2-methylglutaronitrile

Genetic toxicity: in vitro

Administrative data

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:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study performed similarly to OECD guidelines (Nr. 471) but the study was not performed with the GLP. The E. coli or S. thyphimurium TA102 were not tested.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1988

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

E. coli or S. thyphimurium TA102 were not tested

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-methylglutaronitrile

EC Number:

224-923-5

EC Name:

2-methylglutaronitrile

Cas Number:

4553-62-2

Molecular formula:

C6H8N2

IUPAC Name:

2-methylpentanedinitrile

Constituent 2

Reference substance name:

methyl-2-glutaronitrile

IUPAC Name:

methyl-2-glutaronitrile

Details on test material:

- Name of test material (as cited in study report): Methyl Glutaronitrile
- Purity: 85 %
No other data were available

Method

Target gene:

gene of Histidine

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 1537, TA1535, TA100, TA97, TA98

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S-9 fractions of Aroclor 1254-induced males Sprague-Dawley rat and male Syrian hamsters livers (5, 10 or 30 %)

Test concentrations with justification for top dose:

0; 100; 333; 1000; 3333 and 10000 µg/plate.
The appropriate dose range was evaluated in a toxicity assay with TA100.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9: sodium azide (TA1535 and TA100), 9-aminoacridine (TA97 and TA1537), 4-nitro-o-phenylenediamine (TA98). With S9: 2-aminoanthracene.

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation (variation of Haworth et al. protocol, 1983)

DURATION
- Incubation period: 48 hours at 37°C

NUMBER OF REPLICATIONS: triplicate.
For some conditions (some strains and some condition of metabolic activation), the test was performed twice or three times to try to confirm the results.

Evaluation criteria:

Chemicals were judged mutagenic (+), weakly mutagenic (+W), questionable (?) or nonmutagenic (-), depending on the magnitude of the increase in
his+ revertants, and the shape of the dose-response. A trial was considered questionable if the dose-response was judged insufficiently high to
support a call of “+W”, if only a single dose was elevated over the control, or if a weak increase was not dose-related. The distinctions between a
questionable response and a nonmutagenic response, and between a weakly mutagenic response and mutagenic response are highly subjective. It
was not necessary for a response to reach two-folder over background for a trial to be judged mutagenic.
A chemical was judged mutagenic pr weakly mutagenic if it produced a reproducible, dose-related response over the solvent control, under a single metabolic activation condition, in replicate trials. A chemical was judged questionable if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a “+W” response, or if only single doses produced increases in his+ revertants in repeat trials. Chemicals were judged nonmutagenic if they did not meet the criteria for a mutagenic or questionable response.

Statistics:

No data

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

For the TA100, TA1535, TA1537 and TA98, the results are clearly negative with and without metabolic activation. See results in tables 7.6.1/1 to 7.6.1/4. For the TA97 strain, the author judged the results as ambigous. See results in tables 7.6.1/5 to 7.6.

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No additional information.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/1:Results in terms of mutagenicity for the strain TA100.The mean of the number of revertants (triplicate) is presented for each condition. The interpretation of the publication author is written in red.

 

Dose (µg/plate)	Without metabolic activation		10%HLI		10%RLI
	Mean	SD	Mean	SD	Mean	SD
	-		-		-
0	63	2.5	89	11.4	78	3.6
100	78	7.2	81	4.7	88	6.2
333	78	4.2	82	7.1	72	0.3
1000	66	4.7	84	4.1	83	5.8
3333	81	3.2	80	1.2	71	12.6
10000	73	2.1	87	7.8	75	5.5
POS	260	16.8	389	60.0	346	12.0

HLI=Aroclor 1254-induced hamster liver

HRI=Aroclor 1254-induced rat liver

POS: Positive control

?: questionable mutagenic response

-: nonmutagenic

+W: weakly mutagenic

S: slight clearing of bacterial lawn

 

Table 7.6.1/2:Results in terms of mutagenicity for the strain TA1535.The mean of the number of revertants (triplicate) is presented for each condition. The interpretation of the publication author is written in red.

