Polyamide wax

Registration Dossier

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The available data from three in vitro assays suggest that the substance does not have a genotoxic potential.

Link to relevant study records

Reference 1

Endpoint:: in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:: experimental study
Adequacy of study:: key study
Reliability:: 1 (reliable without restriction)
Qualifier:: according to
Guideline:: OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:: 1997
Deviations:: no
Target gene:: n/a
Species / strain / cell type:: lymphocytes:
Details on mammalian cell type (if applicable):: Cultures of human lymphocytes are primary cell cultures recommended by international regulations for the mammalian chromosome aberration test; they have a stable karyotype with 46 chromosomes and an average cell cycle time of 13-14 hours.
Cultures of human lymphocytes were prepared from whole blood pooled from healthy males donors.
Additional strain / cell type characteristics:: not applicable
Test concentrations with justification for top dose:: Mitotic index analysis:
Test 1 (3-hour treatment without activation): 0, 500, 1000, 1500, 2000, 2500, 2750, 3000, 3250, 3500, 3750 and 4000 µg/mL
Test 1 (3-hour treatment with activation): 0, 625, 1250, 2500, 3500, 4000, 4500 and 5000 µg/mL
Test 2 (20-hr without activation ): 0, 500, 1000, 1250, 1500, 1750, 2000, 2500, 3000, 3500, 4000, 4500 and 5000 µg/mL
Test 2 (3-hour treatment with activation):0, 625, 1250, 2500, 3500, 4000, 4500 and 5000 µg/mL

Metaphase analysis
Test 1 (3-hour treatment without activation): 0, 1000, 2000, 3250 and 3500 µg/mL,
Test 1 (3-hour treatment with activation): 0, 1250, 2500 and 5000 µg/mL,
Test 2 (20-hour without activation ): 0, 1500, 2000 and 2500 µg/mL,
Test 2 (3-hour treatment with activation):0, 1250, 2500 and 4500 µg/mL,
: Key result
Species / strain:: lymphocytes: human
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Vehicle controls validity:: valid
Untreated negative controls validity:: not examined
Positive controls validity:: valid

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)
Qualifier:: according to
Guideline:: OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:: 1997
Deviations:: no
Target gene:: Histidine operon
Species / strain / cell type:: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:: E. coli WP2 uvr A pKM 101
Test concentrations with justification for top dose:: First test
Concentration range (with and without metabolic activation): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate Second test
Concentration range (with and without metabolic activation): 50, 150, 500, 1500 and 5000 µg/plate
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:: valid
Untreated negative controls validity:: not examined
Positive controls validity:: valid
Species / strain:: E. coli WP2 uvr A pKM 101
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:: valid
Untreated negative controls validity:: not examined
Positive controls validity:: valid

Reference 3

Endpoint:: in vitro gene mutation study in mammalian cells
Type of information:: experimental study
Adequacy of study:: key study
Reliability:: 1 (reliable without restriction)
Qualifier:: according to
Guideline:: OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:: 1997
Deviations:: no
Target gene:: Thymidine Kinase
Species / strain / cell type:: mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):: - Type and identity of media: RPMI A, 10 and 20
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Test concentrations with justification for top dose:: In the cytotoxicity Range-Finder Experiment six concentrations were tested ranging from 1.18 to 58.0 µg/mL.
In experiment 1 ten concentrations were tested, ranging from 10 to 58 µg/mL.
In experiment 2, six concentrations were tested, ranging from 10 to 58 µg/mL.
Species / strain:: mouse lymphoma L5178Y cells
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
Positive controls validity:: valid

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed (negative)

Additional information

Gene mutation in bacteria

In a reverse gene mutation assay in bacteria, performed according to the OECD guideline N° 471, in compliance with Good Laboratory Practices, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 100 et TA 98) and Escherichia Coli (WP2uvrA/pKM101) were exposed to the test item at concentrations of 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in two independent experiments both in presence and absence of metabolic activation.

No substantial increases in revertant colony numbers over control count were obtained with any of the tester strains following exposure to the test item at any concentrations in either presence or absence of S9 mix.

