Registration Dossier
Registration Dossier
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EC number: 614-503-3 | CAS number: 68441-62-3
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In vitro, positive results were observed in the presence of S9 mix: in the Ames test (in strains TA 1535, TA 98 and TA 100 and in the Escherichia coli strain WP2uvrA) and the MLA test (mouse lymphoma cells).
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Data waiving:
- study scientifically not necessary / other information available
- Justification for data waiving:
- an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP compliant, guideline study, no restrictions, fully adequate for assessment
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- thymidine kinase (TK) locus on chromosome 11
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix
- Test concentrations with justification for top dose:
- 0.37, 0.31, 0.26, 0.18, 0.13, 0.06, 0.03, 0.02, 0.01 and 0 mmol/L in the absence of S9-mix
0.7, 0.6, 0.5, 0.37, 0.26, 0.06, 0.03, 0.02, 0.01 and 0 mmol/L in the presence of S9-mix - Vehicle / solvent:
- DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 3-methylcholanthrene and methylmethanesulfonate
- Details on test system and experimental conditions:
- Prior to the main study two dose range finding studies were performed. In the first dose range finding study single cultures were exposed for 24 hours in the absence of S9-mix to 5 concentrations of IXOL B350 ranging from 0.63 to 10 mmol/l. In the second dose range finding study single cultures were exposed for 24 hours in the absence and 4 hours in the presence of S9-mix to concentrations of IXOL B350 ranging from 0.016 to 0.25 mmol/l or 0.25 to 1.0 mmol/l, respectively.
In the main study single cultures were exposed for 24 hours in the absence and 4 hours in the presence of S9-mix to 15 or 13 concentrations of IXOL B350 ranging from 0.004 to 0.70 mmol/l or 0.0019 to 1.0 mmol/l, respectively. Finally, 9 concentrations were evaluated for mutagenicity in both the absence and presence of S9-mix.
Cell treatment without metabolic activation
In the assay without metabolic activation the cells were exposed to the test substance according to the following procedure; 0.1 ml test substance solution or 0.1 ml positive control and 4.9 ml culture medium without serum were added to ca. 3,000,000 L5178Y cells in 5 ml culture medium (with 10 % horse serum) to a final volume of 10 ml. Two cultures treated with the vehicle (DMSO) were used as negative controls; one single culture treated with MMS was used as positive control substance at a final concentration of 0.1 mmol/l. Single cultures were used for each concentration of the test substance. The cells were exposed for 24 h at ca. 37 oC and ca. 5 % CO2 in a humidified incubator.
The dose levels of the test substance used ranged from 0.004 to 0.70 mmol/l IXOL B350. At the start and end of the treatment, all cell cultures were checked visually and selected cultures were checked for viability by trypan blue exclusion.
Cell treatment with metabolic activation
In the assay with metabolic activation the cells were exposed to the test substance according to the following procedure; 0.1 ml test substance solution or 0.1 ml positive control and 3.9 ml culture medium without serum were added to 1 ml 20% (v/v) S9-mix and 5 ml culture medium (with 10% horse serum) containing ca. 5,000,000 L5178Y cells to a final volume of 10 ml. Two cultures treated with the vehicle (DMSO) were used as negative controls; one single culture treated with MCA was used as positive control substance at a final concentration of 10 µg/ml. Single cultures were used for each concentration of the test substance. The cells were exposed for 4 h at ca. 37 oC and ca. 5 % CO2 in a humidified incubator.
The dose levels of the test substance used ranged from 0.0019 to 1.0 mmol/l IXOL B350. At the start and end of the treatment, all cell cultures were checked visually and selected cultures were checked for viability by trypan blue exclusion.
Assessment of cytotoxicity
The cytotoxicity of the test substance was determined by measuring the relative initial cell yield, the relative suspension growth (RSG) and the relative total growth (RTG). - Evaluation criteria:
- A response was considered to be positive if the induced mutant frequency (mutant frequency of the test substance minus that of the vehicle negative control) was more than 126 mutants per 1,000,000 clonable cells. A response was considered to be equivocal if the induced mutant frequency was more than 88 mutants (but smaller than 126 mutants) per 1,000,000 clonable cells. Any apparent increase in mutant frequency at concentrations of the test substance causing more than 90% cytotoxicity was considered to be an artefact and not indicative of genotoxicity.
The test substance was considered to be mutagenic in the gene mutation test at the TK-locus if a concentration-related increase in mutant frequency was observed, or if a reproducible positive response for at least one of the test substance concentrations was observed.
The test substance was considered not to be mutagenic in the gene mutation test at the TK-locus if it produced neither a dose-related increase in the mutant frequency nor a reproducible positive response at any of the test substance concentrations. - Statistics:
- No statistical analysis was performed. Both numerical significance and biological relevance were considered together in the evaluation.
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: the maximum concentration was limited by cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Remarks:
- at and above 0.5 mmol/L
- Cytotoxicity / choice of top concentrations:
- other: the maximum concentration was limited by cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- The test substance IXOL B350 was tested and evaluated for mutagenicity in both the absence and presence of metabolic activation (S9-mix). Beside treatment for 4 hours in the presence of a metabolic activation system, an extended treatment for 24 hours in the absence of S9-mix was used. In the presence of S9-mix a dose related increase in mutant frequency was observed. Although the increase was observed at limited data points, since in the Bacterial reverse mutation test with IXOL B350 (8405/18) also a mutagenic response was observed in the presence of S9-mix, it was decided not to perform a second assay to obtain more data points. Since relatively more small then large colonies were observed at concentrations causing a positive response, it can not be excluded that IXOL B350 is clastogenic.
