Registration Dossier
Registration Dossier
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EC number: 226-798-2 | CAS number: 5470-11-1
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames test (OECD 471): negative in S. typhimurium TA 1535, TA 97, TA 98 and TA 100 with and without metabolic activation
Chromosome aberration test (similar to OECD 473): positive in cultured peripheral lymphocytes without metabolic activation
Mouse lymphoma assay (similar to OECD 490): ambigious in Trial 1 and negative in Trial 2 without metabolic activation; negative in Trial 1 and ambiguous in Trial 2 with metabolic activation in mouse lymphoma L5178Y cells
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- / AT reversion site not covered, no historical control data provided, no concentrations of positive controls provided, no data on dose-finding study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 21 July 1997
- Deviations:
- yes
- Remarks:
- / AT reversion site not covered, no historical control data provided, no concentrations of positive controls provided, no data on dose-finding study
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- Species / strain / cell type:
- S. typhimurium TA 97
- Species / strain / cell type:
- S. typhimurium TA 98
- Species / strain / cell type:
- S. typhimurium TA 100
- Species / strain / cell type:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats and male hamsters, treated with Aroclor 1254.
- Test concentrations with justification for top dose:
- main experiment:
TA 97: 33, 100, 251, 333, 667 µg/plate (- S9 mix, + 10% S9 mix (hamster/rat)); 33, 100, 200, 333, 667 µg/plate (+ 30% S9 mix (hamster/rat))
TA 98: 33, 100, 251, 333, 667 µg/plate (- S9 mix, + 10% S9 mix (10%, hamster/rat)); 33, 100, 333, 1000, 2000 µg/plate (+ 30% S9 mix (hamster/rat))
TA 1535: 33, 100, 200, 333, 667 µg/plate (- S9 mix, + 30% S9 mix (hamster/rat)); 33, 100, 251, 333, 667 µg/plate (+ 10% S9 mix (hamster)); 33, 100, 250, 333, 667 µg/plate (+ 10% S9 mix (rat))
TA 100:
test 1: 10, 33, 100, 333, 667 µg/plate (-S9 mix) ; 100, 333, 500, 750, 1000 µg/plate (+ 10% S9 mix (hamster); 33, 100, 333, 1000, 2000 µg/plate (+ 30% S9 mix (hamster/rat) and + 10% S9 mix (rat))
test 2: 10, 33, 100, 333, 667 µg/plate (-S9 mix) ; 33, 100, 251, 333, 667 µg/plate (+ 10% S9 mix (hamster); 33, 100, 200, 333, 667 µg/plate (+ 10% S9 mix (rat)); 33, 100, 333, 1000, 2000 µg/plate (+ 30% S9 mix (hamster/rat))
test 3: 100, 333, 500, 750, 1000 µg/plate (+ 30% S9 mix (hamster) and + 10% S9 mix (rat)); 33, 100, 200, 333, 667 µg/plate (+ 30% S9 mix (rat))
test 4: 33, 100, 251, 333, 667 µg/plate (+ 30% S9 mix (hamster) + 10% S9 mix (rat)); 100, 333, 500, 750, 1000 µg/plate (+ 30% S9 mix (rat))
test 5: 33, 100, 251, 333, 667 µg/plate (+ 30% S9 (rat)) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: distilled water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- other: 4-nitro-o-phenylenediamine (4-NPD), 2-aminoanthracene (2AA)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation
DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: The main assay was carried out in triplicate.
DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number his+ colonies and/or clearing of the bacterial background lawn - Rationale for test conditions:
- Reference to "Zeiger E, Drake JW (1980): An environmental mutagenesis test development programme. In Montesano R, Bartsch H, Tomatis L (eds):
“Molecular and Cellular Aspects of Carcinogen Screening Tests." Lyon: IARC Scientific Publications, No. 27, pp 303-313." - Evaluation criteria:
- The test article is judged to be mutagenic or weakly mutagenic, if it produced a reproducible dose-related increase in his+ revertants compared to the corresponding solvent control under a single metabolic activation condition, in replicate trials.
- Statistics:
- Mean values and standard error of means were calculated.