The positive control induced the appropriate responses in the corresponding strains.There was no evidence of induced mutant colonies over background.

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

Gene mutation in mammalian cells

In a mammalian cell gene mutation assay performed according to OECD 476 and in compliance with Good Laboratory Practices, the test item diluted in DiMethylFormamide (heated at 80°C) was tested in L5178Y mouse lymphoma cells.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested, in the absence and presence of S9, ranging from 12.5 to 400 µg/mL (limited by solubility in culture medium).

Two independent mutation experiments were performed in the absence and presence of S9. In Experiment 1, ten concentrations, ranging from 10.0 to 58.0 µg/mL were tested. In Experiment 2, six concentrations of the test item were tested, ranging from 10.0 to 58 µg/mL.

The positive controls induced the appropriate response and negative controls were valids.

When the test item was tested up to precipitating concentrations in Experiments 1 and 2 no increases in mutant frequency (that exceeded the Global Evaluation Factor of 126) were observed at any concentration tested, indicating a negative result. There was no reduction in Relative Total Growth and no linear trends were observed. Hence, the test item was not mutagenic in the absence and presence of S-9 when tested up to precipitating concentrations, for 3 hours, in this test system.

Under the test consitions employed in this study, the test item did not induce mutation at the tk locus of L5178Y mouse lymphoma cells.

Chromosomal aberrations in mammalian cells

An in vitro chromosomal aberration assay was conducted to evaluate the clastogenic potential of the test substance in human lymphocytes according to the OECD Guideline 473 and in compliance with Good Laboratory Practices.

Human lymphocytes, in whole blood culture, were exposed to the test substance both in the absence and presence of S9 mix derived from rat livers. Three hours before the end of the incubation period, cell division was arrested using Colcemid®. The cells were then harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage. On the basis of the mitotic index data obtained from a toxicity test, the following concentrations were selected for two independent tests evaluating the metaphase analysis: in the Test 1, for 3-h treatment without S9 mix dose levels of 0, 1000, 2000, 3250 and 3500 µg/mL and for 3-h treatment with S9 mix dose levels of 0, 1250, 2500 and 5000 µg/ml ; in the test 2, for 20 -hr treatment without S9 mix dose levels of: 0, 1500, 2000 and 2500 µg/mL and for 3-h treatment with S9 mix dose levels of 0, 1250, 2500 and 4500 µg/mL .

Concurrent solvent and positive controls (mitomycin-C (in the absence of S9 mix) and cyclophosphamide (in the presence of S9 mix)) were also included. In both tests, the substance caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations at any concentration in the absence and presence of S9 mix when compared with the vehicle control. Also, no statistically significant increases in the proportion of polyploid or endoreduplicated metaphase cells were observed during metaphase analysis when compared with the vehicle control. All positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix. Under the study conditions, the test substance was therefore not considered to be clastogenic to human lymphocytes in the in vitro chromosomal aberration assay.

Justification for classification or non-classification

Based on the results from three in vitro guideline compliant assays, the substance is not classified for genotoxicity according to regulation (EC) No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures.

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

Substance concentration (µg/mL)	Cells with aberrations excluding gaps			Cells with aberrations including gaps			Relative cell count
Substance concentration (µg/mL)	Individual values per 100 cells observed (%)		Mean (%)	Individual values per 100 cells observed (%)		Mean (%)	Relative cell count
Method without S9 mix / 3 h. time exposure
0 (culture medium)	0	0	0.0	0	0	0.0	100
1000	0	0	0.0	1	0	0.5	115
2000	0	1	0.5	0	1	0.5	109
3250	1	1	1.0	1	3	2.0	91
3500	2		2.0	2		2.0	25
0.1 (Mitomycin C)	10	12	11.0***	11	13	12.0***	-
Method with S9 mix / 3 h. time exposure
0 (Culture medium)	0	0	0.0	1	0	0.5	100
1250	0	1	0.5	1	2	1.5	110
2500	1	2	1.5	4	3	3.5	100
5000	0	0	0.0	1	3	2.0	70
6 (Cyclophosphamide))	15	16	15.5***	23	20	21.5***	-

