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Registration Dossier
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EC number: 230-386-8 | CAS number: 7085-19-0
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In Vitro Gene Mutation Study in Bacteria (Ames Test): Engelhardt (1981)
Under the conditions of this study, the test material was not mutagenic.
Genetic Toxicity In Vitro
In Vitro Gene Mutation Study in Mammalian Cells (CHO): Engelhardt (1988)
Under the conditions the test material is assumed to be non-mutagenic.
An in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from in vivo cytogenicity tests are available.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 18 February 1981 to 09 March 1981
- 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 471 (Bacterial Reverse Mutation Assay)
- GLP compliance:
- no
- Remarks:
- Study pre-dates GLP
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine requirement in Salmonella typhimurium strains.
- Species / strain / cell type:
- S. typhimurium TA 1538
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- Source of S9: Male Sprague-Dawley rats, 200-300 g
- Method of preparation of S9 mix: To activate the enzymes which metabolise foreign substances, the rats received a single intraperitoneal injection of 500 mg Aroclor 1254 per kg body weight 5 days before sacrifice. On the fifth day the rats were killed, and the livers prepared. The livers were weighed and washed in an equivalent volume of a 150 mM KCl solution (1 mL KCl to 1 g wet Liver), then cut into small pieces and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 g for 10 minutes at +4 °C, 5 mL portions of the supernatant (so-called S-9 fraction) are quickly deep-frozen in dry ice and stored at -70 °C to -80 °C for 2 months at the most.
- Concentration or volume of S9 mix and S9 in the final culture medium: 3 volumes of S-9 fraction are mixed with 7 volumes of S-9 supplement (cofactors). Concentration of co-factors: MgCL 8 mM, KCl 33 mM, glucose-6-phosphate 5 mM, NADP 4 mM, phosphate buffer (pH 7.4) 100 mM. - Test concentrations with justification for top dose:
- 100, 500, 2500 and 5000 µg/plate.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- other:
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Four test plates for each concentration
METHOD OF TREATMENT/ EXPOSURE:
- Test tubes containing 2 mL portions of soft agar which consists of 100 mL agar (0.6 % agar + 0.6 % NaCl) and 10 mL amino-acid solution (minimal amino-acid solution for the determination of mutants: 0.5 mM histidine and 0.5 mM biotin) are kept in water at 45 °C, and the remaining components are added in the following order: 0.1 mL test solution , 0.1 mL bacterial suspension, 0.5 mL S-9 mix (in tests with metabolic activation) or 0.5 mL phosphate buffer (in tests without metabolic activation) After mixing, the samples are poured onto the nutrient plates within approx. 30 seconds.
FOR GENE MUTATION:
- Expression time: After incubation for 48 hours at 37 °C in the dark, the bacterial colonies (his+ revertants) are counted.
- Method used: Agar overlay
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition and reduction in the number of revertant colonies - Evaluation criteria:
- In general, a substance to be characterised as positive in the Ames test has to fulfill the following requirements: doubling of the spontaneous mutation rate (control), dose-response relationship and reproducibility of the results.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- At highest dose 5 000 µg/plate, led to reduced his- background growth
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Withought S-9 mix, slight decrease in the number of revertant colonies at highest dose level of 5 000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- STUDY RESULTS:
- Tests without S-9 mix: Without metabolic activation the test material showed no mutagenicity. The number of his+ revertants was always in the range of that of the control for all strains. The highest dose of 5 000 µg/plate showed a slightly toxic effect and, in some cases, led to a reduced his- background growth (TA 1537) or to a slight decrease in the number of revertant colonies (TA 98)
- Tests with S-9 mix: After metabolic activation no mutagenic effect was detected, the number of revertant colonies was again in the same range as that of the control. Under these experimental conditions, too, a slightly toxic effect was detected at the highest dose level (reduced his- background growth or slight decrease in the number of his+ revertants). - Conclusions:
- Under the conditions of this study, the test material was not mutagenic.
- Executive summary:
The genotoxicity of the test material was investigated, according to a similar method described in OECD Test Guideline 471.
