Reaction mass of 1,5-naphthylene diisocyanate and Amines, hydrogenated tallow alkyl

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:

21 June 2017 - 13 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of 1,5-naphthylene diisocyanate (221-641-4) and cyclohexylamine (203-629-0)

Molecular formula:

C24H32N4O2

IUPAC Name:

Reaction product of 1,5-naphthylene diisocyanate (221-641-4) and cyclohexylamine (203-629-0)

Test material form:

solid

Method

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:

S-9 mix

Test concentrations with justification for top dose:

The eight concentrations of the test item in experiment 1 were: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The maximum concentration was 5000 µg/plate, the maximum recommended dose level.
In experiment 2 concentrations were: 5, 50, 150, 500, 1500 and 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl formide (DMF)
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test item formed the best doseable suspension in dimethyl formamide, therefore, this solvent was selected as the vehicle.

Controls

Untreated negative controls:

yes

Remarks:

Untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMF

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene (2AA); In DMSO solvent; Used in the presence of S9-mix

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: At 37 ± 3 °C for 20 minutes (in experiment 2 only)
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3
- Number of independent experiments: 2

Rationale for test conditions:

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Remarks:

Plate incorporation and pre-incubation methods

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Remarks:

Plate incorporation and pre-incubation methods

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

E. coli WP2 uvr A

Remarks:

Plate incorporation and pre-incubation methods

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Remarks:

Plate incorporation and pre-incubation methods

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1537

Remarks:

Plate incorporation and pre-incubation methods

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None reported
- Effects of osmolality: None reported
- Evaporation from medium: None reported
- Water solubility: The test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test item formed the best doseable suspension in dimethyl formamide, therefore, this solvent was selected as the vehicle.
- Precipitation: In Experiment 1, (plate incorporation method) a test item precipitate (greasy in appearance) was observed at and above 1500 μg/plate. In Experiment 2, (pre-incubation method) a greasy test item precipitate was again observed from 1500 μg/plate with a white particulate precipitate also noted at 5000 μg/plate. None of these observations prevented the scoring of revertant colonies.
- Other confounding effects: None reported

RANGE-FINDING/SCREENING STUDIES: The results of experiment 1 (plate incorporation method) were used to inform the dose selection for experiment 2 (pre-incubation method)

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: See tables 6 and 7
- Negative (solvent/vehicle) historical control data: See tables 8 and 9

Any other information on results incl. tables

Table 2: Test Results: Experiment 1 - Without Metabolic Activation (Plate Incorporation)

Test Period		From: 28 June 2017					To: 01 July 2017
S9 -Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMF)	101	(94) 5.9#	12	(13)  1.2	33	(35)  2.6	16	(15)  1.2	12	(11) 1.0
		92		14		34		16		11
		90		14		38		14		10
	1.5 µg	89	(83)  5.6	10	(12)  1.5	30	(29)  1.7	19	(16)  4.6	10	(11)  1.2
		82		12		27		19		10
		78		13		30		11		12
	5 µg	87	(84) 9.8	18	(15) 2.3	31	(29) 2.6	18	(14) 4.0	13	(14) 2.1
		73		14		30		10		16
		92		14		26		14		12
	15 µg	103	(92)  10.5	8	(11)  3.8	25	(27)  3.8	10	(11)  2.6	9	(10)  1.0
		82		9		24		14		10
		91		15		31		9		11
	50 µg	93	(95)  4.4	14	(13)  2.3	31	(30)  1.2	14	(14)  0.0	10	(13)  5.2
		100		14		31		14		19
		92		10		29		14		10
	150 µg	100	(95) 5.6	8	(11)  2.6	25	(24)  2.1	15	(16)  2.1	12	(12)  2.5
		96		13		22		14		10
		89		12		26		18		15
	500 µg	101	(89)  10.2	13	(13)  0.6	35	(30)  7.0	16	(16)  0.6	10	(11)  1.2
		82		12		22		16		12
		85		13		33		15		10
	1500 µg	92 P	(98) 6.5	13 P	(12)  0.6	34 P	(31)  2.3	18 P	(16) 1.5	10 P	(10)  0.0
		98 P		12 P		30 P		16 P		10 P
		105 P		12 P		30 P		15 P		10 P
	5000 µg	102 P	(102)  2.5	13 P	(12)  1.7	32 P	(32)  1.0	11 P	(14)  2.9	12 P	(13)  1.2
		105 P		13 P		31 P		16 P		12 P
		100 P		10 P		33 P		16 P		14 P
Positive controls S9 -Mix (-)	Name	ENNG		ENNG		ENNG		4NQO		9AA
	Dose Level	3 µg		5 µg		2 µg		0.2 µg		80 µg
	No. of Revertants	799	(820) 32.4	449	(450) 16.0	866	(878) 10.6	103	(118)  14.1	479	(495) 30.9
		857		435		886		131		476
		803		467		882		120		531

