Morpholine, 4-(6-hydrazinyl-4-pyrimidinyl)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Salmonella/microsome test (Ames test): positive with strains TA 98, TA 102 (+ S9 mix) and TA 100 (+/- S9 mix) and negative with strains TA 1535 and TA 1537 (+/- S9 mix)

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:

other information

Study period:

March 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: guideline study with restriction (non-GLP)

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced male rat liver S9 mix

Test concentrations with justification for top dose:

0, 16, 50, 158, 500, 1581, 5000 µg/plate (first test, +/-S9 mix, all strains)
0, 500, 1000, 2000, 3000, 4000, 5000 and 6000 µg/plate (repeat test, +/-S9 mix, all strains)

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

Remarks:

No solvent control was used since sufficient evidence was available in the literature and from testing laboratory experience, indicating that the solvents used had no influence on the spontaneous mutant counts of the used strains.

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (only TA 1535), nitrofurantoin (only TA 100), 4-nitro-1,2-phenylene diamine (TA 1537 and TA 98), mitomycin C (only TA 102), 2-aminoanthracene (all strains).

Remarks:

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine and mitomycin C were only used without S9 mix; the positive control 2-aminoanthracene was only used with S9 mix.

Details on test system and experimental conditions:

METHOD: each concentration including the controls was tested in triplicate.

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537 at least a threefold increase should be reached. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these criteria may be overruled by good scientific judgment. In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Statistics:

not specified

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: TA 98, TA 102

Remarks:

Migrated from field 'Test system'.

Table 1: Summary of results from the Salmonella mutagenicity assay (first test) with 4 -Hydrazo-6 -morph-pyrimidin (mean values of revertants per plate)

Dose (µg per plate)	Without metabolic activation
	TA 1535	TA 100	TA 1537	TA 98	TA 102
0	9	110	9	24	245
16	5	98	6	28	234
50	9	107	6	25	228
158	8	116	5	25	235
500	7	114	6	29	186
1581	7	100	9	35	273
5000	7	217	10	41	318
Positive control	1016	435	44	97	1397
Dose ( µg per plate )	With metabolic activation (liver S9 mix)
	TA 1535	TA 100	TA 1537	TA 98	TA 102
0	7	191	10	38	350
16	9	177	7	31	309
50	6	148	6	34	277
158	5	121	10	36	336
500	12	150	7	48	378
1581	11	194	13	56	403
5000	14	311	17	84	518
Positive control	84	2472	139	2253	1025

 

Table 2: Summary of results from the Salmonella mutagenicity assay (repeat test with additional concentrations) with 4 -Hydrazo-6 -morph-pyrimidin (mean values of revertants per plate)

Dose (µg per plate)	With metabolic activation  (liver S9 mix)
	TA 1535	TA 100	TA 1537	TA 98	TA 102
0	10	146	9	38	333
500	6	148	10	40	340
1000	8	133	9	43	334
2000	7	166	12	59	327
3000	11	170	8	52	354
4000	16	210	13	56	457
5000	14	227	17	49	525
6000	18	291	20	68	534
Positive control	71	2337	122	2305	1296
Dose ( µg per plate )	Without metabolic activation
	TA 1535	TA 100	TA 1537	TA 98	TA 102
0	8	119	7	28	244
500	9	127	7	39	220
1000	10	123	6	24	226
2000	6	170	7	28	248
3000	9	157	7	38	191
4000	8	181	5	39	260
5000	7	208	9	44	285
6000	1150	245	8	46	284
Positive control	84	366	58	89	1128

 

There was no indication of a bacteriotoxic effect of 4-Hydrazo- 6-morph-pyrimidin at doses of up to and including 6000 µg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well.

Three of the five strains concerned revealed a relevant increase in mutant counts. Strains TA 100, TA 98 and TA 102 were affected. The findings for Salmonella typhimurium TA 100, TA 98 and TA 102 were confirmed by further repeat tests. The lowest dose at which this finding was observed was 4000 µg per plate for Salmonella typhimurium TA 102 and 6000 µg per plate for Salmonella typhimurium TA l 00 and T A 98. Positive findings for TA 98 and TA l02 were obtained only with S9 mix. Positive findings for TA l00 were obtained with and without S9 mix, the effects being comparable.

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, mitomycin C and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Executive summary:

The mutagenic potential of the test material was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of S9 mix according to OECD TG 471. Doses up to and including 6000 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. Evidence of mutagenic activity of the test material was seen. On Salmonella typhimurium TA 102 and TA 98, a biologically relevant increase was found in the mutant count compared to the corresponding negative control. Positive response was found only with S9 mix. For TA 100 there was a positive response without and with S9 mix, and the effect was comparable. The lowest effective dose was 4000 µg per plate for Salmonella typhimurium TA 102 and 6000 µg per plate for TA 100 and TA 98. The Salmonella/microsome test thus showed the test material to have a mutagenic effect.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

Additional information from genetic toxicity in vitro:

The mutagenic potential of the test material was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of S9 mix according to OECD TG 471. Doses up to and including 6000 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. Evidence of mutagenic activity of the test material was seen. On Salmonella typhimurium TA 102 and TA 98, a biologically relevant increase was found in the mutant count compared to the corresponding negative control. Positive response was found only with S9 mix. For TA 100 there was a positive response without and with S9 mix, and the effect was comparable. The lowest effective dose was 4000 µg per plate for Salmonella typhimurium TA 102 and 6000 µg per plate for TA 100 and TA 98. The Salmonella/microsome test thus showed the test material to have a mutagenic effect.

Justification for selection of genetic toxicity endpoint
The second study is restricted to three Salmonella typhimurium L T2 mutants (strains TA 98; TA 100 and TA 102) which showed a positive response in a previous study (= rel 2, Ames, Herbold 2011)

Justification for classification or non-classification

Based on the available information (restricted to in vitro studies detecting gene mutations in bacteria) a classification according to Directive 67/548/EEC or Regulation (EC) No. 1272/2008 (CLP) is not required although the test result is positive.