- Key result
- Species / strain:
- S. typhimurium TA 97
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- number of revertant colonies decreased to less than half compared to solvent control starting at 333 μg/ plate without S9 mix (52%) and at 667 μg/ plate with 10% S9 mix (hamster, 51%)
- Vehicle controls validity:
- other: no historical control data given
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- other: no historical control data given
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- number of revertant colonies decreased to less than half compared to solvent control starting at 1000 μg/ plate with 30% S9 mix (hamster, 100%)
- Vehicle controls validity:
- other: no historical control data given
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- other: no historical control data given
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- number of revertant colonies decreased to less than half compared to solvent control in exp.1 starting at 750 μg/plate with 10% S9 mix (hamster, 100%) and in exp.1 and 2 at 2000 μg/plate with 30% S9 mix (hamster, 63% and 55% respectively)
- Vehicle controls validity:
- other: no historical control data given
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- other: no historical control data given
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- number of revertant colonies decreased to less than half compared to solvent control starting at 333 μg/plate without S9 mix (59%)
- Vehicle controls validity:
- other: no historical control data given
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- other: no historical control data given
- Remarks on result:
- other: Cytotoxicity was furthermore detected in exp.3 at 1000 μg/plate with 30% S9 mix (hamster, 85%) and starting at 750 μg/plate with 10% S9 mix (rat, 89%), in exp. 4 it started at 750 μg/plate with 30% S9 mix (rat, 85%)
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Version / remarks:
- adopted Jul 2016
- GLP compliance:
- no
- Type of assay:
- other: in vitro mammalian cell gene mutation test
- Target gene:
- TK locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Remarks:
- clone 3.7.2C
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Research Triangle Park, NC, USA
- Methods for maintenance in cell culture if applicable: New stock cultures were thawed every two months to avoid a slight shift in the distribution of chromosome numbers in the cells.
MEDIA USED
- Type and identity of media: Fischer`s powdered medium (Gibco, NY, USA) was mixed with purified deionized water and filter-sterilized. Medium was supplemented with horse serum (Flow Laboratories, VA, USA), L-glutamine, penicillin and streptomycin (Gibco, NY, USA), pluronic F-68 (BASF Wyandotte Corp., MI, USA) and Noble agar (Difco Laboratories, MI, USA).
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.
- Test concentrations with justification for top dose:
- Experiment I
4 h without metabolic activation: 67, 84, 105, 131, 164 and 205 µg/mL
4 h with metabolic activation: 262, 328, 410, 512 and 640 µg/mL
Experiment II
4 h without metabolic activation: 84, 105, 131, 164 and 205 µg/mL
4 h with metabolic activation: 425, 472, 525 and 583 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: distilled water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- other: 3-Methylcholanthrene: +S9: 5 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar
- Cell density at seeding: 6 x 10E06 cells
DURATION
- Exposure duration: 4 h
- Expression time: 2 d
- Selection time: 11 - 12 d
SELECTION AGENT (mutation assays): Triflurothymidine
NUMBER OF REPLICATIONS: Duplicate cultures in each 1 and 2 independent experiments, respectively (without and with methabolic activation)
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - Evaluation criteria:
- The results of this study were evaluated according to the current OECD 490 Guideline: An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above concurrent control), designated the Global Evaluation Factor (GEF), which is based on the analysis of the distribution of the negative control MF data from participating laboratories. For the agar version of the MLA the GEF is 90 x 10E06 and for the microwell version of the MLA the GEF is 126 x 10E06. A test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g. using a trend test). The test chemical is then considered able to induce mutation in this test system. A test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.
- Statistics:
- Mean values were calculated.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- without
- Genotoxicity:
- ambiguous
- Remarks:
- Biological significance unclear (please refer to "any other information on results incl. tables"
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 164 µg/mL
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 205 µg/mL
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with
- Genotoxicity:
- ambiguous
- Remarks:
- Biological significance unclear (please refer to "any other information on results incl. tables"
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 640 µg/mL
- Vehicle controls validity:
- valid
- Remarks:
- Some of the individual CE and MF values of the vehicle control of Experiment I were slightly below the acceptable criteria as defined according to OECD 490.