Substance concentration (µg/mL)	Cells with aberrations excluding gaps			Cells with aberrations including gaps			Relative cell count
Substance concentration (µg/mL)	Individual values per 100 cells observed (%)		Mean (%)	Individual values per 100 cells observed (%)		Mean (%)	Relative cell count
Method without S9 mix / 3 h. time exposure
0 (Culture medium)	2	2	2.0	5	5	5.0	100
1500	2	2	2.0	3	2	2.5	79
2000	2	1	1.5	4	2	3.0	56
2500	1	2	1.5	2	4	3.0	45
0.1 (Mitomycin C)	19	21	20.0***	28	24	26.0***	-
Method with S9 mix / 20 h. time exposure
0 (Culture medium)	1	0	0.5	1	1	1.0	100
1250	2	0	1.0	3	1	2.0	100
2500	2	0	1.0	4	1	2.5	95
4500	2	1	1.5	2	2	2.0	58
6 (Cyclophosphamide))	13	11	12.0***	19	14	16.5***	-

Test substance concentration (µg/plate)	TA 1535		TA 1537		WP2uvrA/ PKM101		TA 98		TA 100
Test substance concentration (µg/plate)	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation
Solvent*	19	2	17	2	128	3	36	2	113	4

5	15	1	19	3	128	7	32	3	109	4
15	14	1	15	4	121	8	34	3	100	11
50	16	4	13	3	125	15	35	4	95	8
150	13	2	16	6	130	8	34	6	97	6
500	12	3	21	1	139	9	34	7	102	8
1500	13	2	15	1	136	9	32	3	105	2
5000	13	2	19	3	122	8	33	2	103	4
Positive control***	410	36	182	16	486	30	536	66	569	8

Test substance concentration (µg/plate)	TA 1535		TA 1537		WP2uvrA/ PKM101		TA 98		TA 100
Test substance concentration (µg/plate)	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation
Solvent*	14	2	18	5	149	13	44	5	113	12

5	15	3	19	3	156	1	41	3	104	9
15	16	5	18	2	162	8	43	7	104	10
50	12	3	15	1	154	12	36	7	10	98
150	15	2	14	2	151	16	37	5	95	7
500	15	1	17	4	144	8	42	6	103	6
1500	12	3	13	3	140	8	39	2	104	3
5000	11	3	17	3	142	8	35	3	98	1
Positive control***	94	2	249	24	403	16	752	24	688	61

Test substance concentration (µg/plate)	TA 1535		TA 1537		WP2uvrA/ PKM101		TA 98		TA 100
Test substance concentration (µg/plate)	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation
Solvent*	19	2	17	4	125	14	34	2	111	10

50	17	3	14	2	137	11	36	8	107	6
150	17	3	12	2	138	13	35	0	105	9
500	20	6	14	4	138	11	37	7	103	12
1500	18	2	13	3	145	1	28	1	107	16
5000	21	1	11	4	132	12	32	3	99	15
Positive control***	400	48	190	13	548	49	402	9	463	4

Treatment (µg/mL)	-S-9 % RTG	+S-9 % RTG
0	100	100
1.81	101	125
3.63	86	104
7.25	94	142
14.5	92	81
29.0	89	89
58.0	71	114

Treatment (µg/mL)	-S-9		Treatment (µg/mL)	+S-9
	%RTG	MF§		%RTG	MF§
0	100	68.33	0	100	83.62
UTC	103	86.58	UTC	104	61.06
10.0	91	86.58	15	99	62.20
15.0	94	72.65	20	112	66.13
20.0	116	71.06	25	82	81.82
25.0	102	79.77	30	104	64.32
30.0	105	68.67	35	88	64.08
35.0	89	108.64	40	107	56.97
40.0	100	87.52	45	97	79.61
45.0	94	81.66	50	90	69.07
50.0	108	80.52	58	80	74.89
58.0	108	76.96
Linear trend		NS	Linear trend		NS
MMS			B[a]P
15	38	1012.19	2	47	896.47
20	20	1652.89	3	27	891.84