It is concluded that under the conditions used in this study, the test substance IXOL B350 is mutagenic at the TK-locus of mouse lymphoma L5178Y cells. - Conclusions:
- Under the conditions of this study, Polyol IXOL B350 is mutagenic (clastogenic) at the TK-locus of mouse lymphoma L5178Y cells.
- Executive summary:
Polyol IXOL B350 was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells, in both the absence and the presence S9-mix (GLP study according to OECD guideline 478). One assay was conducted in which single cultures were treated for 24 hours and 4 hours in the absence and presence of S9-mix, respectively (TNO, 2010d).
The highest concentrations of the substance evaluated for mutagenicity were 0.37 and 0.7 mmol/L in the absence and presence of S9-mix, respectively. The maximum concentrations were limited by cytotoxicity. In the absence of S9-mix no increase in mutant frequency was observed at any test substance concentration evaluated. In the presence of S9-mix a dose related increase in mutant frequency was observed at and above 0.5 mmol/L. In presence of S9-mix at the concentrations causing an increase in mutant frequency, relatively more small than large colonies were formed. The mean percentage of small colonies formed was 64%. Based on these results it cannot be excluded that Polyol IXOL B350 is clastogenic.The negative controls were within historical background ranges and treatment with the positive control yielded the expected significant increase in mutant frequency compared to the negative controls.
It is concluded that under the conditions used in this study, Polyol IXOL B350 is mutagenic (clastogenic) at the TK-locus of mouse lymphoma L5178Y cells.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP compliant, guideline study, no restrictions, fully adequate for assessment
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 liver homogenate
- Test concentrations with justification for top dose:
- 62, 185, 556, 1667 and 5000 µg/plate
- Vehicle / solvent:
- DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: benzo(a)pyrene and 2-aminoanthracene
- Details on test system and experimental conditions:
- One bacterial reverse mutation test was performed. The test substance was dissolved in DMSO at a concentration of 50 mg/ml based on a purity of 99%. A clear, slight-brown solution was obtained. Serial dilutions in DMSO were made. Five concentrations were tested, ranging from 62 to 5000 µg/plate. Negative controls (DMSO) and positive controls were run simultaneously with the test substance.
Fresh bacterial cultures were prepared by inoculation of nutrient broth with a thawed aliquot of the stock culture and subsequent incubation for approximately 10-16 h at 37°C while shaking. Briefly, the mutagenicity assay was carried out as follows. To 2 ml molten top agar (containing 0.6 % agar, 0.5 % NaCl and 0.05 mM L-histidine.HCl/0.05 mM biotin or 0.05 mM tryptophane for the S. typhimurium strains, and E. coli WP2 uvrA strain, respectively), maintained at ca. 46 oC, were added subsequently: 0.1 ml of a fully grown culture of the appropriate strain, 0.1 ml of the test substance or of the negative control or of the positive control substance solution, and 0.5 ml S9-mix for the experiments with metabolic activation or 0.5 ml sodium phosphate 100 mM (pH 7.4) for the experiments without metabolic activation. The ingredients were thoroughly mixed and the mix was immediately poured onto minimal glucose agar plates (1.5 % agar in Vogel and Bonner medium E with 2 % glucose). All determinations were made in triplicate. The plates were incubated at ca. 37 oC for approximately 48-72 hours. Subsequently, the his+ and trp+ revertants were counted. Toxicity is defined as a reduction (by at least 50%) in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth as compared to the negative (vehicle) control and/or the occurrence of pinpoint colonies. - Evaluation criteria:
- A test substance is considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates shows a
concentration-related increase or if a reproducible two-fold or more increase is observed compared the negative controls.
A test substance is considered to be negative in the bacterial gene mutation test if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points. - Statistics:
- Both numerical significance and biological relevance were considered together in the evaluation. No statistical analysis was performed.
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Remarks:
- from 556 µg/plate
- 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
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Remarks:
- at 5000 µg/plate
- 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
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Remarks:
- from 185 µg/plate
- 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
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with
- 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
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Remarks:
- at 5000 µg/plate
- 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
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- 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
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- 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
- Additional information on results:
- In the presence of S9-mix, in strains TA 1535, TA 98, TA 100 and WP2 uvrA, IXOL B350 did induce a dose related increase in the mean number of revertants compared to the background spontaneous reversion rate observed with the negative control. A minimal 2-fold increase was observed in strain TA 1535, at and above 185 µg/plate; in strain TA 98, at 5000 µg/plate; in strain TA 100, at and above 556 µg/plate and in strain WP2 uvrA, at 5000 µg/plate. The maximal increase observed was 27-fold in strain TA 1535 at 5000 µg/plate.
In the absence of S9-mix in all strains, IXOL B350 did not induce a minimal 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control. - Conclusions:
- Positive results were observed in the presence of S9 mix: in the Ames test (in strains TA 1535, TA 98 and TA 100 and in the Escherichia coli strain WP2uvrA).
- Executive summary:
Polyol IXOL B350 was examined for mutagenic activity in the bacterial reverse mutation test (GLP compliant and according to OECD guideline 471) using the histidine-requiringSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100 and the tryptophan-requiringEscherichia colistrain WP2uvrA, in the absence and presence of S9 -mix (TNO, 2010c). All strains were used, with five concentrations of the test substance, ranging from 62 to 5000 µg/plate. Negative controls (DMSO) and positive controls were run simultaneously with the test substance.
