Reaction mass of 1-Hydroxyoctane, 2-methanoate and 1,2-Dihydroxyoctane, dimethanoate and 2-Hydroxyoctane 1-methanoate and 1,2-Dihydroxyoctane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 - 21 Mar 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 Jul 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1-Hydroxyoctane, 2-methanoate and 1,2-Dihydroxyoctane, dimethanoate and 2-Hydroxyoctane 1-methanoate and 1,2-Dihydroxyoctane

EC Number:

937-960-3

Molecular formula:

not applicabe (reaction mass)

IUPAC Name:

Reaction mass of 1-Hydroxyoctane, 2-methanoate and 1,2-Dihydroxyoctane, dimethanoate and 2-Hydroxyoctane 1-methanoate and 1,2-Dihydroxyoctane

Method

Target gene:

his operon (for S. typhimurium strains)
trp operon (for E. coli strain)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

Pre-experiment: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation

The pre-experiment is reported as Experiment 1.

Experiment 2:
3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative low toxicity to bacteria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA)

Remarks:

+S9: 2-AA (2.5 µg/plate, TA1535, TA1537, TA98, TA100; 10 µg/plate, WP2 uvrA); -S9: NaN3 (10 µg/plate, TA1535, TA100); 4-NOPD (10 µg/plate, TA98; 50 µg/plate, TA1537); MMS (2 µL/plate, WP2 uvrA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (Experiment 1); preincubation (Experiment 2)

DURATION
- Preincubation period: 1 h
- Exposure duration: at least 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn

Rationale for test conditions:

The test conditions were chosen according to OECD 471.

Evaluation criteria:

A test substance is considered as a mutagenic 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.
A dose-dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
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.

Statistics:

Mean values and standard deviations were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 1: +S9: at 5000 µg/plate; Exp. 2: -S9: starting at 1000 µg/plate, + S9: starting at 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 1: -S9: starting at 2500 µg/plate, +S9: at 5000 µg/plate; Exp. 2: -S9: starting at 1000 µg/plate, + S9: starting at 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 1: -S9 and +S9: at 5000 µg/plate; Exp. 2: -S9 and +S9: starting at 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 1 and 2: -S9 and +S9: starting at 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 1: +S9: at 5000 µg/plate; Exp. 2: -S9: at 5000 µg/plate, + S9: starting at 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate in the presence of metabolic activation. Precipitation of the test substance in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate in the absence of metabolic activation and at 5000 µg/plate in the presence of metabolic activation in both experiments. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment is reported as Experiment I (all strains were tested in the pre-experiment).

ADDITIONAL INFORMATION ON CYTOTOXICITY: The plates incubated with the test substance showed normal background growth up to 5000 µg/plate with and without metabolic activation in all strains used. Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed in all strains with and without metabolic activation.

Any other information on results incl. tables

Table 1. Test results of Experiment 1 (plate incorporation).

With or without S9-Mix	Test substance concentration [μg/plate]	Mean number of revertant colonies per plate (average of 3 plates ± SD)
		Base-pair substitution type			Frameshift type
		TA1535	TA100	WP2 uvrA	TA1537	TA98
-	0 (DMSO)	13 ± 4	171 ± 14	45 ± 9	9 ± 4	24 ± 6
-	0	8 ± 2	198 ± 4	50 ± 4	10 ± 3	33 ± 7
-	3	12 ± 4	175 ± 15	38 ± 4	8 ± 2	20 ± 4
-	10	11 ± 6	174 ± 15	50 ± 6	8 ± 2	25 ± 8
-	33	11 ± 5	167 ± 6	41 ± 4	10 ± 4	20 ± 4
-	100	14 ± 2	182 ± 6	38 ± 10	8 ± 2	25 ± 3
-	333	11 ± 4	90 ± 22	53 ± 3	10 ± 4	23 ± 8
-	1000	10 ± 2	75 ± 19	35 ± 6	11 ± 3	19 ± 6
-	2500	13 ± 6^P	71 ± 5^P	24 ± 8^P	3 ± 1^P, M	14 ± 5^P
-	5000	9 ± 2^P, M	14 ± 2^P, M	23 ± 6^P	2 ± 1^P, M	6 ± 1^P, M
Positive controls, –S9	Name	NaN3	NaN3	MMS	4-NOPD	4-NOPD
	Concentration [μg/plate]	10	10	2.0 µL	50	10
	Mean No. of colonies/plate (average of 3 plates ± SD)	1219 ± 76	2247 ± 214	1050 ± 118	71 ± 11	377 ± 9
+	0 (DMSO)	12 ± 3	157 ± 23	48 ± 2	11 ± 6	29 ± 8
+	0	10 ± 3	182 ± 6	52 ± 15	10 ± 5	34 ± 2
+	3	12 ± 3	138 ± 9	50 ± 8	10 ± 3	38 ± 13
+	10	10 ± 2	151 ± 18	56 ± 10	10 ± 2	33 ± 5
+	33	14 ± 5	150 ± 8	51 ± 8	12 ± 6	29 ± 6
+	100	11 ± 3	161 ± 19	60 ± 11	11 ± 3	27 ± 3
+	333	14 ± 5	172 ± 6	53 ± 9	11 ± 2	37 ± 3
+	1000	13 ± 4	65 ± 4	34 ± 3	14 ± 4	28 ± 5
+	2500	9 ± 2	35 ± 4	27 ± 6	11 ± 4	30 ± 7
+	5000	4 ± 2^P, M	11 ± 4^P, M	9 ± 2^P, M	2 ± 1^P, M	12 ± 3^P, M
Positive controls, +S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentration [μg/plate]	2.5	2.5	10	2.5	2.5
	Mean No. of colonies/plate (average of 3 plates ± SD)	510 ± 23	3056 ± 141	413 ± 83	103 ± 4	4678 ± 203