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 583 µg/mL
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at 640 µg/mL in Experiment I and at 648 µg/mL in Experiment II. - Remarks on result:
- other: Experiment I
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- (only limited data on the cytogenetic potential given)
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- adopted Jul 2016
- Deviations:
- yes
- Remarks:
- (only limited data on the cytogenetic potential given)
- GLP compliance:
- no
- Target gene:
- Not applicable
- Species / strain / cell type:
- lymphocytes: cultured peripheral lymphocytes from male muntjac
- Details on mammalian cell type (if applicable):
- CELLS USED
- Whole blood was used
- Methods for maintenance in cell culture: 0.1 mL phytohaemagglutinin P, at 37 °C
MEDIA USED
- Type and identity of media including CO2 concentration: Parker 1999 with 20% heat-inactivated human AB serum - Metabolic activation:
- without
- Test concentrations with justification for top dose:
- 18 h treatment without metabolic activation: 50 µg/mL
- Untreated negative controls:
- yes
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 18 h
- Fixation time (start of exposure up to fixation or harvest of cells): 18 h
SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.01 µg/mL)
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The slides were finally processed for G- and C-bandings by the modified techniques of Seabright (1971) and Summer (1972). - Key result
- Species / strain:
- lymphocytes: cultured peripheral lymphocytes from male muntjac
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Remarks:
- at 50 µg/mL
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- not applicable
- Additional information on results:
- The majority of the damage consisted of constrictions, gaps and chromatid breaks.
Referenceopen allclose all
Table 1: Summary of test results (main assay)
With or without S9 -mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates) |
|||||||
Frameshift type |
Base-pair substitution type |
||||||||
TA97 |
TA98 |
TA100 |
TA1535 |
||||||
exp. 1 |
exp. 2 |
exp. 3 |
exp. 4 |
exp. 5 |
|||||
– S9 |
Solvent control (water) |
86 ± 7.8 |
28 ± 4.3 |
118 ± 6.6 |
160 ± 11.3 |
- |
27 ± 2.1 |
||
10 |
- |
103 ± 3.8 |
147 ± 11.6 |
- |
|||||
33 |
105 ± 5.8 |
23 ± 0.0 |
102 ± 5.0 |
138 ± 3.3 |
28 ± 3.2 |
||||
100 |
82 ± 7.4 |
24 ± 2.8 |
100 ± 3.7 |
174 ± 3.1 |
22 ± 3.6 |
||||
200 |
- |
17 ± 2.0 |
|||||||
250 |
- |
||||||||
251 |
64 ± 5.5 |
24 ± 0.3 |
- |
||||||
333 |
41 ± 4.0 s |
20 ± 1.7 |
126 ± 2.6 |
173 ± 4.9 |
11 ± 1.8 |
||||
500 |
- |
- |
- |
- |
- |
||||
667 |
18 ± 1.5 |
21 ± 0.0 |
64 ± 3.2 |
94 ± 10.8 |
9 ± 2.0 |
||||
750 |
- |
- |
|||||||
1000 |
|||||||||
2000 |
|||||||||
Positive controls |
