Cyclohexanepropanal, 4-(2-methylpropyl)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genotoxic potential of the test substance, TM 10-202, was assessed as negative in the presence and absence of metabolic activation according to OECD Test Guideline 471 using the Bacterial Reverse Mutation method.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 25 June 2010 and 22 July 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 471 using the Bacterial Reverse Mutation method and in compliance with GLP.

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:

Salmonella typhimurium: Histidine
Escherichia coli: Tryptophan

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.

Mutation Test – Experiment 1 (Range-finding Test):
Salmonella strains (without S9-mix): 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate.
Salmonella strains (with S9-mix): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
WP2uvrA- (with and without S9-mix): 50, 150, 500, 1500 and 5000 µg/plate.

Mutation Test – Experiment 2 (Main Test)
Salmonella strain TA100 (without S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 µg/plate.
Salmonella strains TA1535 and TA1537 (without S9-mix): 0.15, 0.5, 1.5, 5, 15, 50 and 150 µg/plate.
Salmonella strain TA98 (without S9-mix): 0.015, 0.05, 0.15, 0.5, 5 and 50 µg/plate.
All Salmonella strains (with S9-mix): 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate.
WP2uvrA- (with and without S9-mix): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.

Vehicle / solvent:

In solubility checks performed in house, the test item was noted to be immiscible in sterile distilled water and only partially miscible in dimethyl sulphoxide at 50 mg/ml. However, the test item was fully miscible in acetone at the same concentration (50 mg/ml) therefore, acetone was selected as the vehicle.

Untreated negative controls:

yes

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:

Tester strains
The four strains of Salmonella used in the test were obtained either from the University of California, Berkeley, on culture discs, on 04 August 1995 or from Syngenta CTL, Alderley Edge, as frozen vials, on 20 March 2007. E. coli strain WP2uvrA- was obtained from the British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987. All of the strains were stored at approximately ¯196°C in a Statebourne liquid nitrogen freezer, model SXR 34. Prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate (Mortelmans and Zeiger (2000)).
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37ºC for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Preparation of Test Item
The test item was accurately weighed and appropriate dilutions prepared in acetone by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity content (6.8%) of the test item. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino silicate pellets with a nominal pore diameter of 4 x E-04 microns. All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Preliminary toxicity test
In order to select appropriate dose levels for use in the main test, a preliminary assay was carried out to determine the toxicity of the test item. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The assay was performed by mixing 0.1 ml of bacterial culture (TA100 or WP2uvrA-), 0.1 ml of test item formulation, 0.5 ml of S9-mix or phosphate buffer and 2 ml of molten, trace histidine or tryptophan supplemented, top agar and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Ten concentrations of the test item and a vehicle control (acetone) were tested. In addition, 0.1 ml of the maximum concentration of the test item and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test item. After approximately 48 hours incubation at 37ºC the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Mutation test - Experiment 1 (Range-Finding test).
Up to seven concentrations of the test item (ranging between 0.5 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method. The dose ranges were allocated as follows:
Salmonella strains (without S9-mix): 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate.
Salmonella strains (with S9-mix): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
WP2uvrA- (with and without S9-mix): 50, 150, 500, 1500 and 5000 µg/plate.
Additional dose levels and expanded dose range were selected (where applicable) in order to achieve both four non-toxic doses and the toxic limit of the test item.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test item formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test item both with and without S9-mix.
All of the plates were incubated at 37ºC for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

Mutation test - Experiment 2 (Main test).
The second experiment was performed using fresh bacterial cultures, test item and control solutions. The test item dose range was amended following the results of the range-finding test and ranged between 0.015 and 5000 µg/plate. The dose ranges were allocated as follows:
Salmonella strain TA100 (without S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 µg/plate.
Salmonella strains TA1535 and TA1537 (without S9-mix): 0.15, 0.5, 1.5, 5, 15, 50 and 150 µg/plate.
Salmonella strain TA98 (without S9-mix): 0.015, 0.05, 0.15, 0.5, 5 and 50 µg/plate.
All Salmonella strains (with S9-mix): 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate.
WP2uvrA- (with and without S9-mix): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Additional dose levels and expanded dose range were again selected in order to achieve both four non-toxic doses and the toxic limit of the test item.
The test item formulations and vehicle control were dosed using the pre-incubation method as follows:
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 ml of S9-mix or phosphate buffer and 0.05 ml of the vehicle or test item formulation and incubated for 20 minutes at 37°C with shaking at approximately 130 rpm prior to the addition of 2 ml of molten, trace histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test item both with and without S9-mix.
All of the standard plates were incubated at 37ºC for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

Evaluation criteria:

The reverse mutation assay may be considered valid if the following criteria are met:

Tester Strain Integrity
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Although the number of spontaneous revertants can be expected to fall within the ranges, they may occasionally fall outside these. The experiment may, however, still be considered valid if the organisms respond normally to their respective positive controls.