The mean number of his+and trp+revertant colonies of the negative controls were within the acceptable range and the positive controls gave the expected increase in the mean number of revertant colonies. The test substance was not toxic to any strain, in both the absence and presence of S9-mix, as neither a decrease in the mean number of revertants or a clearing of the background lawn of bacterial growth compared to the negative controls was observed.
In the presence of S9-mix, in strains TA 1535, TA 98, TA 100 and WP2uvrA, Polyol IXOL B350 did induce a dose related increase in the mean number of revertants compared to the background spontaneous reversion rate observed with the negative control. A minimal 2-fold increase was observed in strain TA 1535, at and above 185 µg/plate; in strain TA 98, at 5000 µg/plate; in strain TA 100, at and above 556 µg/plate and in strain WP2uvrA, at 5000 µg/plate. The maximal increase observed was 27-fold in strain TA 1535 at 5000 µg/plate.
In the absence of S9-mix in all strains, Polyol IXOL B350 did not induce a minimal 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control.
It is concluded that the results obtained with the test substance inSalmonella typhimuriumstrains TA 1535, TA 98 and TA 100 and in theEscherichia colistrain WP2uvrA, indicate that Polyol IXOL B350 is mutagenic in the presence of the S9-mix and all strains used indicate that Polyol IXOL B350 is not mutagenic in the absence of S9-mix under the conditions employed in this study.
Referenceopen allclose all
Table: Summary of the results:
Dose (mmol/l) |
absence of S9-mix (24h) |
Dose (mmol/l) |
presence of S9-mix (4h) |
||
MF |
RTG |
MF |
RTG |
||
0.37 |
83 |
12 |
0.7 |
407 |
8 |
0.31 |
90 |
18 |
0.6 |
298 |
19 |
0.26 |
67 |
47 |
0.5 |
129 |
48 |
0.18 |
70 |
51 |
0.37 |
123 |
58 |
0.13 |
74 |
56 |
0.26 |
77 |
82 |
0.06 |
66 |
103 |
0.06 |
59 |
103 |
0.03 |
64 |
93 |
0.03 |
70 |
98 |
0.02 |
39 |
94 |
0.02 |
58 |
105 |
0.01 |
60 |
92 |
0.01 |
60 |
82 |
0 |
44* |
100* |
0 |
49* |
100* |
* Mean of duplicate cultures RTG: relative total growth is a measure for the cytotoxicity of the test substance.
Table: Number of revertants counted in bacterial reverse mutation test with IXOL B350
|
|
TA 1535 |
|
TA 1537 |
|
TA 98 |
|
TA 100 |
|
E. Coli |
|
|||||
|
|
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|||||
0 µg/plate |
|
34 |
20 |
14 |
17 |
37 |
43 |
139 |
128 |
36 |
23 |
|||||
|
|
34 |
12 |
11 |
24 |
35 |
50 |
118 |
133 |
30 |
38 |
|||||
|
|
26 |
26 |
11 |
24 |
28 |
59 |
127 |
131 |
20 |
26 |
|||||
|
|
|
|
|
|
|
|
|
|
|
|
|||||
|
Mean |
31 |
19 |
12 |
22 |
33 |
51 |
128 |
131 |
29 |
29 |
|||||
|
StDev |
5 |
7 |
2 |
4 |
5 |
8 |
11 |
3 |
8 |
8 |
|||||
62 µg/plate |
|
35 |
34 |
18 |
19 |
40 |
49 |
133 |
174 |
25 |
28 |
|||||
|
|
32 |
22 |
20 |
22 |
42 |
53 |
153 |
159 |
22 |
38 |
|||||
|
|
26 |
25 |
14 |
26 |
28 |
56 |
145 |
186 |
24 |
36 |
|||||
|
|
|
|
|
|
|
|
|
|
|
|
|||||
|
Mean |
31 |
27 |
17 |
22 |
37 |
53 |
144 |
173 |
24 |
34 |
|||||
|
StDev |
5 |
6 |
3 |
4 |
8 |
4 |
10 |
14 |
2 |
5 |
|||||
185 µg/plate |
|
31 |
66 |
8 |
14 |
25 |
59 |
143 |
241 |
35 |
29 |
|||||
|
|
28 |
56 |
16 |
18 |
36 |
67 |
136 |
233 |
28 |
37 |
|||||
|
|
26 |
50 |
20 |
18 |
37 |
53 |
143 |
277 |
20 |
40 |
|||||
|
|
|
|
|
|
|
|
|
|
|
|
|||||
|
Mean |
28 |
57 |
15 |
17 |
33 |
60 |
141 |
250 |
28 |
35 |
|||||
|
StDev |
3 |
8 |
6 |
2 |
7 |
7 |
4 |
23 |
8 |
6 |
|||||
556 µg/plate |
|
34 |
88 |
16 |
16 |
36 |
61 |
146 |
429 |
46 |
37 |
|||||
|
|
29 |
80 |
12 |
22 |
35 |
77 |
130 |
519 |
35 |
36 |
|||||
|
|
41 |
107 |
13 |
22 |
38 |
61 |
135 |
458 |
42 |
25 |
|||||
|
|
|
|
|
|
|
|
|
|
|
|
|||||
|
Mean |
35 |
92 |
14 |
20 |
36 |
66 |
137 |
469 |
41 |
33 |
|||||
|
StDev |
6 |
14 |
2 |
3 |
2 |
9 |
8 |
46 |
6 |
7 |
|||||
1667 µg/plate |
|
26 |
244 |
18 |
19 |
23 |
75 |
117 |
813 |
32 |
48 |
|||||
|
|
38 |
268 |
12 |
35 |
31 |
78 |
119 |
930 |
36 |
55 |
|||||
|
|
29 |
342 |
14 |
34 |
44 |
86 |
143 |
978 |
23 |
46 |
|||||
|
|
|
|
|
|
|
|
|
|
|
|
|||||
|
Mean |
31 |
285 |
15 |
29 |
33 |
80 |
126 |
907 |
30 |
50 |
|||||
|
StDev |
6 |
51 |
3 |
9 |
11 |
6 |
14 |
85 |
7 |
5 |
|||||
5000 µg/plate |
|
40 |
512 |
7 |
29 |
40 |
123 |
139 |
776 |
42 |
89 |
|||||
|
|
38 |
470 |
19 |
25 |
36 |
123 |
159 |
1575 |
35 |
84 |
|||||
|
|
35 |
608 |
10 |
37 |
37 |
129 |
147 |
1781 |
35 |
86 |
|||||
|
|
P |
P |
P |
P |
P |
P |
P |
P |
P |
P |
|||||
|
