Amides, C8-10, N-(hydroxyethyl)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD Guideline 471 (Bacterial Reverse Mutation Assay): negative with and without metabolic activation.

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:

May 24 - June 21, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his and trp operon

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: agar medium contained: 7 g/L purified agar, 6 g/L NaCl. The agar plates also contained 12.2 mg/l biotin and 10.5 mg/l histidine.
Strains were regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Type and identity of media: Agar medium contained: 7 g/L purified agar, 6 g/L NaCl. The agar plates also contained 10.2 mg/l tryptophan.
The sensitivity of the strain to a wide variety of mutagens was enhanced by permeabilization of the strain using Tris-EDTA treatment. The strain was regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital/-naphthoflavone

Test concentrations with justification for top dose:

Experiment 1 : 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate in the presence and abscence of S9-mix (all strains);

Experiment 2:
without S9 mix:
Salmonella strains: 0.3; 1; 3; 10; 33; 100; 333; and 1000 µg/plate
WP2 uvrA: 1; 3; 10; 33; 100; 333; 1000, and 2500 µg/plate
with S9 mix all strains: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (test substance), DMSO and deionised water (positive controls)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see "Details on test system and conditions"

Details on test system and experimental conditions:

Positive control substances:
-S9: sodium azide (10 μg/plate in deionised water) for TA1535 and TA100; 4-nitro-o-phenylene-diamine (10 μg/plate in DMSO) for TA98; 4-nitro-o-phenylene-diamine (50 μg/plate in DMSO) for TA1537; methylmethanesulfonate (3.0 μg/plate in deionised water) for WP2uvrA
+S9: 2-aminoanthracene in DMSO for all strains (TA1535, TA1537, TA98, TA100: 2.5 µg/plate; WP2uvrA: 10 µg/plate)

METHOD OF APPLICATION: in agar (plate incorporation and pre-incubation)

DURATION
- Exposure duration: 48 ± 4h at 37°C in the dark

NUMBER OF REPLICATIONS: 3 replications for each experiment

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A test substance was considered negative (not mutagenic) in the test if:
The total number of revertants in tester strains TA 98, TA 100, and WP2 uvrA was not greater than two times the concurrent control, and the total number of revertants in tester strains TA 1535 and TA 1537 was not greater than three times the concurrent vehicle control.
The negative response was reproducible in at least one independently repeated experiment.

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed (3).
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration (2).
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100; E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see "Additional information on results"

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: yes

RANGE-FINDING/SCREENING STUDIES: In a dose range finding test (reported as part of experiment 1), the test substance was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in all. The test substance precipitated on the plates at the highest dose level.
Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups.
In tester strains TA1535 and TA98, toxicity was observed at dose levels of 333-5000 μg/plate in the absence of S9-mix and at 2500-5000 µg/plate in the presence of S9-mix. In tester strains TA1537 and TA100, toxicity was observed at dose levels of 333-5000 μg/plate in the absence of S9-mix and at 2500-5000 µg/plate in the presence of S9-mix. In tester strain WP2uvrA toxicity was observed at dose levels of 2500-5000 μg/plate in the absence of S9-mix and at 5000 µg/plate in the presence of S9-mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment 2, cytotoxicity was observed in tester strains TA1535, TA98 and TA100 at dose levels of 333-1000 μg/plate in the absence of S9-mix and at 21000-5000 µg/plate in the presence of S9-mix. In tester strain TA1537, toxicity was observed at dose levels of 100-1000 μg/plate in the absence of S9-mix and at 1000-5000 µg/plate in the presence of S9-mix. In tester strain WP2uvrA toxicity was observed at dose levels of 1000-2500 μg/plate in the absence of S9-mix and at dose levels of 2500-5000 µg/plate in the presence of S9-mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1 (Experiment 1): Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain (the S9 -mix contained 5% (v/v) S9 fraction)