The test material was tested for mutagenicity in the Ames test in a dose range of 20 µg - 5 000 µg/plate using the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100. The test was carried out directly and in the presence of a mammalian metabolising system (9 000 g supernatant of liver homegenate from rats treated with Aroclor 1254, the so-called S-9 mix) in order to register possible mutagenic metabolites. Under all these experimental conditions no mutagenicity was detected for the test material. The number of his+ revertants was always in the range of that of the control, both using the base pair strains TA 1535 and TA 100 and using the frameshift strains TA 1537, TA 1538 and TA 98. The highest dose of 5000 µg/plate was slightly toxic for some strains and, in some cases, led to a reduced his- background growth or to a slight decrease in the number of mutant colonies.
Under the conditions of this study, the test material was not mutagenic.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 16 March 1987 to 12 August 1987
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Specific details on test material used for the study:
- TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Stock solution: 25 mg/mL 1.25 g test material is mixed with Ham's F12 medium, set to pH 7 using aqueous NaHCO3 and filled up to the final volume of 50 mL. Aliquots of this solution are used to obtain the target concentration.
STABILITY TEST MATERIAL
The test material was proven to be stable for more than 4 years, therefore its stability over the test period can be assumed. - Target gene:
- HGPRT locus
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type of cells: CHO (Chinese Hamster Ovary Cells)
- Sub-strain: K 1
- Karyotype: 18 - 22 Chromosomes
For cell lines:
- Number of passages during the expression period: Four passages. At the last passage the cells are transferred into the 6-TG containing selection medium.
- Methods for maintenance in cell culture: Cells were routinely grown in monolayers at 37 °C in an atmosphere containing 5 % CO2. For each subculture cells were trypsinisend and reseeded at a
density of ca. 100 000 cells in 25 cm^2 flasks and covered with 5 mL medium. At every second passage antibiotics were added.
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: The basic medium used was Ham's F 12 supplemented with 200 μM glutamine and - with the exception of exposure time - with 10 Vol. % FCS.
- During the whole test procedure and for all cytotoxicity determinations the culture media contained the following antibiotics: Penicillin: 50 IU/mL and Streptomycin: 50 μg/mL.
- Selection medium: Glutamine and FCS supplemented Ham's F 12, hypoxanthine-free, 10 μM 6- Thioguanine (TG) throughout selection time (7 days to colony formation) without medium change.
- "HAT" for the preselection (elimination of TG-resistant mutants): Glutamine and FCS supplemented Ham's F 12 medium containing:
2 x 10^-4 mole glycine
5 x 10^-6 mole thymidine
1 x 10^-5 mole hypoxanthine
3.2 x 10^-6 mole aminopterine - Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9: 500 mg Aroclor 1254 (solution in arachis oil - 20% w/v) per kg body weight was administered once intraperitoneally to 5 male Sprague-Dawley rats 5 days before sacrifice. At the 5th day the animals were sacrificed, and the livers prepared. All the preparation steps for recovering the microsome enzymes were carried out using sterile solvents and vessels at a temperature of + 4 °C. The livers were weighed and washed in an equivalent volume of a 150 mM CaCl2 solution (1 mL = 1 g wet liver), then cut into small pieces and homogenized in a 3-fold volume of 150 mM CaCl2 solution. After the homogenate had been centrifuged at 9 000 g for 10 minutes at + 4 °C, the supernatant (so-called S-9 fraction) was quickly deep frozen in 3 mL portions in dry ice and stored for a maximum of 6 months at -70 ° to -80 °C.
- method of preparation of S9 mix: A sufficient amount of S-9 fraction was thawed at room temperature before the beginning of the experiment and 3 parts of S-9 fraction were mixed with 7 parts of S-9 supplement (cofactors) in stock solution (7 - 9). The concentrations of the cofactors in the stock solution were as follows:
KCl: 30 mM
MgCl2: 10 mM
CaCl2: 10 mM
Glucose-6-phosphate: 5 mM
NADP: 4 mM
Na-phosphate (pH 7.4): 50 μM
- concentration or volume of S9 mix and S9 in the final culture medium: 1 mL of this S-9 mix was used for the test. - Test concentrations with justification for top dose:
- Test concentrations: 5.0, 2.5, 1.0, 0.5, 0.25, 0.1, 0.05, 0.025 mg/mL
PRELIMINARY RANGE-FINDING STUDY
- Preliminary range-finding cytotoxicity tests were performed to determine the effect of the test material on cell survival (as measured by relative cloning efficiencies; "detachment assay"). The dose levels used hereby covered a range from 0.1 to 10.0 mg/mL and were tested in the presence and absence of S-9 activation. Exposure periods were 16 hours.