Mean |
38 |
530 |
12 |
30 |
38 |
125 |
148 |
1377 |
37 |
86 |
|||||
|
StDev |
3 |
71 |
6 |
6 |
2 |
3 |
10 |
531 |
4 |
3 |
|||||
Pos. Control |
|
609 |
743 |
2301 |
319 |
822 |
1984 |
719 |
2525 |
231 |
1463 |
|||||
|
|
578 |
767 |
2319 |
389 |
840 |
2198 |
822 |
2647 |
191 |
1727 |
|||||
|
|
589 |
805 |
3485 |
384 |
645 |
2232 |
791 |
2382 |
223 |
1681 |
|||||
|
|
|
|
|
|
|
|
|
|
|
|
|||||
|
Mean |
592 |
772 |
2702 |
364 |
769 |
2138 |
777 |
2518 |
215 |
1624 |
|||||
|
StDev |
16 |
31 |
678 |
39 |
108 |
134 |
53 |
133 |
21 |
141 |
|||||
Mean Average number of revertants per plate
StDev Standard deviation
S9 Liver homogenate from rats treated with aroclor
Pos. Control Positive control; see text for actual concentrations of reference mutagens
P Slight precipitation of the test substance on the agar plates
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Genetic toxicity in vivo
Description of key information
In vivo, as no increased micronucleated
erythrocytes (clastogenicity) and no unscheduled DNA synthesis (UDS,
gene mutations) in hepatocytes were observed, the substance is not
considered mutagenic in vivo.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP compliant, near-guideline study, no restrictions, fully adequate for assessment.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- GLP compliance:
- yes
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- Swiss
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: TNO, Zeist, the Netherlands
- Age at study initiation: sex weeks
- Diet: free access
- Water: ad libitum
- Acclimation period: five days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24
- Humidity (%): 60-90
- Air changes (per hr): 16
- Photoperiod (hrs dark / hrs light): 12 / 12 - Route of administration:
- oral: gavage
- Vehicle:
- 1% tragacanth
- Details on exposure:
- The total dosages (3000, 1500 and 750 mg/kg bw) were given as two equal administrations separated by an interval of 24 hours. The mice were fasted overnight only prior to the first dosing. The test compound and vehicle controL were dosed by oral gavage in a volume of 0.1 ml/10 g body weight. Mitomycin C, the positive reference compound, was dosed by intraperitoneal injection in a volume of 0.1 ml/10 g body weight.
Total dosages of 3000, 1500 and 750 mg/kg bw were chosen for the micronucleus test. The top dose of 3000 mg/kg bw would be expected to
cause one or two deaths out of ten, within the 30 hours of the duration of the experiment. - Duration of treatment / exposure:
- two equal administrations separated by an interval of 24 hours
- Frequency of treatment:
- twice
- Post exposure period:
- 6 hours after the second dose (in total 30 hours after the first dose)
- Remarks:
- Doses / Concentrations:
750, 1500 and 3000 mg/kg bw
Basis:
actual ingested - No. of animals per sex per dose:
- 5 male and 5 female animals
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Mitomycin C
- Tissues and cell types examined:
- bone marrow, erythrocytes
- Details of tissue and slide preparation:
- After each administration, the animals were observed. All mortalities during the experiment were recorded.
The animals were killed six hours after the second administration by cervical dislocation and one femur was dissected from each animal. By injection of New Born calf serum in the femur the bone marrow was removed. The suspension of serum and bone marrow was centrifuged for 5 min at 800 rpm. The supernatant was taken off, a bone marrow smear was made of the pellet. After air-drying overnight the smears were fixed in methanol for 5 minutes. The smears were succesively placed in May-Grunwald (1.25%) for 15 minutes and in Giemsa (3%) for 20 minutes. After rinsing in buffered distilled water, the slides were air-dried and mounted in Depex. The smears were examined by light microscopy to determine the incidence of micronucleated cells per 1000 polychromatic erythrocytes per animal and the ratio of normochromatic to polychromatic erythrocytes. - Evaluation criteria:
- Incidence of micronucleated cells per 1000 polychromatic erythrocytes per animal and the ratio of normochromatic to polychromatic erythrocytes.
- Statistics:
- Significance levels of the ratios of normochromatic to-polychromatic erythrocytes were determined by the Mann-Whitney formulation of the Wilcoxon rank sum procedure using pairwise comparison with the control.
