solvent control cultures fell within the normal range. Increases were obtained with the positive control treatments, indicating the correct functioning of the assay system. It was concluded that 1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether induces mutations at the TK locus of L5178Y mouse lymphoma cells in the presence of S9 metabolic activation. Colony size analysis of the concentrations resulting in a positive response showed an increase in the proportion of small colonies, indicative of gross structural changes at the chromosomal level.

In vivo Micronucleus Test

The clastogenic potential of 1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether to induce micronuclei in the mouse bone marrow erythropoietic cells was evaluated in CD-1 mice received a single intraperitoneal dose of 100, 200 and 400 mg/kg and sacrified 24, 48 and 72 hours post-dose. Analysis of the data indicated there was not a statistically significant increase in the frequency of MPCEs

in any group of mice treated with the test item when compared to the concurrent negative control group. Statistically significant increases in the PCE/NCE ratios were detected at the 48 hour sacrifice

time in the 100 and 400 mg/kg group of mice treated with 1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether, but the biological significance of this increase (if any) is unknown, since no dose response was observed and such an increase was not noticed at 24 or 72hours ethier.

Discussion of in vitro and in vivo data

The data from the mouse lymphoma assay showed an increase in the proportion of small colonies, consistent with chromosomal effects; this positive response was seen in the presence of metabolic activation. Although the clastogenicity assay reports a negative result, it is notable that there is some indication of posible activity at the highest concentration in the presence of metabolic activation. The two in vitro studies therefore indicated potential clastogenic activity which was investigated in an in vivo mamalian erythrocyte micronucleus test (similar to OECD 474). No statistically significant increase in the frequency of MPCEs in any group of mice treated with 1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether ( 100, 200 and 400 mg/kg) was observed when compared to the concurrent negative control group. Statistically significant increases in the PCE/NCE ratios were detected at the 48 hour sacrifice time in the 100 and 400 mg/kg group but was unlikely to be biological significance. The substance was considered non-clastogenic in the microucleus test. Therefore 1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether is considered not to be genotoxic based on the overall information above.

Justification for classification or non-classification

No classification is proposed based on the in virto and in vivo data available.

The data from the mouse lymphoma assay showed an increase in the proportion of small colonies, consistent with chromosomal effects; this positive response was seen in the presence of metabolic activation. Although the clastogenicity assay reports a negative result, it is notable that there is some indication of posible activity at the highest concentration in the presence of metabolic activation. The two in vitro studies therefore indicated potential clastogenic activity which was investigated in anin vivomamalian erythrocyte micronucleus test (similar to OECD 474). No statistically significant increase in the frequency of MPCEs in any group of mice treated with 1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether ( 100, 200 and 400 mg/kg) was observed when compared to the concurrent negative control group. Statistically significant increases in the PCE/NCE ratios were detected at the 48 hour sacrifice time in the 100 and 400 mg/kg group but was unlikely to be biological significance. The substance was considered non-clastogenic in the microucleus test. Therefore 1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether is considered not to be genotoxic based on the overall information above.