Reaction products of 12-hydroxyoctadecanoic acid with ethane-1,2-diamine and hexane-1,6-diamine and 1,3-phenylenedimethanamine

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:

From 2012-05-16 to 2012-06-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of 12-hydroxyoctadecanoic acid with ethane-1,2-diamine and hexane-1,6-diamine and 1,3-phenylenedimethanamine

EC Number:

700-825-2

Molecular formula:

Not applicable (UVCB substance).

IUPAC Name:

Reaction products of 12-hydroxyoctadecanoic acid with ethane-1,2-diamine and hexane-1,6-diamine and 1,3-phenylenedimethanamine

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Method

Target gene:

Histidine operon

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:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

- First mutagenicity experiment with and without S9 mix: 62.5, 125, 250, 500 and 1000µg/plate
- Second mutagenicity experiment with and without S9 mix: 62.5, 125, 250, 500 and 1000µg/plate.

Vehicle / solvent:

- Vehicle used: dimethylsulfoxide
- Justification for choice: test item was soluble in the vehicle at 100 mg/mL

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar.
The test item was tested in a preliminary test and two mutagenicity experiments.
The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate
incorporation method. The second experiment with S9 mix was performed according to the preincubation method.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 or 72 hours.

SELECTION AGENT (mutation assays):
- Agar containing traces of histidine and biotin, maintained at 45°C for Salmonella strains.

NUMBER OF REPLICATIONS
- Two independent mutagenicity experiments each using three plates/dose-level

DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies and/or thinning of the bacterial lawn.

Evaluation criteria:

A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account.

Statistics:

No statistical analysis performed.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Tested up to limit concentrations recommended by the test guideline

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Tested up to limit concentrations recommended by the test guideline

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

- The number of revertants for the vehicle and positive controls were as specified in the acceptance criteria. The study was therefore considered as valid.

- Since the test item was found poorly soluble and non-cytotoxic in the preliminary test, the choice of the highest dose-level was based on the level of precipitate, according to the criteria specified in the international guidelines.

- A moderate to strong precipitate was observed at dose-levels > or = to 125 µg/plate, this did not prevent the scoring of revertant colonies.

- No toxicity was noted at any dose-level in any strain.

- The test item did not induce any noteworthy increase in the number of revertants, in any of the strains, either with or without S9 mix.

Table 7.6.1/1 Experiment 1- Without metabolic activation

Strain	Compound	Dose-level (µg/plate)	With or without S9 mix	Number of revertant per plate	Mean revertant colony count (SD)
TA1535	DMSO		-	16-20-17	18 (2)
	TEST ITEM	62.5	-	19-17-24	20 (4)
		125	-	17-13-14	15 (2)
		250	-	10Mp-26Mp-16Mp	17 (8)
		500	-	23Mp-17Mp-16Mp	19 (4)
		1000	-	14Sp-17Sp-19Sp	17 (3)
	NAN3	1	-	761-623-614	666 (82)
TA1537	DMSO		-	11-6-5	7 (3)
	TEST ITEM	62.5	-	11-7-8	9 (2)
		125	-	18Mp-11Mp-8Mp	12 (5)
		250	-	8Mp-7Mp-6Mp	7 (1)
		500	-	14Mp-10Mp-7Mp	10 (4)
		1000	-	4Sp-7Sp-4Sp	5 (2)
	9 AA	50	-	404-242-362	336 (84)
TA 98	DMSO		-	14-20-28	21 (7)
	TEST ITEM	62.5	-	22-16-14	17 (4)
		125	-	23Mp-32Mp-25Mp	27 (5)
		250	-	18Mp-16Mp-16Mp	17 (1)
		500	-	16Mp-13Mp-18Mp	16 (3)
		1000	-	12Sp-12Sp-6Sp	10 (3)
	2 NF	0.5	-	116-116-84	105 (18)
TA 100	DMSO		-	117-153-127	132 (19)
	TEST ITEM	62.5	-	128-119-145	131 (13)
		125	-	198Mp-131Mp-149Mp	159 (35)
		250	-	164Mp-144Mp-138Mp	149 (14)
		500	-	162Mp-159Mp-125Mp	149 (21)
		1000	-	90Sp-134Sp-110Sp	111 (22)
	NAN3	1	-	429-515-563	502 (68)
TA102	DMSO		-	269-369-347	328 (53)
	TEST ITEM	62.5	-	358-309-320	329 (26)
		125	-	338Mp-307Mp-332Mp	326 (16)
		250	-	302Mp-340Mp-311Mp	318 (20)
		500	-	388Mp-398Mp-325Mp	370 (40)
		1000	-	305Sp-364Sp-374Sp	348 (37)
	MMC	0.5	-	1872-1804-1820	1832 (36)