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Mortalities
After administration of 1%-tragacanth (the vehicle control) and Mitomycin C (the positive reference compound) no mortalities were observed.
In the top dose of B251 (the test compound) two male and two female mice died. In the middle dose one female mouse died.
Micronucleus counts
After administration of B251 at all dosages, the group mean micronucleated cell counts were in the same range as the concurrent control value (vehicle; range 0-3 per 1000 polychromatic erythrocytes)). The positive reference group, administered intraperitoneally with Mitomycin C, gave a mean count of 25.2 micronucleated cells per 1000 polychromatic erythrocytes (range 13-26).
Normochromatic to polychromatic erythrocyte ratios
In this experiment the vehicle control group gave a mean ratio normochromatic to polychromatic erythrocytes of 0.63 (range 0.31-1.53). The positive reference group, dosed with Mitomycin C intraperitoneally, gave a mean ratio of 0.87 normochromatic to polychromatic erythrocytes (range 0.46-1.70). The mean ratio of normochromatic to polychromatic erythrocytes of the top dose level of B251 was 0.78 (range 0.59-1.17). Both were statistically significantly different from the mean ratio of the control compound, indicating toxicity of the test compound and the positive reference compound. - Conclusions:
- Polyol IXOL B251 was negative in a micronucleus study (clastogenicity) in which mice were exposed to doses of 3000, 1500 and 750 mg/kg bw by oral gavage, in two equal dosages, separated by an interval of 24 hours. Toxicity (mortality) was observed at the mid and high dose.
- Executive summary:
In a study performed according to a protocol similar to OECD guideline 474 and under GLP, the effect of Polyol IXOL B251 on the incidence of micronucleated polychromatic erythrocytes in mice was assessed (Duphar B.V., 1984). Doses of 3000, 1500 and 750 mg/kg bw were administered by oral gavage, in two equal dosages, separated by an interval of 24 hours. A control group was dosed in an identical manner with the vehicle, 1%-tragacanth. A positive control group was dosed by intraperitoneal injection with Mitomycin C, at a total dosage of 6.6 mg/kg bw. Each of these groups of mice consisted of five females and five males. All the mice were killed six hours after the second treatment, bone marrow smears were examined for the presence of micronuclei in 1000 polychromatic erythrocytes per mouse. At all doses of Polyol IXOL B251, the mean micronucleated cell counts were in the same range as the counts obtained for the negative control group. The positive control compound, Mitomycin C, produced a large increase in the group mean micronucleated cell count. It was concluded that Polyol IXOL B251 did not show any evidence of mutagenic potential for polychromatic erythrocytes of mice in this test.
Regarding the occurrence of toxicity, in the top dose of Polyol IXOL B251 two male and two female mice died. In the middle dose one female mouse died.
- Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP compliant, guideline study, no restrictions, fully adequate for assessment
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- unscheduled DNA synthesis
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source:Charles River Laboratories
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 205.9 g for males, and 153.0 g for females
- Assigned to test groups randomly: yes
- Housing: housed three to five animals to a cage, except during exposure: housed individually in the holders.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 13-14 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C):20-24
- Humidity (%):45-65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- inhalation: vapour
- Vehicle:
- none
- Details on exposure:
- TYPE OF INHALATION EXPOSURE: nose only
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
The animals of groups 1-4 were exposed to the test atmosphere in nose-only exposure units (a modification of the chamber manufactured by ADG Developments Ltd., Codicote Hitchin, Herts, SG4 8UB, United Kingdom; see Figure 1). The inhalation chamber consisted of a cylindrical polypropylene (group 1) or aluminium (groups 2-4) column, surrounded by a transparent cylinder. The column had a volume of ca. 60 (groups 2 and 4) or 70 (group 3) liters and consisted of a top assembly with the entrance of the unit, a rodent tube section and at the bottom the base assembly with the exhaust port. The rodent tube section had 20 ports for animal exposure. Several empty ports were used for test atmosphere sampling and measurement of temperature and relative humidity.
TEST ATMOSPHERE
- Brief description of analytical method used:
The inhalation equipment was designed to expose the animals to a continuous supply of fresh test atmosphere. To generate the test atmosphere, the test material was diluted with water (80% test material/20% water). The solution was nebulized with an air-driven atomizer (Schlick type 970/S, Coburg, Germany). The amount nebulized was controlled by a peristaltic pump (Minipulse, Gilson, Velliers le Bel, France). The atomizer was supplied with humidified compressed air, the flow of which was measured using a mass stream meter (Bronkhorst Hi Tec, Ruurlo, The Netherlands). The resulting test atmosphere was led to the top inlet of the exposure chamber and from there to the noses of the animals, and exhausted at the bottom. The control atmosphere (group 1) consisted of a mass flow controlled (Bronkhorst Hi Tec) stream of humidified compressed air.
ANALYSIS OF TEST ATMOSPHERE
The actual concentration (by weight) of the non-volatile fraction of the aerosol present in the test atmosphere was determined three times during each exposure by means of gravimetric analysis.
To establish the dry matter content of the test material, known amounts (17.26, 17.93 and 17.23 mg) of undiluted test material were applied to glass fiber filters (Whatman, GF10, Ø 47 mm). The evaporation of the test material was measured after drying with ambient air for 5 to 6 days until a stable filter weight was reached. The percentage of captured weight remaining on the filters during that period was determined to be 95.42% on average. Hence, the weight of test material on the filters captured from the test atmosphere during exposure was multiplied by 100/95.42 to compensate for evaporation of volatile components.