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA 9-Aminoacridine

P Test item precipitate

# Standard deviation

Table 3: Test Results: Experiment 1 - With Metabolic Acitvation (Plate Incorporation)

Test Period		From: 28 June 2017					To: 01 July 2017
S9 -Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMF)	95	(98) 7.9#	11	(12)  1.7	38	(41)  4.6	18	(22)  4.7	8	(9) 1.2
		92		11		46		20		10
		107		14		38		27		8
	1.5 µg	84	(103)  16.5	7	(9)  2.9	48	(38)  9.5	23	(18)  6.1	14	(12) 2.5
		114		12		37		11		9
		111		7		29		19		12
	5 µg	100	(100) 3.5	12	(12) 1.0	37	(38) 2.3	18	(18) 7.0	10	(9) 1.2
		97		13		41		25		10
		104		11		37		11		8
	15 µg	102	(91)  14.7	9	(13)  3.5	37	(39)  4.7	19	(23)  3.8	8	(9)  0.6
		74		16		44		25		9
		96		13		35		26		9
	50 µg	96	(94)  16.1	15	(15)  3.0	41	(39)  2.9	33	(26)  9.9	11	(9)  1.5
		109		12		41		31		9
		77		18		36		15		8
	150 µg	104	(96) 12.7	11	(15)  3.6	38	(35)  3.1	20	(24)  6.4	9	(11)  1.5
		81		18		32		31		12
		102		16		36		20		11
	500 µg	102	(97)  7.8	11	(14)  5.8	40	(41)  1.5	20	(21)  1.0	8	(9)  1.5
		101		11		43		22		9
		88		21		41		21		11
	1500 µg	85 P	(93) 8.5	15 P	(13)  1.5	27 P	(30)  9.3	20 P	(20) 0.6	10 P	(9)  1.2
		92 P		13 P		40 P		20 P		8 P
		102 P		12 P		22 P		21 P		8 P
	5000 µg	100 P	(101)  0.6	13 P	(13)  1.5	38 P	(41)  4.2	24 P	(22)  2.1	5 P	(10)  4.6
		101 P		15 P		46 P		20 P		13 P
		101 P		12 P		40 P		21 P		13 P
Positive controls S9 -Mix (+)	Name	2AA		2AA		2AA		BP		2AA
	Dose Level	1 µg		2 µg		10 µg		5 µg		2 µg
	No. of Revertants	1993	(2032) 111.2	293	(304) 22.9	245	(243) 15.1	223	(228)  4.2	552	(532) 37.3
		2157		330		257		229		555
		1945		288		227		231		489

BP Benzo(a)pyrene

2AA 2-Aminoanthracene

P Test item precipitate

# Standard deviation

Table 4: Test results; Experiment 2 - Without Metabolic Activation (Pre-Incubation)