Table 7.6.1/2 Experiment 1- With metabolic activation

Strain	Compound	Dose-level (µg/plate)	With or without S9 mix	Number of revertant per plate	Mean revertant colony count (SD)
TA1535	DMSO		+	23-26-23	24 (2)
	TEST ITEM	62.5	+	22-13-23	19 (6)
		125	+	25Mp-22mp-17mp	21 (4)
		250	+	31Mp-25Mp-17Mp	24 (7)
		500	+	19Mp-35Mp-20Mp	25 (9)
		1000	+	20Sp-28Sp-30Sp	26 (5)
	2 AM	2	+	246-268-253	256 (11)
TA1537	DMSO		+	6-12-4	7 (4)
	TEST ITEM	62.5	+	6-5-4	5 (1)
		125	+	4Mp-10Mp-12Mp	9 (4)
		250	+	16Mp-16Mp-8Mp	13 (5)
		500	+	8Mp-22Mp-10Mp	13 (8)
		1000	+	8Sp-8Sp-1Sp	6 (4)
	2 AM	2	+	195-182-205	194 (12)
TA 98	DMSO		+	14-20-28	19(3)
	TEST ITEM	62.5	+	22-16-18	24 (7)
		125	+	23Mp-32Mp-25Mp	29 (7)
		250	+	18Mp-16Mp-16MP	23 (1)
		500	+	16Mp-13Mp-18MP	29 (10)
		1000	+	12sp-12Sp-6Sp	15 (8)
	2 AM	2	+	116-116-84	1353 (54)
TA 100	DMSO		+	165-135-126	142 (20)
	TEST ITEM	62.5	+	151-145-141	146 (5)
		125	+	129Mp-156Mp-149Mp	145 (14)
		250	+	125Mp-131Mp-137Mp	131 (6)
		500	+	131Mp-113Mp-133Mp	126 (11)
		1000	+	103Sp-104Sp-99Sp	102 (3)
	BAP	5	+	704-473-440	539 (144)
TA102	DMSO		+	467-534-519	507 (35)
	TEST ITEM	62.5	+	399-493-381	424 (60)
		125	+	556Mp-466mp-419Mp	480 (70)
		250	+	501Mp-381Mp-453Mp	445 (60)
		500	+	344Mp-389Mp-331Mp	355 (30)
		1000	+	301Sp-293Sp-328Sp	307 (18)
	2 AM	10	+	3477-4079-4423	3993 (479)

Table 7.6.1/3 Experiment 2- Without metabolic activation

Strain	Compound	Dose-level (µg/plate)	With or without S9 mix	Number of revertant per plate	Mean revertant colony count (SD)
TA1535	DMSO		-	10-12-16	13 (3)
	TEST ITEM	62.5	-	17-24-16	19 (4)
		125	-	16-19-19	18 (2)
		250	-	14Mp-13Mp-16Mp	14 (2)
		500	-	5Mp-14Mp-19Mp	13 (7)
		1000	-	18Sp-25Sp-18Sp	20 (4)
	NAN3	1	-	719-752-733	735 (17)
TA1537	DMSO		-	12-5-16	11 (6)
	TEST ITEM	62.5	-	22-16-24	21 (4)
		125	-	12Mp-20Mp-29Mp	20 (9)
		250	-	22Mp-16Mp-18Mp	19 (3)
		500	-	25Mp-12Mp-20Mp	19 (7)
		1000	-	13Sp-16Sp-16Sp	15 (2)
	9 AA	50	-	166-187-141	165 (23)
TA 98	DMSO		-	20-17-23	20 (3)
	TEST ITEM	62.5	-	16-12-16	15 (2)
		125	-	20Mp-25Mp-17Mp	21 (4)
		250	-	20Mp-20Mp-22Mp	21 (1)
		500	-	20Mp-32Mp-18Mp	23 (8)
		1000	-	23Sp-16sp-20Sp	20 (4)
	2 NF	0.5	-	139-134-104	126 (19)
TA 100	DMSO		-	137-156-149	147 (10)
	TEST ITEM	62.5	-	140-163-149	151 (12)
		125	-	186Mp-153Mp-157Mp	165 (18)
		250	-	158Mp-171Mp-144Mp	158 (14)
		500	-	149Mp-190Mp-162Mp	167 (21)
		1000	-	147Sp-159Sp-174Sp	160 (14)
	NAN3	1	-	507-502-450	486 (32)
TA102	DMSO		-	313-315-357	328 (25)
	TEST ITEM	62.5	-	335-390-335	353 (32)
		125	-	320Mp-304Mp-285Mp	303 (18)
		250	-	353Mp-335Mp-343Mp	34 (9)
		500	-	299Mp-343Mp-357Mp	333 (30)
		1000	-	346Sp-296Sp-316Sp	319 (25)
	MMC	0.5	-	1423-1850-1871	1715 (253)