Particle size measurements were carried out weekly for each of the three test atmospheres using a 10-stage cascade impactor (Andersen, Atlanta, USA). The Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were calculated. - Duration of treatment / exposure:
- 28 days
- Frequency of treatment:
- 6 hours/day, 5 days/week
- Post exposure period:
- no
- Remarks:
- Doses / Concentrations:
1 and 3 g/m3
Basis:
nominal conc. - Remarks:
- Doses / Concentrations:
1.03 and 3.14 g/m3
Basis:
analytical conc. - No. of animals per sex per dose:
- 5 males
- Control animals:
- yes
- Positive control(s):
- 2-acetylaminofluorene
- Route of administration: oral gavage 12-16 hours prior to sacrifice
- Doses / concentrations: 50 mg/kg bw ( 2.5 mg/mL in corn oil) - Tissues and cell types examined:
- liver hepatocytes
- Details of tissue and slide preparation:
- Within 12-16h after exposure, animals were sacrificed for isolation of hepatocytes. Hepatocytes were isolated from the liver using the perfusion technique described by Williams et al. (1977) with minor modifications. Briefly, the liver of each rat was perfused in situ with a Ca2+and Mg2+ free HEPES buffer (0.01 M) whilst under anaesthesia with sodium pentobarbital and exsanguination from the abdominal aorta, fol¬lowed by an in vitro perfusion with a HEPES-buffered (0.1 M) collagenase solution.
Directly after the start of the perfusion with the Ca2+ and Mg2+free HEPES buffer to remove the blood from the tissue, a small part of the caudate lobe was tied off using a ligature. Subsequently, part of the lobe was removed and preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde (10 % solution of formalin) for histopathological examination.
After isolation, the dissociated cells were incubated for 5-10 minutes in a shaking water bath at 37 ºC. Thereafter, they were filtered over a 200 mesh nylon filter, centrifuged and resuspended in WEC medium [= Williams medium E complete, which consisted of Williams medium E containing Glutamax supple¬mented with 10 % foetal calf serum and gentamycin (50 µg/mL)]. Cell counts were made. The viability of the hepatocytes was deter¬mined by trypan blue exclusion. The viability of the hepatocytes of the negative control animals was at least 50 %, and therefore considered sufficient. Suspensions containing 5 x 105 cells/mL were prepared in WEC medium. Aliquots (1 mL) were seeded onto Thermanox 25 mm round plastic cover slips in 35 mm 6-well dishes, which already contained 1 mL of WEC medium. The cultures were then incubated at ca 37 ºC in a humidified incubator containing ca 5 % CO2 and 95 % air to allow cells to attach (2 – 4 h).
Labelling of hepatocyte cultures
Two to four hours after seeding of the cells, the medium was removed and cells were washed twice with Williams E medium leaving only attached viable cells. Immediately after washing, 2 mL WEI [= Williams E medium Incomplete, which consisted of Williams medium E containing Glutamax, gentamycin (50 µg/mL), insulin (8 µg/mL), hydrocortisone (36 µg/mL) and ca 10 µCi [methyl-3H]thymidine (specific activity: 49 Ci.mmol-1 [1.81 TBq/mmol]) per mL was added to the cultures.
The hepatocyte cultures were incubated for 16 to 20 hours at ca 37 ºC. There¬after, the cover slips were rinsed in three successive washes with Williams E medium. The cover slips were then immersed in 2 mL of a 1 % sodium citrate solution for 10 min to allow cells to swell. Subsequently, cells were fixed in three 30 min changes of absolute ethanol-glacial acetic acid (3:1), air-dried, and mounted on glass slides.
Autoradiography
Slides were processed for autoradiography using Ilford K5D emulsion. At two time points, 7 and 10 days of exposure at < –18 ºC, slides were developed, fixed and washed with water. Slides were stained with haematoxylin and eosin and coded by a qualified person not involved in analysing the slides to enable ‘blind’ scoring. The slides that were developed after 10 days of exposure were chosen to be analyzed.
Grain counting and calculations
Sorcerer UDS software (Perceptive Instruments, UK) with microscopic attachment (Zeiss microscope connected to a high resolution camera) was used for grain counting of nuclei and cytoplasm. Fifty cells (randomly selected from each quadrant of the cover slip) per slide and 2 (out of 3) slides per animal were counted. The remaining slide of each animal was kept in reserve. Cells with abnormal morphology (pyknotic or lysed nuclei), or heavily-labelled S-phase cells were not counted. Cytoplasmic labelling was determined by counting two nuclei-sized area of cytoplasm adjacent to the nucleus.
The mean cytoplasmic count was subtracted from the nuclear count to give the net nuclear grains (NNG).
The following calculations were made for each slide:
the population average NNG +-SD (cell to cell)
the percent of cells in repair
the population average NNG +-SD for the subpopulation of the cells in repair
The following calculations were made for each animal:
the population average NNG +- SD (slide to slide)
the percent of cells in repair (slide to slide)
The following calculations were made for each data point:
the population average NNG +- SD (animal to animal)
the percent of cells in repair +- SD (animal to animal) - Evaluation criteria:
- The study will be considered valid if the positive controls give a positive response and if the negative control is non-genotoxic.
A response at a data point is considered positive if the population average NNG ≥ 5, and if at least 20 % of the cells are “in repair”.
A response is considered weakly positive if the population average NNG is between 0 and 5.
