R-AMINR

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two seperate Ames tests are available covering all the standard strains required in the OECD 471 guideline. All available results were negative for mutagenic activity.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Preceeds OECD guideline

Deviations:

no

Remarks:

Test missing the strains with AT base pair at primary reversion site.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Batches were checked for strain characteristics of histidine dependence, rfa character and resistance to ampicillin (TA98 and TA100).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
The S9 liver fraction was prepared from male Sprague Dawley rats induced with Aroclor 1254 and obtained from Molecular Toxicology Incorporated, USA. Batches were stored frozen and thawed just prior to incorporation into the top agar. Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens and CYP450 catalysed enzyme activities.

Test concentrations with justification for top dose:

Range-finder and Mutation experiment 1: 8, 40, 200, 1000 and 5000 µg/plate.
Mutation experiment 2: 312.5, 625, 1250, 2500 and 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: standard as per OECD guideline

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

Range-finder: The test substance was tested in TA100. Triplicate plates without and with S9 mix were used. Negative and positive controls were included in quintuplicate and triplicate respectively without and with S9 mix. These platings were achieved by the following sequence of additions to 2.5 mL molten agar at 46°C: 0.1 mL bacterial culture, 0.1 mL test article solution or control and 0.5 mL 10% S9 mix or buffer solution. This was followed by rapid mixing and pouring on to Minimal Davis agar plates. When set, the plates were inverted and incubated at 37°C in the dark for 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn and where possible revertant colonies were counted.

Main experiment: The test substance was tested in S. typhimurium strains TA98, TA100, TA1535 and TA1537 using triplicate plates with and without S9. Negative (solvent) controls wre included in both assays, in quintuplicate with and without S9. In each experiment, bacterial strains were treated with diagnostic mutagens in triplicate in the absence of S9. The activity of the S9 mix used ine ach experiment was confirmed by AAN treatments (in triplicate) of at least 1 strain in the presence of S9. Platings were achieved as described for the range finder.

As the results of the first experiment were negative, treatments in the presence of S9 in experiment 2 included a pre-incubation step, where the quantities of test article or control solution, bacteria and S9 mixwere mixed together and incubated for 1 hour at 37°C, before tha ddition of 2.5 mL molten agar at 46°C. Plating of these treatments then proceeded as for the normal plate-incorporation rpocedure. This was performed to help increase the sensitivity of the assay.

Colony counting:
Colonies were counted electronically using a Seescan Colony Counter and the background lawn inspected for signs of toxicity.

Rationale for test conditions:

Standard as per OECD guideline

Evaluation criteria:

Treatment of data: Individual plate counts from both experiments were recorded seperately and the mean and SD of the plate counts for each treatment were determined.

The assay was considered valid if the following criteria were met:
1) The mean negative control counts fell within the normal ranges.
2) The positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains and an active S9 preparation.
3) No more than 5% of the plates were lost through contamination or some other unforeseen event.

The test article was considered to be mutagenic if:
1) The assay was valid.
2) Dunnett's test gave a significant response (P 3) The positive responses described were reproducible.

Statistics:

The m-statistic was calculated to check that the data were Poisson-distributed and Dunnett's test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was checked by linear regression analysis.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Toxicity at 5000 µg/plate (+/- S9)

Remarks:

Slight thinning of background lawn

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True 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:

other: Toxicity at 5000 µg/plate (+S9)

Remarks:

Slight thinning of background lawn

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True 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:

other: Toxicity at 5000 µg/plate (+S9)

Remarks:

Slight thinning of background lawn

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True 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:

other: Toxicity at 5000 µg/plate (+/- S9)

Remarks:

Slight thinning of background lawn

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Toxicity and dose selection:
The range-finder was carried out with strain TA100 only at concentrations of 8, 40, 200, 1000 and 5000 µg/plate with negative and positive controls. No evidence of toxicity was observed, so the results were considered acceptable as mutagenicity data for this strain. Treatments of the remaining strains were performed using the same doses. Evidence of toxicity in the form of a thinning of teh background lawn was observed following 5000 µg/plate treatments with strain TA1537 ain the absence and presence of S9, and of strain TA98 in the presence of S9.

As evidence of toxicity was observed in 2 strains and solely at the maximum dose, this dose was retained as the maximum test dose for all the second experiment treatments (this dose being the best estimate of the limit of toxicity). A narrowed dose range was employed in order to more closely investigate those doses of the substance most likely to induce mutation. In addition, all experiment 2 treatments in the presence of S9 incorporate a pre-incubation step. Evidence of toxicity (in the form of a slight thinning of the background lawn) was observed in all strains at 5000 µg/plate in the presence of S9 and in TA98 and TA1537 at 5000 µg/plate in the absence of S9.