Table 7.6.1/4 Experiment 2- With metabolic activation

Strain	Compound	Dose-level (µg/plate)	With or without S9 mix	Number of revertant per plate	Mean revertant colony count (SD)
TA1535	DMSO		+	20-29-23	24 (5)
	TEST ITEM	62.5	+	20-25-24	23 (3)
		125	+	23Mp-28Mp-23Mp	25 (3)
		250	+	18Mp-23Mp-31Mp	24 (7)
		500	+	26Mp-28Mp-19Mp	24 (5)
		1000	+	26Sp-22Sp-18Sp	22 (4)
	2 AM	2	+	719-752-733	176 (27)
TA1537	DMSO		+	12-11-11	11 (1)
	TEST ITEM	62.5	+	22-16-24	25 (5)
		125	+	12Mp-20Mp-29Mp	16 (2)
		250	+	22Mp-16Mp-18Mp	16 (4)
		500	+	25Mp-12Mp-20Mp	14 (4)
		1000	+	13Sp-16Sp-16Sp	19 (5)
	2 AM	2	+	166-187-141	192 (22)
TA 98	DMSO		+	29-23-29	27 (3)
	TEST ITEM	62.5	+	34-29-31	31 (3)
		125	+	30Mp-40Mp-35Mp	35 (5)
		250	+	30Mp-34Mp-46Mp	37(8)
		500	+	29Mp-40Mp-28Mp	32 (7)
		1000	+	23Sp-16Sp-20Sp	25 (4)
	2 AM	2	+	1011-950-1016	992 (37)
TA 100	DMSO		+	180-99-135	138 (41)
	TEST ITEM	62.5	+	139-140-147	142 (4)
		125	+	140Mp-159Mp-141Mp	147 (11)
		250	+	160Mp-171Mp-133Mp	155 (20)
		500	+	164Mp-164Mp-134Mp	154 (17)
		1000	+	145Sp-138Sp-133Sp	139 (6)
	BAP	5	+	366-496-572	478 (104)
TA102	DMSO		+	315-438-514	422 (100)
	TEST ITEM	62.5	+	386-335-390	370 (31)
		125	+	523mp-519Mp-466Mp	503 (32)
		250	+	335Mp-332Mp-394Mp	354 (35)
		500	+	411Mp-352Mp-376Mp	380 (30)
		1000	+	361Sp-340Sp-331Sp	344 (15)
	2 AM	10	+	1484-1460-1310	1418 (94)

 

NAN3: Sodium azide

BAP: Benzo(a)pyrene

9AA: 9 -Aminoacridine

2NF: 2 -nitrofluorene

MMC: Mitomycine C

2AM: 2 -anthramine

Mp: Moderate precipitate

Sp: Strong precipitate

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, either in the presence or in the absence of a rat liver metabolizing system.

Executive summary:

The potential of the substance to induce reverse mutation in bacteria was assessed using five strains of Salmonella typhimurium according to the OECD guideline 471.The study was conducted in compliance with Good Laboratory Practices.

A preliminary toxicity test was performed to define the dose-levels of the test substance to be used for the mutagenicity study. The test item ( suspended in dimethylsulfoxide) was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

Both experiments were performed according to the direct plate incorporation.After 48 -72 hours of incubation at 37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

Since the test item was found poorly soluble and non-cytotoxic in the preliminary test, the choice of the highest dose-level was based on the level of precipitate, according to the criteria specified in the international guidelines. The selected treatment-levels were 62.5, 125, 250, 500 and 1000 µg/plate for the five strains used in both mutagenicity experiments.

In the main experiment, the number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

A moderate to strong precipitate was observed in the petri plates at dose-levels equal or higher than 125 µg/plate. No noteworthy toxicity was noted at any dose-levels towards the five strains used, either with or without S9 mix.

In the two independent assays, no significant increase in the mean number of revertants was noted in the bacterial strains tested in the presence of the test substance neither with nor without metabolic activation.

Under these experimental conditions,the substance did not show any mutagenic activity in the bacterial reverse mutation test.