Tester strain Culture Density
To demonstrate that appropriate numbers of bacterial cells have been plated, the bacterial cell count for each tester strain culture should be in the range of 0.9 to 9 E+09 bacteria per ml.

Positive Control Values
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study must induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.

Cytotoxicity
Generally, a minimum of four non-toxic test item dose levels is required for the assay to be acceptable.

Contamination
There should be no evidence of excessive contamination. However, if a small number of plates are affected by sporadic airborne contaminants then the experiment will be assessed and accepted provided that the integrity of the study is not compromised and that the contamination is not systematic.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: A toxic response was noted for the Salmonella strains with and without S9-mix. No toxicity was observed to E.coli strain WP2uvrA- in the presence of S9 although weakened lawns were noted in the absence of S9-mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test
The test item was toxic at and above 50 µg/plate to TA100 and non toxic to WP2uvrA-. The test item formulation and S9-mix used in this experiment were both shown to be sterile.

Mutation test
Prior to use, the properties of the S typhimurium strains and the E coli strain regarding the membrane permeability, ampicillin resistance, biotin/tryptophan dependence, normal spontaneous mutation rates and viability was performed as defined by Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

In the range-finding test (plate incorporation), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains initially from 50 and 500 µg/plate in the absence and presence of S9-mix, respectively. No toxicity was noted to Escherichia coli strain WP2uvrA- at any dose level either in the absence or presence of S9-mix. In the main test (pre-incubation), a stronger test item induced toxic response was noted with visible reductions in the growth of the bacterial background lawns noted for the Salmonella strains from 5 and 150 µg/plate in the absence and presence of S9-mix, respectively. No toxicity was observed to E.coli strain WP2uvrA- in the presence of S9 although weakened lawns were noted in the absence of S9-mix from 500 µg/plate. The sensitivity of the bacterial tester strains to the toxicity of the test item varied between bacterial strain type, exposures with and without S9-mix and methodology. The test item was, therefore, either tested up to the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial strain type, exposure to S9 mix and experiment number. An oily, globular precipitate was observed at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Preliminary toxicity test

The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	143	116	131	134	97	105	90 *	108 *	33 *	8 *	11*P
+	TA100	88	87	73	100	100	69	81	94	29 *	15 *	8 *P
-	WP2uvrA^-	34	21	24	20	24	36	35	20	25	20	27 P
+	WP2uvrA^-	33	20	26	26	30	28	26	38	20	29	32 P

* Partial absence of bacterial background lawn

P Precipitate

Spontaneous Mutation Rates (Concurrent Negative Controls)

Range-finding Test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
98		20		35		27		12
100	(99)	21	(20)	51	(44)	22	(24)	14	(13)
98		18		46		22		13

Main Test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
93		13		47		22		10
99	(93)	18	(16)	55	(47) ††	15	(18)	7	(9)
86		18		39		17		9
102						31
97	(96) †					15	(25) †
89						28

†         Experintal procedure perford at a later date (without S9-mix only) due to excessive toxicity in the original test

††       Experintal procedure perford at a later date (with and without S9-mix) due to elevated solvent control values in the original test

 

Test Results: Range-Finding Test– Without Metabolic Activation

Test Period		From: 01 July 2010					To: 04 July 2010
With or 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	101 106 101	(103) 2.9#	20 18 13	(17) 3.6	43 42 51		(45) 4.9	15 26 22		(21) 5.6	7 13 13		(11) 3.5
-	0.5	107 98 107	(104) 5.2	12 24 23	(20) 6.7	N/T			25 18 25		(23) 4.0	12 11 12		(12) 0.6
-	1.5	103 101 98	(101) 2.5	23 25 21	(23) 2.0	N/T			22 25 31		(26) 4.6	13 5 14		(11) 4.9
-	5	107 92 95	(98) 7.9	20 30 16	(22) 7.2	N/T			23 26 18		(22) 4.0	15 14 11		(13) 2.1
-	15	99 108 108	(105) 5.2	18 12 16	(15) 3.1	N/T			20 21 11		(17) 5.5	10 12 8		(10) 2.0
-	50	106 * 100 * 113 *	(106) 6.5	13 13 8	(11) 2.9	46 41 51		(46) 5.0	24 12 22		(19) 6.4	10 11 12		(11) 1.0
-	150	111 * 96 * 99 *	(102) 7.9	19 * 12 * 14 *	(15) 3.6	44 40 47		(44) 3.5	10 * 15 * 10 *		(12) 2.9	7 * 10 * 10 *		(9) 1.7
-	500	84 * 67 * 78 *	(76) 8.6	16 * 14 * 21 *	(17) 3.6	48 47 34		(43) 7.8	11 * 14 * 9 *		(11) 2.5	5 * 10 * 7 *		(7) 2.5
-	1500	N/T		N/T		41 27 42		(37) 8.4	N/T			N/T
-	5000	N/T		N/T		26 P 30 P 43 P		(33) 8.9	N/T			N/T
Positive controls   S9-Mix   -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO			9AA
		3		5		2			0.2			80
		515 521 441	(492) 44.6	145 146 145	(145) 0.6	263 258 214		(245) 27.0	143 128 120	(130) 11.7		2614 1283 1645	(1847) 688.2

 ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

N/T     Not tested at this dose level

P        Precipitate

*         Partial absence of bacterial background lawn

#        Standard deviation

 

Test Results: Range-Finding Test– With Metabolic Activation

Test Period		From: 01 July 2010							To: 04 July 2010
With or 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	92 79 104	(92) 12.5#		11 15 9	(12) 3.1		60 42 53		(52) 9.1	29 24 29	(27) 2.9	15 13 18	(15) 2.5
+	5	70 73 100	(81) 16.5		13 16 16	(15) 1.7		N/T			27 30 30	(29) 1.7	10 12 15	(12) 2.5
+	15	86 97 54	(79) 22.3		10 18 16	(15) 4.2		N/T			34 16 30	(27) 9.5	10 14 14	(13) 2.3
+	50	81 82 90	(84) 4.9		18 12 15	(15) 3.0		53 56 55		(55) 1.5	27 29 18	(25) 5.9	13 13 12	(13) 0.6
+	150	73 85 82	(80) 6.2		14 5 18	(12) 6.7		56 47 52		(52) 4.5	24 27 18	(23) 4.6	7 12 11	(10) 2.6
+	500	85 * 67 * 86 *	(79) 10.7		12 16 14	(14) 2.0		52 38 53		(48) 8.4	26 * 13 * 21 *	(20) 6.6	5 5 10	(7) 2.9
+	1500	34 * 53 * 52 *	(46) 10.7		13 * 11 * 10 *	(11) 1.5		42 44 37		(41) 3.6	9 * 14 * 4 *	(9) 5.0	4 * 1 * 9 *	(5) 4.0
+	5000	35 P * 30 P * 34 P *		(33) 2.6	13 P * 13 P * 14 P *		(13) 0.6	37 P 40 P 41 P		(39) 2.1	7 P * 0 P * 4 P *	(4) 3.5	2 P * 1 P * 2 P *	(2) 0.6
Positive controls   S9-Mix   +	Name Concentration (μg/plate) No. colonies per plate	2AA			2AA			2AA			BP		2AA
		1			2			10			5		2
		1530 1286 1379	(1398) 123.1		187 144 159	(163) 21.8		256 274 239		(256) 17.5	200 185 177	(187) 11.7	167 143 142	(151) 14.2

BP      Benzo(a)pyrene

2AA    2-Aminoanthracene

N/T     Not tested at this dose level

P        Precipitate

*         Partial absence of bacterial background lawn

#         Standard deviation

 

Conclusions:

Interpretation of results (migrated information):
negative

The test item, TM 10-202, was considered to be non-mutagenic under the conditions of this test.

Executive summary:

The genotoxic potential of the test substance, TM 10-202, was assessed as negative in the presence and absence of metabolic activation according to OECD Test Guideline 471 using the Bacterial Reverse Mutation method.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Justification for selection of genetic toxicity endpoint
The study was conducted on the target substance in appropriate test species and according to internationally recognised guidelines.

Justification for classification or non-classification

This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.

To arrive at a classification, test results are considered from experiments determining mutagenic and genotoxic effects in germ and/or somatic cells of exposed animals and in in vitro tests.

The system is hazard based, classifying substances on the basis of their intrinsic ability to induce mutations in germs cells, and does not give a quantitative assessment of the risk.

To this end, the test substance has been assessed according to internationally recognized guidelines in an in vitro gene mutation study in bacteria (Ames test). 

 

In the Ames test, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains tested with any dose of the test item, either with or without metabolic activation.

 

Based on the negative results in vitro, the test item is considered non-mutagenic.