NaN3: sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

M: manual count

P: precipitate

 

Table 2. Test results of Experiment 2 (preincubation).

With or without S9-Mix	Test substance concentration [μg/plate]	Mean number of revertant colonies per plate (average of 3 plates ± SD)
		Base-pair substitution type			Frameshift type
		TA1535	TA100	WP2 uvrA	TA1537	TA98
-	0 (DMSO)	11 ± 2	154 ± 20	31 ± 2	8 ± 1	21 ± 4
-	0	17 ± 4	195 ± 14	37 ± 10	9 ± 3	27 ± 8
-	3	13 ± 4	132 ± 13	27 ± 3	7 ± 2	21 ± 1
-	10	13 ± 4	144 ± 11	32 ± 2	6 ± 2	25 ± 4
-	33	13 ± 3	152 ± 6	30 ± 7	7 ± 4	23 ± 6
-	100	11 ± 3	86 ± 17	30 ± 6	5 ± 2	21 ± 9
-	333	11 ± 4	79 ± 15	30 ± 1	7 ± 1	28 ± 8
-	1000	4 ± 3	10 ± 4	22 ± 1	2 ± 1	17 ± 3
-	2500	4 ± 1^P	2 ± 1^P	29 ± 5^P	2 ± 1^P	1 ± 0^P
-	5000	1 ± 1^P	0 ± 0^P	1 ± 1^P, M	0 ± 0^P, M	0 ± 0^P
Positive controls, –S9	Name	NaN3	NaN3	MMS	4-NOPD	4-NOPD
	Concentration [μg/plate]	10	10	2.0 µL	50	10
	Mean No. of colonies/plate (average of 3 plates ± SD)	1288 ± 37	2038 ± 84	695 ± 35	102 ± 20	430 ± 35
+	0 (DMSO)	11 ± 4	148 ± 20	41 ± 8	10 ± 3	29 ± 5
+	0	14 ± 6	182 ± 15	41 ± 7	11 ± 2	35 ± 3
+	3	11 ± 5	125 ± 10	35 ± 8	12 ± 6	32 ± 8
+	10	11 ± 5	151 ± 24	42 ± 5	9 ± 1	29 ± 1
+	33	13 ± 6	131 ± 4	36 ± 6	10 ± 3	27 ± 5
+	100	13 ± 2	129 ± 9	37 ± 4	10 ± 2	28 ± 6
+	333	13 ± 3	124 ± 5	43 ± 1	11 ± 2	36 ± 8
+	1000	12 ± 3	46 ± 3	36 ± 8	9 ± 3	27 ± 9
+	2500	1 ± 1	3 ± 1	5 ± 3	1 ± 1	1 ± 1
+	5000	0 ± 0^P, M	0 ± 0^P, M	1 ± 1^P, M	0 ± 0^P, M	0 ± 0^P, M
Positive controls, +S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentration [μg/plate]	2.5	2.5	10	2.5	2.5
	Mean No. of colonies/plate (average of 3 plates ± SD)	454 ± 33	3078 ± 144	369 ± 36	118 ± 24	4837 ± 627

NaN3: sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

M: manual count

P: precipitate

Applicant's summary and conclusion

Conclusions:

Under the conditions of the conducted test the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) tested with and without metabolic activation.

Executive summary:

The plates incubated with the test item showed normal background growth up to 5000μg/plate with and without S9 mix in all strains used.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with 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.