9AA |
4-NPD |
SAZ |
SAZ |
|||||
Mean No. of colonies/plate (average of 3 plates) |
692 ± 11.7 |
582 ± 21.3 |
440 ± 3.1 |
570 ± 42.9 |
- |
257 ± 33.5 |
|||
+ 10% S9 (hamster) |
Solvent control (water) |
208 ± 7.5 |
32 ± 2.6 |
118 ± 7.3 |
124 ± 2.3 |
- |
15 ± 2.4 |
||
10 |
- |
- |
|||||||
33 |
216 ± 6.0 |
29 ± 1.5 |
- |
110 ± 4.2 |
16 ± 1.5 |
||||
100 |
207 ± 6.9 |
26 ± 2.9 |
117 ± 6.4 |
120 ± 1.3 |
14 ± 1.5 |
||||
200 |
- |
- |
|||||||
250 |
|||||||||
251 |
197 ± 5.2 |
26 ± 0.9 |
- |
151 ± 8.4 |
21 ± 1.0 |
||||
333 |
156 ± 1.8 |
25 ± 3.2 |
139 ± 3.3 |
155 ± 0.6 |
19 ± 2.7 |
||||
500 |
- |
62 ± 2.3 |
- |
- |
|||||
667 |
101 ± 11.0 |
29 ± 3.0 |
- |
198 ± 6.1 |
24 ± 0.9 |
||||
750 |
- |
0 ± 0 s |
- |
- |
|||||
1000 |
0 ± 0 s |
||||||||
2000 |
- |
||||||||
Positive control |
2AA |
||||||||
Mean No. of colonies/plate (average of 3 plates) |
646 ± 8.1 |
244 ± 12.5 |
440 ± 12.5 |
383 ± 16.7 |
- |
111 ± 6.7 |
|||
+ 30% S9 (hamster) |
Solvent control (water) |
161 ± 9.4 |
31 ± 0.9 |
123 ± 5.8 |
132 ± 4.8 |
132 ± 5.8 |
176 ± 18.0 |
- |
12 ± 1.3 |
10 |
- |
- |
|||||||
33 |
183 ± 9.0 |
41 ± 1.5 |
126 ± 7.0 |
164 ± 4.1 |
- |
141 ± 5.4 |
17 ± 0.9 |
||
100 |
169 ± 7.4 |
31 ± 2.0 |
100 ± 2.6 |
158 ± 3.5 |
132 ± 1.9 |
115 ± 7.4 |
11 ± 0.9 |
||
200 |
154 ± 23.8 |
- |
- |
13 ± 1.5 |
|||||
250 |
- |
- |
|||||||
251 |
146 ± 9.9 |
||||||||
333 |
138 ± 18.2 |
27 ± 7.6 s |
166 ± 4.6 |
210 ± 3.5 |
216 ± 8.0 |
158 ± 9.5 |
15 ± 0.9 |
||
500 |
- |
- |
201 ± 6.9 |
- |
- |
||||
667 |
105 ± 11.1 |
- |
214 ± 11.9 |
14 ± 2.3 |
|||||
750 |
- |
91 ± 7.1 |
- |
- |
|||||
1000 |
0 ± 0.0 s |
104 ± 7.4 |
130 ± 7.8 s |
20 ± 6.3 s |
|||||
2000 |
t |
45 ± 12.3 s |
60 ± 3.2 s |
- |
|||||
Positive control |
2AA |
||||||||
Mean No. of colonies/plate (average of 3 plates) |
898 ± 51.3 |
171 ± 13.5 |
856 ± 40.5 |
748 ± 11.3 |
578 ± 14.0 |
503 ± 13.5 |
- |
130 ± 7.6 |
|
+ 10% S9 (rat) |
Solvent control (water) |
145 ± 6.0 |
50 ± 2.7 |
126 ± 10.5 |
121 ± 1.8 |
132 ± 4.2 |
136 ± 12.1 |
- |
12 ± 1.5 |
10 |
- |
||||||||
33 |
136 ± 6.2 |
51 ± 4.6 |
128 ± 7.1 |
148 ± 9.2 |
- |
156 ± 6.7 |
- |
15 ± 1.5 |
|
100 |
136 ± 6.6 |
49 ± 2.3 |
169 ± 7.8 |
151 ± 3.6 |
122 ± 4.2 |
162 ± 8.8 |
19 ± 3.0 |
||
200 |
- |
- |
182 ± 4.5 |
- |
- |
- |
|||
250 |
- |
17 ± 4.1 |
|||||||
251 |
159 ± 6.1 |
46 ± 5.5 |
193 ± 13.7 |
- |
|||||
333 |
156 ± 4.0 |
54 ± 5.9 |
188 ± 3.5 |
206 ± 5.9 |
170 ± 7.7 |
195 ± 11.0 |
17 ± 3.1 |
||
500 |
- |
- |
- |
105 ± 3.8 |
- |
- |
|||
667 |
84 ± 2.6 |
55 ± 7.4 |
185 ± 8.5 |
- |
229 ± 13.5 |
15 ± 2.3 |
|||
750 |
- |
- |
14 ± 2.3 |
- |
- |
||||
1000 |
142 ± 9.6 |
0 ± 0.0 s |
|||||||
2000 |
74 ± 3.2 s |
- |
|||||||
Positive control |
2AA |
||||||||
Mean No. of colonies/plate (average of 3 plates) |
1421 ± 8.7 |
118 ± 9.5 |
1215 ± 17.1 |
854 ± 155.7 |
1582 ± 29.0 |