Test Period		From: 10 July 2017					To: 13 July 2017
S9 -Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMF)	67	(76)  10.5#	9	(9) 2.0	13	(16)  3.1	11	(14) 3.0	8	(11)  3.0
		86		11		19		14		11
		77		7		15		17		14
	15 µg	69	(70)  7.5	16	(14)  2.9	19	(16)  4.4	14	(15)  0.6	13	(13) 4.5
		63		16		11		15		8
		78		11		18		15		17
	50 µg	74	(76)  4.9	7	(11) 4.6	18	(17)  5.6	19	(17) 8.6	12	(14) 4.7
		73		10		22		25		19
		82		16		11		8		10
	150 µg	75	(74)  5.1	10	(14)  7.8	21	(18)  3.5	20	(17)  3.0	7	(12)  5.0
		78		9		14		17		12
		68		23		18		14		17
	500 µg	80	(79) 4.2	9	(15)  7.2	15	(15)  0.6	13	(15)  3.8	19	(15) 4.5
		74		23		15		12		15
		82		13		16		19		10
	1500 µg	73 P	(83)  9.0	16 P	(15)  2.3	18 P	(14)  3.5	10 P	(16)  6.6	13 P	(16) 3.1
		88 P		16 P		12 P		23 P		19 P
		89 P		12 P		12 P		15 P		17 P
	5000 µg	69 P	(81)  10.8	7 P	(8)  1.0	17 P	(15)  2.1	13 P	(10) 3.1	9 P	(9) 1.5
		86 P		9 P		14 P		7 P		8 P
		89 P		8 P		13 P		11 P		11 P
Positive controls S9 -mix (-)	Name	ENNG		ENNG		ENNG		4NQO		9AA
	Dose Level	3 µg		5 µg		2 µg		0.2 µg		80 µg
	No. of revertants	872	(906)  72.6	136	(141)  10.1	647	(626)  20.5	406	(437)  33.7	202	(182)  30.7
		989		135		626		433		198
		856		153		606		473		147

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA 9-Aminoacridine

P Test item precipitate

# Standard deviation

Table 5: Test results; Experiment 2 - With Metabolic Activation (Pre-incubation)

Test Period		From: 10 July 2017					To: 13 July 2017
S9 -Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMF)	82	(76)  7.9#	12	(11) 3.2	16	(20)  3.2	19	(18) 1.2	14	(14)  1.5
		67		7		22		17		12
		79		13		21		19		15
	15 µg	75	(77)  8.7	11	(13)  4.7	20	(18)  4.7	24	(21)  3.5	13	(13) 0.6
		87		9		22		17		13
		70		18		13		21		14
	50 µg	75	(79)  8.4	11	(10)  2.6	23	(18)  5.0	17	(19) 2.1	13	(14) 2.6
		89		12		17		21		17
		74		7		13		20		12
	150 µg	80	(71)  8.5	12	(11)  2.1	13	(20)  9.5	18	(22)  3.5	16	(16)  2.5
		71		13		31		22		14
		63		9		17		25		19
	500 µg	95	(84) 10.0	13	(17)  4.7	15	(16)  1.7	29	(24)  4.7	13	(9) 3.2
		76		22		18		22		8
		80		15		15		20		7
	1500 µg	77 P	(92)  14.2	13 P	(13)  0.6	23 P	(26)  2.6	25 P	(21)  3.6	10 P	(10) 1.5
		105 P		13 P		28 P		18 P		11 P
		95 P		12 P		27 P		20 P		8 P
	5000 µg	79 P	(77)  1.7	11 P	(12)  1.0	16 P	(19)  5.8	14 P	(14) 2.5	7 P	(8) 1.5
		76 P		13 P		16 P		12 P		10 P
		76 P		12 P		26 P		17 P		8 P
Positive controls S9 -mix (+)	Name	2AA		2AA		2AA		BP		2AA
	Dose Level	1 µg		2 µg		10 µg		5 µg		2 µg
	No. of revertants	1839	(1720)  114.4	229	(238)  14.2	143	(143)  19.5	195	(175)  26.3	319	(352)  31.6
		1709		254		123		184		354
		1611		230		162		145		382