A test substance is considered to cause DNA damage and induce DNA repair in liver cells if the dose levels result in a positive or weakly positive response.
A test substance is considered non-genotoxic under the conditions of the test if the dose levels produce NNG ≤ 0. - Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- The mean NNG determined for the test substance groups (-8.03 and -11.30) were comparable to the mean NNG determined for the negative control (-10.58) and clearly lower than the mean NNG determined for the positive control 2-AAF (-0.25). In addition, the percentage ‘cells in repair’ of the test substance groups (both 2.00%) was also comparable to the negative control group (2.80% 'cells in repair’) and clearly lower than the percentage found in the positive control group (30.20%). The analysis of bromide – as a marker for the test substance (see below) – in blood, together with the observed systemic toxicity indicated systemic availability of the test compound, and thus it can be concluded that the test substance was able to penetrate the liver. Therefore, there is no reason to assume that the responses observed in the groups treated with the test substance were not correct.
Bromide analysis in blood
Bromide concentrations (± standard deviation) in blood sampled immediately after exposure on day 15/16 were 8.5 (± 0.3) and 9.9 (± 0.6) mg/kg for males and females of the control group, and 640 (± 59) and 820 (± 75) mg/kg for males and females of the high concentration group, respectively. Blood sampled the next day before the start of exposure contained 9.3 (± 0.4) and 9.9 (± 0.4) mg/kg bromide for males and females of the control group, and still 632 (± 76) and 776 (± 89) mg/kg for males and females of the high concentration group, respectively. This indicates the systemic availability of the test compound upon inhalation, as measured by the blood bromide concentration.
It is concluded that the test substance IXOL B251 did not induce unscheduled DNA synthesis (UDS) in liver cells of male rats, exposed to the test substance by inhalation, under the conditions used in this study. - Conclusions:
- Polyol IXOL B251 did not induce unscheduled DNA synthesis (UDS, gene mutations) in liver cells of male rats, exposed to the test substance by inhalation to 1 and 3 g/m3.
- Executive summary:
An UDS test was performed with Polyol IXOL B251 as part of a sub-acute (28-day, 6 hours/day, 5 days/week) inhalation toxicity studyin rats (TNO, 2010e). The male groups (n=5/group) exposed at 1 and 3 g/m3 (nose-only) and the control group (clean air) were used in the UDS test (GLP compliant and according to OECD guideline 486). Animals were sacrificed on the day after the last exposure. Six animals (including 1 reserve animal) were treated as positive control group. The animals were dosed by gavage (12-16 hour prior to sacrifice) with the mutagen 2-AAF (2-acetylaminofluorene) at 50 mg/kg bw.
The mean NNG determined for the test substance groups (-8.03 and -11.30) were comparable to the mean NNG determined for the negative control (-10.58) and clearly lower than the mean NNG determined for the positive control 2-AAF (-0.25). In addition, the percentage ‘cells in repair’ of the test substance groups (both 2.00%) was also comparable to the negative control group (2.80% ‘cells in repair’) and clearly lower than the percentage found in the positive control group (30.20%). The analysis of bromide – as a marker for the test substance – in blood, together with the observed systemic toxicity indicated systemic availability of the test compound, and thus it can be concluded that the test substance was able to penetrate the liver.
It is concluded that Polyol IXOL B251 did not induce unscheduled DNA synthesis (UDS) in liver cells of male rats, exposed to the test substance by inhalation.
Referenceopen allclose all
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Four genotoxicity studies are available for assessment: an Ames test, an in vitro MLA, an in vivo UDS test and an in vivo micronucleus test. The two in vivo studies were performed with Polyol IXOL B251. Polyol IXOL B251 is a mixture consisting of 93-94% Polyol IXOL B350 and 6-7% triethyl phosphate (CAS number: 78-40-0). Based on the available data on triethyl phosphate as summarised in the OECD SIDS of that substance, it is not expected that 6-7% triethyl phosphate as present in Polyol IXOL B251 changes the toxicity profile significantly, i.e., the toxicity profiles of Polyol IXOL B350 and Polyol IXOL B251 are considered similar.
Polyol IXOL B350 was examined for mutagenic activity in the bacterial reverse mutation test (GLP compliant and according to OECD guideline 471) using the histidine-requiringSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100 and the tryptophan-requiringEscherichia colistrain WP2uvrA, in the absence and presence of S9 -mix (TNO, 2010c). All strains were used, with five concentrations of the test substance, ranging from 62 to 5000 µg/plate. Negative controls (DMSO) and positive controls were run simultaneously with the test substance.
The mean number of his+and trp+revertant colonies of the negative controls were within the acceptable range and the positive controls gave the expected increase in the mean number of revertant colonies. The test substance was not toxic to any strain, in both the absence and presence of S9-mix, as neither a decrease in the mean number of revertants or a clearing of the background lawn of bacterial growth compared to the negative controls was observed.
In the presence of S9-mix, in strains TA 1535, TA 98, TA 100 and WP2uvrA, Polyol IXOL B350 did induce a dose related increase in the mean number of revertants compared to the background spontaneous reversion rate observed with the negative control. A minimal 2-fold increase was observed in strain TA 1535, at and above 185 µg/plate; in strain TA 98, at 5000 µg/plate; in strain TA 100, at and above 556 µg/plate and in strain WP2uvrA, at 5000 µg/plate. The maximal increase observed was 27-fold in strain TA 1535 at 5000 µg/plate.
In the absence of S9-mix in all strains, Polyol IXOL B350 did not induce a minimal 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control.