Dose (µg/plate)	S9-mix	TA1535	TA1537	TA98	TA100	WP2uvrA
Positive control	-	1328 ± 77	66 ± 3	363 ± 23	1266 ± 64	774 ± 36
Solvent control	-	15 ± 3	14 ± 1	30 ± 2	138 ± 6	60 ± 11
Untreated	-	20 ± 1	13 ± 3	31 ± 6	159 ± 6	60 ± 5
3	-	19 ± 3	14 ± 2	26 ± 7	130 ± 18	53 ± 7
10	-	16 ± 2	14 ± 2	26 ± 3	145 ± 11	63 ± 11
33	-	14 ± 3	14 ± 2	29 ± 2	130 ± 25	64 ± 6
100	-	15 ± 4	12 ± 2	33 ± 4	108 ± 19	57 ± 9
333	-	4 ± 2^M R	2 ± 1^M R	19 ± 5^M R	17 ± 3^M R	47 ± 8
1000	-	0 ± 0 R	0 ± 0 R	3 ± 1^M R	2 ± 1^M R	51 ± 2
2500	-	0 ± 0 R	0 ± 0 R	0 ± 0 R	0 ± 0 R	6 ± 2 M R
5000	-	0 ± 0 P R	0 ± 0 P R	0 ± 0 P R	0 ± 0 P R	0 ± 0 P R
Positive control	+	317 ± 24	196 ± 20	1851 ± 498	2056 ± 26	334 ± 20
Solvent control	+	21 ± 2	21 ± 3	39 ± 6	173 ± 6	70 ± 9
Untreated	+	25 ± 3	27 ± 1	47 ± 7	164 ± 6	71 ± 8
3	+	22 ± 1	24 ± 6	48 ± 4	147 ± 8	57 ± 8
10	+	25 ± 2	26 ± 6	36 ± 4	161 ± 27	63 ± 2
33	+	21 ± 4	22 ± 4	41 ± 8	157 ± 24	72 ± 5
100	+	24 ± 5	23 ± 1	45 ± 3	162 ± 25	68 ± 8
333	+	22 ± 7	26 ± 3	47 ± 10	147 ± 17	61 ± 10
1000	+	12 ± 2	9 ± 1 R	34 ± 6	62 ± 5 R	54 ± 8
2500	+	3 ± 1 MR	0 ± 1 MR	6 ± 2 MR	0 ± 1 R	23 ± 5
5000	+	0 ± 0 PMR	0 ± 0 PMR	0 ± 0 PMR	0 ± 0 PMR	4 ± 2 PMR

(testing 3-5000 µg/plate in all strains represent the dose-range finding test of the study and were reported as the first experiment)

solvent control: DMSO

M: Manual count

R: Reduced background growth

P: Precipitate

Table 2 (Experiment 2): Mean number of revertant colonies/3 replicate plates (± S.D.) with two strains of Salmonella typhimurium

and one Escherichia coli strain (the S9 -mix contained 5% (v/v) S9 fraction)

Dose (µg/plate)	S9-mix	TA1535	TA1537	TA98	TA100	WP2uvrA
Positive control	-	2020 ± 123	94 ± 15	424 ± 19	2049 ± 57	481 ± 24
Solvent control	-	17 ± 5	22 ± 5	26 ± 1	118 ± 12	59 ± 6
Untreated	-	14 ± 5	17 ± 7	25 ± 3	154 ± 7	63 ± 3
0.3	-	16 ± 3	18 ± 4	25 ± 3	112 ± 12
1	-	14 ± 3	19 ± 2	28 ± 5	132 ± 9	63 ± 11
3	-	15 ± 5	17 ± 3	24 ± 4	119 ± 1	62 ± 8
10	-	20 ± 6	18 ± 4	21 ± 1	120 ± 2	59 ± 3
33	-	9 ± 2	17 ± 4	26 ± 6	110 ± 4	70 ± 5
100	-	10 ± 2	2 ± 1 M R	16 ± 3	57 ± 8 M R	49 ± 9
333	-	1 ± 1 M R	1 ± 1 M R	3 ± 1 M R	36 ± 5 M R	50 ± 1
1000	-	0 ± 0 M R	0 ± 0 M R	0 ± 0 M R	0 ± 0 M R	9 ± 2 M R
2500	-					0 ± 0 M R
Positive control	+	294 ± 23	216 ± 9	1380 ± 66	1813 ± 49	280 ± 14
solvent control	+	18 ± 5	23 ± 3	31 ± 6	147 ± 20	62 ± 4
Untreated	+	22 ± 3	27 ± 3	45 ± 12	194 ± 15	70 ± 3
3	+	20 ± 1	22 ± 0	36 ± 6	130 ± 6	67 ± 2
10	+	17 ± 1	27 ± 3	38 ± 5	143 ± 14	69 ± 11
33	+	20 ± 7	22 ± 8	32 ± 5	152 ± 13	64 ± 9
100	+	23 ± 3	23 ± 3	33 ± 8	160 ± 18	72 ± 7
333	+	21 ± 1	24 ± 2	34 ± 5	132 ± 13	80 ± 6
1000	+	5 ± 1 M R	4 ± 1 M R	7 ± 2 M R	13 ± 2 M R	52 ± 8
2500	+	0 ± 0 M R	0 ± 0 M R	2 ± 0 M R	0 ± 0 M R	10 ± 1 R
5000	+	0 ± 0 P M R	0 ± 0 P M R	0 ± 0 P M R	0 ± 0 P M R	0 ± 0 P M R

solvent control: DMSO

M: Manual count

R: Reduced background growth

P: Precipitate

Conclusions:

Interpretation of results (migrated information):
negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

One Bacterial Reverse Mutation Assay according to OECD Guideline 471 was performed in two independent experiments both with and without liver microsomal activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation

rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Therefore, Amides, C8-10 (even numbered) is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.