 

Dose (µg/plate)	Without metabolic activation		10%HLI		10%RLI
	Mean	SD	Mean	SD	Mean	SD
	-		-		-
0	17	3.9	5	1.7	8	2.1
100	13	0.0	8	1.9	6	0.3
333	11	3.5	6	0.9	7	1.8
1000	10	2.6	8	1.5	6	1.5
3333	13	1.9	7	1.7	6	1.2
10000	15	2.1	6	1.2	7	0.9
POS	95	13.9	39	1.5	72	3.0

 HLI=Aroclor 1254-induced hamster liver

HRI=Aroclor 1254-induced rat liver

POS: Positive control

?: questionable mutagenic response

-: nonmutagenic

+W: weakly mutagenic

S: slight clearing of bacterial lawn

 

Table 7.6.1/3:Results in terms of mutagenicity for the strain TA1537.The mean of the number of revertants (triplicate) is presented for each condition.

 

Dose (µg/plate)	Without metabolic activation		10%HLI		30%HLI		10%RLI		30%RLI
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
	-		-		-		-		-
0	7	0.9	5	0.9	6	1.2	6	0.7	5	1.2
100	6	0.7	5	0.9	4	0.9	6	1.7	5	1.2
333	4	0.3	4	1.2	5	0.7	5	3.2	5	1.3
1000	4	1.2	5	0.6	3	0.3	6	1.2	6	3.1
3333	5	1.2	5	1.2	3	0.6	6	1.2	5	0.3
10000	6	0.3	2	0.3	5	1.2	3	0.3	5	2.1
POS	23	1.7	734	31.7	1213	19.2	69	7.4	23	3.9

 HLI=Aroclor 1254-induced hamster liver

HRI=Aroclor 1254-induced rat liver

POS: Positive control

?: questionable mutagenic response

-: nonmutagenic

+W: weakly mutagenic

S: slight clearing of bacterial lawn

 

Table 7.6.1/4:Results in terms of mutagenicity for the strain TA98.The mean of the number of revertants (triplicate) is presented for each condition. The interpretation of the publication author is written in red.

 

Dose (µg/plate)	Without metabolic activation		10%HLI		10%RLI
	Mean	SD	Mean	SD	Mean	SD
	-		-		-
0	14	2.9	26	4.0	20	1.2
100	13	1.8	20	0.9	17	1.2
333	12	0.9	18	4.9	18	3.2
1000	11	1.5	19	1.7	21	1.9
3333	12	1.8	21	0.9	18	2.6
10000	11	2.3	23	4.3	21	3.6
POS	98	11.3	90	4.4	140	15.1

HLI=Aroclor 1254-induced hamster liver

HRI=Aroclor 1254-induced rat liver

POS: Positive control

?: questionable mutagenic response

-: nonmutagenic

+W: weakly mutagenic

S: slight clearing of bacterial lawn

Table 7.6.1/5:Results in terms of mutagenicity for the strain TA97 without metabolic activation. The mean of the number of revertant (triplicate) is presented for each condition. The interpretation of the publication author is written in red.3 independent experiments are presented in the table.

 

Dose (µg/plate)	Experiment No.1		Experiment No.2		Experiment No.3
	Mean	SD	Mean	SD	Mean	SD
	-		-		-
0	62	3.6	72	4.7	103	4.7
100	51	8.5	75	3.8	112	10.6
333	53	8.0	73	4.3	112	5.5
1000	54	3.3	77	9.3	81	11.0
3333	55	2.0	77	2.1	87	11.6
10000	68	4.4	80s	2.1	89	5.7
POS	101	9.9	803	66.2	295	6.3

HLI=Aroclor 1254-induced hamster liver

HRI=Aroclor 1254-induced rat liver

POS: Positive control

?: questionable mutagenic response

-: nonmutagenic

+W: weakly mutagenic

S: slight clearing of bacterial lawn

 

Table 7.6.1/6:Results in terms of mutagenicity for the strain TA97 with metabolic activation (HLI). The mean of the number of revertant (triplicate) is presented for each condition. The interpretation of the publication author is written in red.Two independant experiments are presented in the table for the HLI concentrations 10 and 30%.