Mutation: The mean solvent controls fell within the normal historical ranges and the positive control chemicals all induced large increases in revertant numbers in the appropriate strains and that less than 5% of plates were lost, leaving adequate numbers of plates at all treatments. The study was accepted as valid.

No test substance treatment of any of the test strains in the absence or presence of S9 producted an increase in revertant numbers that was statistically significant at the 1% level. There was, therefore, no evidence of mutagenic activity of the substance in this study.

Conclusions:

The test substance was unable to induce mutations in 4 strains of S. typhimurium when tested at concentrations up to 5000 µg/plate both in the absence and presence of a metabolic activation system.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

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:

yes

Remarks:

Only 1 strain used which was not included in original Ames test

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

BACTERIAL CELLS USED
- The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using
Tris-EDTA treatment. The strain is checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants at least every year.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1°C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/mL). Freshly grown cultures of each strain were used for testing.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
Each S9 batch is characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 µg/plate and 2.5 µg/plate, respectively.

S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL or 5.0 mL Milli-Q water (first or second experiment respectively) (Millipore Corp., Bedford, MA., USA); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution (Merck); 1 mL 0.33 M KCl solution (Merck). The above solution was filter (0.22 µm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 mL of S9-mix components 1.0 mL S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment.

Test concentrations with justification for top dose:

First experiment: Control, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate. The high dose is the higest recommended in the test guideline.
Second experiment: Control, 492, 878, 1568, 2800 and 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: Standard vehicle as per OECD guideline

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

other: 2-Aminoanthracene (2AA)

Details on test system and experimental conditions:

Dose preparation: A solubility test was performed based on visual assessment. The test item formed a clear colourless solution in DMSO. The stock solution was treated with ultrasonic waves until the test item had completely dissolved in the dose range finding study. Test item concentrations were used within 2.5 hours after preparation.

Procedure: Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test item in DMSO and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (Trp+) for Escherichia coli were counted.

Colony counting: The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Rationale for test conditions:

Standard as per OECD guidelines.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
- The total number of revertants in the tester strain WP2uvrA is not greater than two times the concurrent vehicle control.
- The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
- The total number of revertants in the tester strain WP2uvrA is greater than two times the concurrent vehicle control.
- In case a follow up experiment is performed when a positive response is observed, the positive response should be reproducible in at least one follow up experiment.

Statistics:

No statistical analysis was conducted.

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

R-Amine was tested in the tester strain WP2uvrA at concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the absence and presence of 5% S9-mix. Based on the results of the first experiment, the following dose-range was selected for the second mutation experiment with the tester strain, WP2uvrA in the absence and presence of 10% S9-mix: 492, 878, 1568, 2800 and 5000 μg/plate. No precipitation of the substance on the plates was not observed.

To determine the toxicity of the test substance, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. No increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.

Mutagenic response of the test substance in the E.coli reverse mutation assay:

Dose (µg/plate)	Mean no. of revertant colonies/3 replicate plates (+/- SD) with E.coli strain
	Experiment 1 (without 5% S9-mix)					Experiment 1 (with 5% S9-mix)
Positive control	1437		+/-	126		246	+/-	65
Solvent control	15		+/-	1		16	+/-	9
1.7	19		+/-	4		22	+/-	6
5.4	19		+/-	7		18	+/-	2
17	16		+/-	3		19	+/-	3
52	18		+/-	3		23	+/-	6
164	14		+/-	7		20	+/-	6
512	16		+/-	2		22	+/-	8
1600	20		+/-	3		18	+/-	2
5000	18		+/-	5		21	+/-	3
Dose (µg/plate)	Mean no. of revertant colonies/3 replicate plates (+/- SD) with E.coli strain
	Experiment 2 (without 10% S9-mix)					Experiment 2 (with 10% S9-mix)
Positive control	1242	+/-			171	247	+/-	22
Solvent control	18	+/-			3	25	+/-	2
492	23	+/-			6	18	+/-	7
878	16	+/-			7	21	+/-	4
1568	17	+/-			1	18	+/-	5
2800	15	+/-			3	18	+/-	4
5000	18	+/-			5	25	+/-	5

Conclusions:

In conclusion, based on the results of this study it is concluded that the substance is not mutagenic in the Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Only the results of an Ames test are available and required at REACH Annex VII. The result on all the necessary strains indicated no mutagenic potential of the substance. Based on this classification is not warranted for germ cell mutagenicity in accordance with the CLP Reglation (EC No. 1272/2008, as amended).