Alkenes, C15-18-branched and linear, maleated, hydrolyzed

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genetic toxicity study was carried out according to OECD guideline 471 (Salmonella/Mammalian-Microsome Plate Incorporation Mutagenicity Assay (Ames test) with a confirmatory assay) is negative in the presence and in the absence of metabolic activation.

Endpoint Conclusion: No adverse effect observed (negative)

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-10-31 to 2008-11-21

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Reversion to histidine independence in Salmonella typhimurium and reversion to tryptophan independence in Escherichia coli.

Species / strain / cell type:

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

Species / strain / cell type:

E. coli, other: WP2 uvrA-

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate (10% liver S9 in standard cofactors)

Test concentrations with justification for top dose:

A preliminary test was carried out to determine the toxicity of the test material, where 10 concentrations (0 - 5000 ug/plate) and a vehicle control were tested. For experiment 1, five concentrations of the test material (50, 150, 500, 1500 and 5000 ug/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method. A second experiment was performed using the same methodology and the test material dose range for experiment 1.

Vehicle / solvent:

50 mg/ml Dimethyl formamide.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl formamide

True negative controls:

no

Positive controls:

yes

Remarks:

N-ethyl-N'-nitro-N-nitrosoguaniding (ENNG), 9-Aminoacridine (9AA), 4-Nitroquinoline-1-oxide (4NQO), 2-Aminoanthracene (2AA) and Benzo(a)pyrene (BP)

Evaluation criteria:

The assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed and all tester strain cultures should be in the range of 1 to 9.9 E+09 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of 4 non-toxic test material dose levels and there should be no evidence of excessive contamination.

Statistics:

Statistical methods as recommended by the UKEMS.

Key result

Species / strain:

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

Remarks:

A globular precipitate was observed at 5000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

other: Historical data from 2006-2007

Positive controls validity:

valid

The vehicle control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of the revertant colonies, both with or without metabolic activation. Thus the sensitivity of the assay and the efficacy of the S9-mix were validated.

Table 1 - Test Results: Experiment 1- Without metabolic activation

Test Period		From 10 November 2008 to 13 November 2008
Without S9-Mix	Test Substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base pair substitution type						Frameshift type
		TA100		TA1535		WP2uvrA^-		TA98		TA1537
-	0	90		35		28		13		6
		90	(88)	32	(32)	17	(25)	12	(14)	6	(7)
		83	4.0#	28	3.5	30	7.0	16	2.1	8	1.2
-	50	93		19		14		9		5
		88	(83)	28	(24)	26	(20)	18	(16)	5	(5)
		67	13.8	25	4.6	20	6.0	20	5.9	6	0.6
-	150	89		27		15		17		3
		84	(82)	29	(26)	28	(21)	16	(16)	5	(3)
		73	8.2	21	4.2	20	6.6	15	1.0	2	1.5
-	500	73		25		18		13		6
		95	(84)	18	(21)	21	(18)	13	(14)	2	(3)
		84	11.0	20	3.6	14	3.5	15	1.2	2	2.3
-	1500	89		24		17		17		5
		80	(84)	20	(23)	17	(19)	21	(17)	2	(3)
		83	4.6	24	2.3	24	4.0	13	4.0	3	1.5
-	5000	79 P		16 P		8 P		12 P		4 P
		67 P	(76)	15 P	(14)	14 P	(13)	10 P	(11)	3 P	(4)
		83 P	8.3	10 P	3.2	18 P	5.0	11 P	1.0	5 P	1.0
Positive controls S9-Mix -	Name concentration (µg/plate) No. colonies per plate	ENNG		ENNG		ENNG		4NPQ		9AA
		3		5		2		0.2		80
		336		89		182		108		1425
		405	(362)	111	(103)	197	(182)	114	(109)	812	(975)
		346	37.3	109	12.2	167	15.0	105	4.6	687	395.0

P = precipitate, # = Standard deviation

Table 2 - Test Results: Experiment 1- With metabolic activation

Test Period		From 10 November 2008 to 13 November 2008
With S9-Mix	Test Substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base pair substitution type						Frameshift type
		TA100		TA1535		WP2uvrA^-		TA98		TA1537
-	0	85		22		16		10		5
		87	(87)	26	(22)	22	(21)	14	(13)	5	(5)
		90	2.5#	18	4.0	24	4.2	15	2.6	5	0.0
-	50	90		32		20		11		7
		88	(86)	26	(27)	16	(19)	12	(11)	6	(5)
		79	5.9	24	4.2	20	2.3	11	0.6	2	2.6
-	150	82		25		9		9		3
		90	(89)	12	(19)	16	(15)	8	(8)	3	(3)
		95	6.6	21	6.7	20	5.6	9	0.6	4	0.6
-	500	91		16		12		15		3
		78	(80)	13	(16)	16	(16)	9	(11)	4	(4)
		72	9.7	19	3.0	19	3.5	9	3.5	4	0.6
-	1500	86		19		15		12		4
		75	(78)	17	(16)	18	(17)	10	(10)	2	(3)
		73	7.0	13	3.1	17	1.5	9	1.5	4	1.2
-	5000	81 P		12 P		15 P		17 P		3 P
		81 P	(79)	26 P	(18)	14 P	(14)	17 P	(17)	3 P	(4)
		74 P	4.0	16 P	7.2	14 P	0.6	17 P	0.0	5 P	1.2
Positive controls S9-Mix +	Name concentration (µg/plate) No. colonies per plate	2AA		2AA		2AA		BP		2AA
		1		2		10		5		2
		389		215		269		208		132
		461	(428)	244	(428)	276	(237)	196	(215)	176	(175)
		433	36.3	252	19.5	272	3.5	240	22.7	217	42.5

 

Conclusions:

Interpretation of results: negative with and without metabolic activation

No evidence of mutagenicity was seen under the conditions of this assay. The test material is considered to be non-mutagenic

Executive summary:

The mutagenicity of Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes was investigated in an Ames test (plate incorporation assay) using S. typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2uvrA-. Three replicate plates of each strain were exposed to the test material at concentrations of 50, 150, 500, 1500 and 5000 ug/plate in the presence and absence of an exogenous metabolic activation system (Rat liver homogenate (10% liver S9 in standard cofactors)). There was no evidence of cytotoxicity at the limit concentration; globular precipitation of the test material was seen at 5000 ug/plate. Exposure to the test material did not induce increased numbers of revertant colonies of any strain. Appropriate positive control compounds induced large increases in the numbers of revertant colonies, confirming the sensitivity of the assay. Results were confirmed in an independently-repeated assay. No evidence of mutagenicity was seen under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The mutagenicity test ((Bowles, A.J., 2009) of Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes was investigated in an Ames test (plate incorporation assay) using S. typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2uvrA-. Three replicate plates of each strain were exposed to the test material at concentrations of 50, 150, 500, 1500 and 5000 ug/plate in the presence and absence of an exogenous metabolic activation system. There was no evidence of cytotoxicity at the limit concentration; globular precipitation of the test material was seen at 5000 ug/plate. Exposure to the test material did not induce increased numbers of revertant colonies of any strain. Appropriate positive control compounds induced large increases in the numbers of revertant colonies, confirming the sensitivity of the assay. Results were confirmed in an independently-repeated assay. No evidence of mutagenicity was seen under the conditions of this study.

Justification for classification or non-classification

Based on the limited data available, Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes is not considered to be mutagenic according to Regulation (EC) No 1272/2008.