650 ± 64.0 |
- |
302 ± 7.3 |
|
+ 30% S9 (rat) |
Solvent control (water) |
139 ± 19.7 |
34 ± 3.3 |
110 ± 3.0 |
190 ± 4.3 |
161 ± 3.7 |
138 ± 5.7 |
159 ± 3.7 |
13 ± 0.9 |
10 |
- |
||||||||
33 |
130 ± 7.5 |
33 ± 1.9 |
117 ± 6.7 |
187 ± 11.4 |
164 ± 4.3 |
- |
161 ± 4.5 |
12 ± 3.5 |
|
100 |
138 ± 3.5 |
32 ± 0.9 |
130 ± 3.5 |
191 ± 6.0 |
125 ± 5.5 |
143 ± 8.5 |
174 ± 6.2 |
18 ± 1.5 |
|
200 |
96 ± 4.8 |
- |
143 ± 9.0 |
- |
- |
18 ± 0.9 |
|||
250 |
- |
- |
|||||||
251 |
165 ± 1.7 |
||||||||
333 |
99 ± 11.5 |
36 ± 3.2 |
172 ± 4.9 |
184 ± 5.9 |
175 ± 7.0 |
162 ± 5.5 |
178 ± 6.4 |
18 ± 1.2 |
|
500 |
- |
- |
- |
143 ± 2.8 |
- |
- |
|||
667 |
69 ± 4.2 |
173 ± 12.1 |
- |
242 ± 20.5 |
12 ± 2.6 |
||||
750 |
- |
- |
21 ± 0.3 |
- |
|||||
1000 |
28 ± 1.3 |
211 ± 13.3 |
225 ± 18.1 |
0 ± 0.0 s |
|||||
2000 |
18 ± 3.5 s |
66 ± 3.1 s |
86 ± 3.1 s |
- |
|||||
Positive control |
2AA |
||||||||
Mean No. of colonies/plate (average of 3 plates) |
537 ± 34.9 |
499 ± 10.4 |
1536 ± 1.3 |
673 ± 11.6 |
1536 ± 16.6 |
1525 ± 23.7 |
1161 ± 19.9 |
71 ± 10.3 |
|
2AA = 2-aminoanthracene
4-NPD = 4-nitro-o-phenylenediamine
SAZ = sodium azide
9AA = 9-aminoacridine
s = slight clearing of background lawn
t = complete clearing of background lawn
Interpretation of results:
In Experiment II in the absence of S9 mix and in Experiment I in the presence of S9 mix no concentration related response was observed and no increase in the MF above the concurrent background exceeding the GEF was observed at any tested concentration thus, according to the criteria defined in OECD TG 490 (§64), the test substance is not considered to be mutagenic in these experimental trials. However, in Experiment I a dose related increase in mutation frequency was observed at 131, 164 and 205 µg/mL without metabolic activation, respectively, exceeding the MF above the concurrent background and the GEF, and inducing relative total growth (RTG) values of 29, 15 and 6%, respectively. In the presence of S9 mix, a dose related increase in mutation frequency was observed in Experiment II with the highest concentration applied (583 µg/mL) exceeding the MF above the concurrent background and the GEF. However, this concentration induces a RTG value of 19.5%. Taking into consideration that 1) care should be taken when interpreting positive results only found between 20 and 10% RTG as defined in OECD TG 490 (§67), 2) the concentration range at the border to cytotoxicity is quite tight (+ S9: between 525 and 583 µg/mL; - S9: between 131 and 164 µg/mL) and thus a RTG near the border of the accepted RTG value of 10 - 20% can be considered as cytotoxicity and 3) the data do not allow a robust statistical analysis of the dose response dependency, the results in these experimental trials are considered as ambiguous.