 

Dose (µg/plate)	5%HLI		10%HLI (Exp No. 1)		10%HLI (Exp No. 2)			30%HLI (Exp No. 1)		30%HLI (Exp No. 2)
	Mean	SD	Mean	SD	Mean	SD	Mean		SD	Mean	SD
	-		?		-			+W		+W
0	102	8.5	93	2.9	95	4.7	222		13.8	154	3.2
100	109	5.8	119	6.7	107	10.5	271		16.9	159	1.3
333	102	6.0	113	3.8	94	4.4	369		25.0	134	10.3
1000	101	4.3	114	6.0	101	6.1	378		6.1	152	14.6
3333	119	14.8	121	9.4	105	5.0	239		11.9	260	22.0
10000	106	6.1	146	3.4	110	8.0	47		12.4	256	33.5
POS	1392	134.9	684	32.0	755	5.8	859		55.2	1037	44.7

 

HLI=Aroclor 1254-induced hamster liver

HRI=Aroclor 1254-induced rat liver

POS: Positive control

?: questionable mutagenic response

-: nonmutagenic

+W: weakly mutagenic

S: slight clearing of bacterial lawn

 

 

Table 7.6.1/7:Results in terms of mutagenicity for the strain TA97 with metabolic activation (RLI). The mean of the number of revertant (triplicate) is presented for each condition. The interpretation of the publication author is written in red.Several independant experiments are presented in the table for the RLI concentrations 5, 10 and 30%.

 

Dose (µg/plate)	5%RLI(Exp No. 1)		5%RLI (Exp No. 2)		10%RLI (Exp No. 1)		10%RLI (Exp No. 2)		10%RLI (Exp No. 3)		30%RLI (Exp No. 1)		30%RLI (Exp No. 2)
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
	?		-		-		?		-		-		-
0	208	21.4	100	8.5	99	8.3	169	21.1	158	4.7	160	4.8	174	7.2
100	287	31.9	103	5.7	75	6.8	176	17.9	145	3.8	126	22.5	184	3.2
333	295	40.2	90	11.7	76	5.8	186	11.8	139	15.3	130	18.4	179	6.1
1000	287	28.0	96	0.3	87	10.3	197	6.2	151	6.4	152	7.1	172	18.6
3333	322	2.6	98	0.7	72	4.8	206	50.47	117	1.7	159	2.8	213	13.3
10000	236	50.8	98	5.0	101	3.2	294	43.7	140	23.8	180	3.9	177	4.6
POS	1517	18.7	1584	18.3	548	25.2	776	22.0	741	9.3	316	9.7	382	6.7

 

HLI=Aroclor 1254-induced hamster liver

HRI=Aroclor 1254-induced rat liver

POS: Positive control

?: questionable mutagenic response

-: nonmutagenic

+W: weakly mutagenic

S: slight clearing of bacterial lawn

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the test conditions, Methylglutaronitrile is not mutagenic in bacteria.

Executive summary:

In a reverse gene mutation assay in bacteria (Zeiger et al., 1988) performed similarly to the OECD test guideline No. 471, strains TA 1535, TA 1537, TA 97, TA 98 and TA 100 of S. typhimurium were exposed to methylglutaronitrile (85%) in DMSO at concentrations of 0; 100; 333; 1000; 3333 and 10000 µg/plate. Tests were conducted in the presence and absence of mammalian metabolic activation (S-9 fractions of Aroclor 1254-induced males Sprague-Dawley rat and male Syrian hamsters livers (5, 10 or 30 %)).

The positive controls induced the appropriate responses in the corresponding strains.  

Methylglutaronitrile was tested up to limit concentration (10 mg/plate) recommended in the OECD guideline No. 471 and no significant cytotoxicity was observed.

For the TA100, TA1535, TA1537 and TA98, the results are clearly negative with and without metabolic activation. For the TA97 strain, the author judged the results as ambigous. However, the number of revertants with the test item for the TA 97 strain with and without metabolic activation did never exceed twice the number of revertants obtained with the vehicule control. Therefore, when the number of revertants with the test item increased, this increase can not be considered as significant (expert judgement).

Under the test conditions, Methylglutaronitrile is not considered as mutagenic in bacteria.

This study is considered as acceptable as the main criteria of the OECD guideline No. 471.