It is concluded that the results obtained with the test substance inSalmonella typhimuriumstrains TA 1535, TA 98 and TA 100 and in theEscherichia colistrain WP2uvrA, indicate that Polyol IXOL B350 is mutagenic in the presence of the S9-mix and all strains used indicate that Polyol IXOL B350 is not mutagenic in the absence of S9-mix under the conditions employed in this study.
Polyol IXOL B350 was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells, in both the absence and the presence S9-mix (GLP study according to OECD guideline 478). One assay was conducted in which single cultures were treated for 24 hours and 4 hours in the absence and presence of S9-mix, respectively (TNO, 2010d).
The highest concentrations of the substance evaluated for mutagenicity were 0.37 and 0.7 mmol/L in the absence and presence of S9-mix, respectively. The maximum concentrations were limited by cytotoxicity. In the absence of S9-mix no increase in mutant frequency was observed at any test substance concentration evaluated. In the presence of S9-mix a dose related increase in mutant frequency was observed at and above 0.5 mmol/L. In presence of S9-mix at the concentrations causing an increase in mutant frequency, relatively more small than large colonies were formed. The mean percentage of small colonies formed was 64%. Based on these results it cannot be excluded that Polyol IXOL B350 is clastogenic. The negative controls were within historical background ranges and treatment with the positive control yielded the expected significant increase in mutant frequency compared to the negative controls.
It is concluded that under the conditions used in this study, Polyol IXOL B350 is mutagenic (clastogenic) at the TK-locus of mouse lymphoma L5178Y cells.
In study performed according to a protocol similar to OECD guideline 474 and under GLP, the effect of Polyol IXOL B251 on the incidence of micronucleated polychromatic erythrocytes in mice was assessed (Duphar B.V., 1984). Doses of 3000, 1500 and 750 mg/kg bw were administered by oral gavage, in two equal dosages, separated by an interval of 24 hours. A control group was dosed in an identical manner with the vehicle, 1%-tragacanth. A positive control group was dosed by intraperitoneal injection with Mitomycin C, at a total dosage of 6.6 mg/kg bw. Each of these groups of mice consisted of five females and five males. All the mice were killed six hours after the second treatment, bone marrow smears were examined for the presence of micronuclei in 1000 polychromatic erythrocytes per mouse. At all doses of Polyol IXOL B251, the mean micronucleated cell counts were in the same range as the counts obtained for the negative control group. The positive control compound, Mitomycin C, produced a large increase in the group mean micronucleated cell count. It was concluded that Polyol IXOL B251 did not show any evidence of mutagenic potential for polychromatic erythrocytes of mice in this test.
Regarding the occurrence of toxicity, in the top dose of Polyol IXOL B251 two male and two female mice died. In the middle dose one female mouse died.
An UDS test was performed with Polyol IXOL B251 as part of a sub-acute (28-day, 6 hours/day, 5 days/week) inhalation toxicity study in rats (TNO, 2010e). The male groups (n=5/group) exposed at 1 and 3 g/m3 (nose-only) and the control group (clean air) were used in the UDS test (GLP compliant and according to OECD guideline 486). Animals were sacrificed on the day after the last exposure. Six animals (including 1 reserve animal) were treated as positive control group. The animals were dosed by gavage (12-16 hour prior to sacrifice) with the mutagen 2-AAF (2-acetylaminofluorene) at 50 mg/kg bw.
The mean NNG determined for the test substance groups (-8.03 and -11.30) were comparable to the mean NNG determined for the negative control (-10.58) and clearly lower than the mean NNG determined for the positive control 2-AAF (-0.25). In addition, the percentage ‘cells in repair’ of the test substance groups (both 2.00%) was also comparable to the negative control group (2.80% ‘cells in repair’) and clearly lower than the percentage found in the positive control group (30.20%). The analysis of bromide – as a marker for the test substance – in blood, together with the observed systemic toxicity indicated systemic availability of the test compound, and thus it can be concluded that the test substance was able to penetrate the liver.
It is concluded that Polyol IXOL B251 did not induce unscheduled DNA synthesis (UDS) in liver cells of male rats, exposed to the test substance by inhalation.
The required studies regarding mutagenicity are available. The substance was mutagenic in the Ames test in the presence of S9 mix in strains TA 1535, TA 98 and TA 100 and in theEscherichia colistrain WP2uvrA. Furthermore, the substance was positive in mouse lymphoma L5178Y cells in the presence of S9-mix. Relatively more small than large colonies were formed in the MLA which may indicate clastogenic effects.
The positive results observed in the in vitro studies were not seen in the in vivo studies. Polyol IXOL B251 was negative in a micronucleus study (clastogenicity) in which mice were exposed to doses of 3000, 1500 and 750 mg/kg bw by oral gavage, in two equal dosages, separated by an interval of 24 hours. Toxicity (mortality) was observed at the mid and high dose.
In addition, Polyol IXOL B251 did not induce unscheduled DNA synthesis (UDS, gene mutations) in liver cells of male rats, exposed to the test substance by inhalation to 1 and 3 g/m3. The analysis of bromide, as a marker for the test substance, in blood, together with the observed systemic toxicity indicated systemic availability of the test compound, showed that the test substance was able to penetrate the liver.
In
conclusion,
Polyol IXOL B350 is
not considered a mutagenic substance in vivo.
Justification for classification or non-classification
Based on the available data, classification for mutagenicity is not needed according to EU Directive 67/584/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.
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.
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