Table 1: Results of Experiment I without metabolic activation
| RTG | Mean RTG | Mutant Frequency (= MF) | Mean MF | Mean MF plus GEF (90x10E06) | GEF exceeded | cytotox (RTG 10 - 20%) | |
| Control | 107 | 100 | 32 | 35 | 125 | - | - |
| 101 | 31 | ||||||
| 88 | 32 | ||||||
| 104 | 45 | ||||||
| 67 µg/mL | 71 | 71.5 | 72 | 60,5 | - | no | no |
| 72 | 49 | ||||||
| 84 µg/mL | 61,00 | 56,00 | 67 | 70 | - | no | no |
| 51,00 | 73 | ||||||
| 105 µg/mL | 42,00 | 43,50 | 97 | 85 | - | no | no |
| 45,00 | 73 | ||||||
| 131 µg/mL | 29,00 | 29,00 | 128 | 128 | - | yes | no |
| 164 µg/mL | 18,00 | 15,00 | 130 | 130,5 | - | yes | yes |
| 12,00 | 131 | ||||||
| 205 µg/mL | 6,00 | 6,00 | 138 | 133,5 | - | yes | yes |
| 6,00 | 129 | ||||||
| MCA | 38,00 | 33,00 | 733 | 796 | - | yes | no |
| 26,00 | 940 | ||||||
| 35,00 | 716 |
MCA: 3-Methylcholanthrene
RTG: Relative total growth
GEF: Global evaluation factor
Table 2: Results of Experiment I with metabolic activation
| RTG | Mean RTG | Mutant Frequency (= MF) | Mean MF | Mean MF plus GEF (90x10E06) | GEF exceeded | Cytotoxicity (RTG 10 - 20%) | |
| Control | 79 | 93 | 48 | 36 | 126 | - | - |
| 95 | 30 | ||||||
| 105 | 30 | ||||||
| 262 µg/mL | 114 | 108 | 50 | 52.5 | - | no | no |
| 102 | 55 | ||||||
| 328 µg/mL | 113 | 105.5 | 28 | 31 | - | no | no |
| 98 | 34 | ||||||
| 410 µg/mL | 92 | 85.5 | 59 | 70.5 | - | no | no |
| 79 | 82 | ||||||
| 512 µg/mL | 50 | 57.5 | 111 | 88.5 | - | no | no |
| 65 | 66 | ||||||
| 640 µg/mL | 0 | 0 | 0 | 0 | - | - | - |
| 0 | 0 | ||||||
| MCA | 39 | 41.33 | 253 | 293.33 | - | yes | no |
| 40 | 328 | ||||||
| 45 | 299 |
MCA: 3-Methylcholanthrene
RTG: Relative total growth
GEF: Global evaluation factor
Table 3: Results of Experiment II without metabolic activation
| RTG | Mean RTG | Mutant Frequency (= MF) | Mean MF | Mean MF plus GEF (90x10E06) | GEF exceeded | Cytotoxicity (RTG 10 - 20%) | |
| Control | 80 | 100 | 50 | 54.25 | 144.25 | - | - |
| 101 | 57 | ||||||
| 115 | 59 | ||||||
| 104 | 51 | ||||||
| 84 µg/mL | 62 | 65.5 | 87 | 87.5 | - | no | no |
| 69 | 88 | ||||||
| 105 µg/mL | 51 | 41.5 | 122 | 111.5 | - | no | no |
| 32 | 101 | ||||||
| 131 µg/mL | 38 | 35.5 | 121 | 122.5 | - | no | no |
| 33 | 124 | ||||||
| 164 µg/mL | 27 | 26.5 | 115 | 114 | - | no | no |
| 26 | 113 | ||||||
| 205 µg/mL | 5 | 5.5 | 188 | 183.5 | - | yes | yes |
| 6 | 179 | ||||||
| EMS | 37 | 38 | 1068 | 1035.66 | - | yes | no |
| 34 | 1088 | ||||||
| 43 | 951 |
EMS: Ethylmethane sulfonate
RTG: Relative total growth
GEF: Global evaluation factor
Table 4: Results of Experiment II with metabolic activation
| RTG | Mean RTG | Mutant Frequency (= MF) | Mean MF | Mean MF plus GEF (90x10E06) | GEF exceeded | cytotox (RTG 10 - 20%) | |
| Control | 108 | 100.33 | 90 | 90.3 | 180.3 | - | - |
| 104 | 82 | ||||||
| 89 | 99 | ||||||
| 425 µg/mL | 96 | 96 | 90 | 83 | - | no | no |
| 96 | 75 | ||||||
| 472 µg/mL | 73 | 81 | 91 | 93 | - | no | no |
| 89 | 95 | ||||||
| 525 µg/mL | 59 | 57 | 147 | 148 | - | no | no |
| 55 | 149 | ||||||
| 583 µg/mL | 20 | 19.5 | 271 | 259 | - | yes | yes |
| 19 | 247 | ||||||
| MCA | 34 | 36.66 | 476 | - | yes | no | |
| 37 | 450 | ||||||
| 39 | 447 |
MCA: 3-Methylcholanthrene
RTG: Relative total growth
GEF: Global evaluation factor
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Micronucleus test (OECD 474, GLP): negative in NMRI mice
(RA from CAS 10039-54-0)
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- refer to analogue justification provided in IUCLID section 13
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- The highest dose level led to evident signs of toxicity, such as irregular respiration, piloerection, apathy, abdominal position, closed eyelids and blueish skin within 30 minutes after test substance administration.