2AA 2-Aminoanthracene

BP Benzo(a)pyrene

P Test item precipitate

# Standard deviation

Table 6: Positive Control Values 2015

Strain S9 -Mix	TA100		TA1535		TA102		WP2uvrA		TA98		TA1537		WP2uvrApKM101		WP2pKM101
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Values*	276	280	252	264	13	13	231	227	262	276	253	261	20	35	20	35
Min	222	250	79	118	953	673	116	103	100	78	164	97	430	494	745	325
Max	2266	2402	2779	457	3140	1655	2769	550	502	705	2318	823	1696	2264	3662	1174
Mean	614	927	472	246	2303	1093	792	266	222	218	911	336	761	1461	2257	569
SD	260.6	452.5	434.8	55.7	815.2	376.5	342.1	97.7	70.2	107.6	412.4	135.7	350.0	382.0	790.7	220.3

SD standard deviation

Min minimum value

Max maximum value

* Number of mean values used to create dataset

Table 7: Positive control values 2016

Strain S9 -Mix	TA100		TA1535		TA102		WP2uvrA		TA98		TA1537		WP2 uvrA pKM101		WP2pKM101
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Values	409	406	381	386	30	28	341	335	388	385	379	381	14	24	8	16
Min	221	284	84	92	897	629	107	102	100	96	95	101	445	574	1674	372
Max	2222	2863	2994	879	2326	2140	1611	637	449	4357	1413	639	1117	1855	2823	945
Mean	724	1264	854	240	1633	950	718	240	186	188	406	290	743	1271	2379	535
SD	320.4	562.9	664.9	62.1	564.5	382.7	338.6	98.2	49.8	230.8	227.0	92.7	214.6	326.5	426.2	143.3

Table 8: Combined Vehicle and Untreated Control Values 2015

Strain S9 -Mix	TA100		TA1535		TA102		WP2uvrA		TA98		TA1537		WP2uvrApKM101		WP2pKM101
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Values	274	278	504	285	26	13	461	229	526	299	506	282	42	51	39	49
Min	60	61	7	7	222	278	10	12	11	10	4	6	87	98	89	93
Max	166	175	31	29	376	388	58	43	45	46	27	27	237	254	174	177
Mean	91	95	16	14	286	333	24	27	21	24	12	13	156	164	123	137
SD	19.3	19.1	4.5	4.0	48.7	37.6	5.6	5.9	6.2	6.1	3.8	3.4	42.2	35.6	23.1	21.2

Table 9: Combined Vehicle and Untreated Control Values 2016

Strain S9 -Mix	TA100		TA1535		TA102		WP2uvrA		TA98		TA1537		WP2uvrApKM101		WP2pKM101
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Values	399	401	758	393	60	30	690	345	788	415	762	398	32	32	16	24
Min	63	66	8	8	216	221	10	13	8	12	3	4	97	104	78	52
Max	154	156	34	39	30	375	53	53	49	51	24	23	268	243	148	166
Mean	90	93	15	15	268	310	22	27	21	25	12	13	161	159	118	110
SD	14.5	14.3	4.5	5.2	26.4	31.1	5.8	6.3	4.8	5.7	3.5	3.5	39.2	32.3	17.0	29.3

Applicant's summary and conclusion

Conclusions:

For the test item there were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). R59 was considered to be non-mutagenic under the conditions of this test.

Executive summary:

The genetic toxicity of the test item was investigated in an OECD 471, EU method B13/14 and US EPA 870.1500 guideline study. The study was conducted with two experiments, Experiment 1 was a plate incorporation method and Experiment 2 was a pre-incubation method with both experiments performed with and without metabolic activation. The study was performed at concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate and 15, 50, 150, 500 and 1500 µg/plate for experiment 1 and 2, respectively, with DMF used as the vehicle solvent. The studies without metabolic activation utilised N-ethyl-N'-nitro-N-nitrosoguanidine, 4 -Nitroquinoline-1 -oxide and 9 -Aminoacridine as positive control substances; the studies with metabolic activation utilised Benzo(a)pyrene and 2 -Aminoanthracene as positive control substances. Untreated test media was used as the negative control in all experiments.

There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). The test item was considered to be non-mutagenic under the conditions of this test.

The study is a GLP compliant guideline experimental study, without restrictions and is fully adequate for assessment of this endpoint.