- Vehicle controls validity:
- not valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
Reference
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Justification for read-across
There are data available regarding genetic toxicity (mutagenicity and cytogenicity) in mammalian cells for hydroxylammonium chloride (CAS 5470-11-1). In addition, read-across from an appropriate substance was conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5. in order to fulfil the standard data requirements, defined in Regulation (EC) No 1907/2006, Annex VIII, 8.4. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).
As there is only supporting data on genetic toxicity (cytogenicity) in mammalian cells available for hydroxylammonium chloride (CAS 5470-11-1), information available for the analogue substance bis(hydroxylammonium)sulfate (CAS 10039-54-0) is taken into account to fulfil the standard data requirements defined in Regulation (EC) No 1907/2006, Annex VIII, 8.4.
Genetic toxicity in bacteria (Ames)
CAS 5470-11-1
The test substance was investigated for mutagenicity to bacteria (Ames test) in a study similar to OECD guideline 471 (Zeiger et al., 1992). The Salmonella typhimurium strains TA 1535, TA 100, TA 98, TA 97 were exposed to concentrations ranging from 10 - 2000 µg/plate in distilled water with and without the addition of a metabolic activation system (cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats and male hamsters, treated with Aroclor 1254) in two independent assays with triplicates each (dose finding study and main experiment). The experiments were conducted according to the preincubation methodology. But only the results of the main assay were depicted in the literature. Cytotoxicity was observed in tester strains TA 97 and TA 1535 in the absence of S9-mix, where the number of revertant colonies decreased to less than half compared to solvent control starting at 333 μg/plate (52 and 59%, respectively) and in TA 97 in the presence of hamster S9-mix (10%) at 667 μg/plate (51%) and in TA 98 in the presence of hamster S9-mix (30%) starting at 1000 μg/plate (100%). For TA 100, results of up to 5 experiments were presented, showing cytotoxicity in presence of the different activation systems at different concentrations and to different extent. In contrast to the other strains TA 100 showed a dose dependent increase of revertants in more than one experiment. But as no doubling of the revertants could be observed in any of the replicates, the result is still considered to be negative. The reference mutagens showed the expected increase in the number of revertant colonies.
Genetic toxicity (cytogenicity) in mammalian cells in vitro
CAS 5470-11-1
An in vitro mammalian chromosome aberration test was performed with the source substance in cultured peripheral lymphocytes from male muntjac similar to OECD guideline 473 in the absence of metabolic activation (Gupta and Sharma, 1980). The cells were treated with 50 µg/mL for 18 h without metabolic activation. An untreated control served as negative control, a positive control was not included. Hydroxylamine hydrochloride induced aberrations in cultured peripheral lymphocytes. The majority of the damage consisted of constrictions, gaps and chromatid breaks. Based on the results of this study, the source substance has cytogenetic properties in mammalian cells.
Genetic toxicity (mutagenicity) in mammalian cells in vitro
CAS5470-11-1
An in vitro mammalian cell gene mutation assay according to OECD guideline 476 and similar to OECD guideline 490 was performed with the test substance in mouse lymphoma L5178Y cells (Mitchell et al., 1988). The cells were treated for 4 hours with and without metabolic activation (cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats, treated with Aroclor 1254). The test substance was tested up to cytotoxicity, the following concentrations were tested 67, 84, 105, 131, 164 and 205 µg/mL (Experiment I without metabolic activation, 4 h exposure), 262, 328, 410, 512 and 640 µg/mL (Experiment I with metabolic activation, 4 h exposure), 84, 105, 131, 164 and 205 µg/mL (Experiment II without metabolic activation, 4 h exposure) and 425, 472, 525 and 583 µg/mL (Experiment II with metabolic activation, 4 h exposure). 3-Methylcholanthrene and ethylmethanesulphonate were used as positive controls with and without S9 mix, respectively. A decrease of the relative total growth (RTG) was observed at the highest concentration of 640 and 583 µg/mL in Experiment I and II (without metabolic activation), respectively, and at the highest concentrations of 205 µg/mL without metabolic activation in Experiment II. Positive control materials induced distinct increases in mutation frequencies indicating the validity of the test and of the activity of the metabolizing system. In Experiment I a dose related increase in mutation frequency was observed at 131, 164 and 205 µg/mL without metabolic activation, respectively, exceeding the MF above the concurrent background and the GEF, and inducing relative total growth (RTG) values of 29, 15 and 6%, respectively. In the presence of S9 mix, a dose related increase in mutation frequency was observed in Experiment II with the highest concentration applied (583 µg/mL) exceeding the MF above the concurrent background and the GEF. However, this concentration induces a RTG value of 19.5%. Taking into consideration that 1) care should be taken when interpreting positive results only found between 20 and 10% RTG as defined in OECD TG 490 (§67), 2) the concentration range at the border to cytotoxicity is quite tight (+ S9: between 525 and 583 µg/mL; - S9: between 131 and 164 µg/mL) and thus a RTG near the border of the accepted RTG value of 10 - 20% can be considered as cytotoxicity and 3) the data do not allow a robust statistical analysis of the dose response dependency, the results in these experimental trials are considered as ambiguous. In Experiment II in the absence of S9 mix and in Experiment I in the presence of S9 mix no concentration related response was observed and no increase in the MF above the concurrent background exceeding the GEF was observed at any tested concentration thus, according to the criteria defined in OECD TG 490 (§64), the test substance is not considered to be mutagenic in these experimental trials.
Genetic toxicity (cytogenicity) in mammalian cells in vivo
CAS 10039-54-0
An in vivo mammalian cell gene cytogenicity assay according to OECD guideline 474 was performed with the source test substance in male and female NMRI mice (reference 7.6.2-1). The maximum tolerated dose was selected based on the results of a pre-test for the determination of acute oral toxicity, thus the animals were exposed to doses of 300, 600 and 1200 mg/kg bw. Control animals treated with the solvent (aqua dest.) and the positive controls (cyclophosphamide and vincristine) were included in the study. The positive controls induced an increase of micronucleated polychromatic erythrocytes indicating the validity of the test. The highest dose level of the test substance revealed evident signs of toxicity, such as irregular respiration, piloerection, apathy, abdominal position, closed eyelids and blueish skin within 30 minutes after test substance administration indicating the systemic availability of the test substance. An increase of micronucleated polychromatic erythrocytes was not observed in any of the dose groups, thus, the test substance was not clastogenic in NMRI mice.
Conclusion for genetic toxicity
The available data indicate that hydroxylammonium chloride (CAS 5470-11-1) was not mutagenic to bacteria. The available mutagenicity data in mammalian cells indicate that the result with and without metabolic activation was ambiguous. The available data of bis(hydroxylammonium)sulfate (CAS 10039-54-0) show that the source substance was not clastogenic in vivo. Therefore, based on common functional groups and structural similarities, hydroxylammonium chloride (CAS 5470-11-1) is also considered not to be clastogenic in vivo.
Justification for classification or non-classification
According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to hydroxylammonium chloride, data will be generated from information on reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.
The available data on genetic toxicity with hydroxylammonium chloride (CAS 5470-11-1) and with the source substance bis(hydroxylammonium)sulfate (CAS 10039-54-0) do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.
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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