- Reduced cloning efficiency was found with and without S-9 mix at 2.5 mg/mL. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Test medium
Soluble in DMSO; poorly soluble in water, but soluble in water upon neutralisation with NaHCO3.
pH of 5 mg/mL suspension in Ham's F 12 Medium: 3.7
Stability in water was analytically proven. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- The same media were used as in the dose groups. Medium was added instead of test material.
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 3-methylcholanthrene
- Remarks:
- In the presence of S-9 mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- The same media were used as in the dose groups. Medium was added instead of test material.
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- Without S-9 mix
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments: 3
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 100 000 cells in 25 cm^2 flasks covered with 5 mL routine medium. This is also assumed to be the number of exposed cells, since only minor cell division occurs during the 24 hours attachment period.
- Number of cells seeded into the selection medium: 300 000 per 80 cm^2 flask (8 - 10 mL medium covering the cells). 3 - 5 flasks per dose.
- Test material added in medium. At the start of the exposure the medium was changed to ham's F12 without FCS supplement. Then the test material was added and the mixture left for 4 h. At the end of the exposure period the monolayer was rinsed and covered with routine medium again.
- Volumes:
Without S9-mix: 4 mL Ham's F12, 1 mL test material solution.
With S9 mix: 3 mL Ham's F12, 1 mL S-9 mix, 1 mL test material solution.
- Fixing and staining of colonies: Methanol/ Giemsa.
TREATMENT AND HARVEST SCHEDULE:
- Pretreatment of the cells: 1 week (2 subcultures) in "HAT medium".
- Exposure duration/duration of treatment: 4 hours
FOR GENE MUTATION:
- Expression period: 8 days
- Attachment period: 24 hours
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 18 - 20 hours after termination of exposure the cells were subcultured for the first time; at this step all the cells of the same dose level were pooled and reseeded at a density of 10^5 cells per 25 cm^2 in duplicates. Duplicate samples of approximately 200 cells (received by aliquot dilution) of each dose level were taken and used for cloning efficiency determination. Until the end of the expression time three more passages are performed. At the last passage cells from the duplicate flasks of each concentration were pooled and seeded into the selection medium. 3 x 10^5 cells per flask (80 cm^2 ). 3 - 5 flasks per dose. At this step the cloning efficiency was checked again.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- 20 hours after treatment termination 200 cells of each dose (cells pooled from 2 flasks) were taken in duplicates, seeded into Ham's F 12 medium and allowed for colony formation. At the end of the expression period a second assay was carried out following the same procedure.
- Colony counting: Colonies were visually scored 7 days after seeding the cells for the determination of cloning efficiency in the cytotoxicity assays.
METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Cell counting: A coulter counter was used. The numbers obtained in the untreated control were rechecked in a Bürker chamber, yielding an interval correction factor.
- Colony counting: Colonies were visually scored 7 days after seeding the cells for final selection of mutants. or for the determination of cloning efficiency in the cytotoxicity assays. - Evaluation criteria:
- The test material is regarded as a mutagen is the corrected mutation rates exceed 15 x 10^-6 and follow a dose response relation.
Isolated increases of the mutation rate above the limit of 15 x 10^-6 in a lower concentration without a dose relation may indicate a biological effect, but are not regarded as sufficient evidence for mutagenicity. - Species / strain:
- Chinese hamster Ovary (CHO)
- Remarks:
- Experiment No. 1.
- Metabolic activation:
- with and without
- Genotoxicity:
- ambiguous
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- Chinese hamster Ovary (CHO)
- Remarks:
- Experiment No. 3.
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Additional information on results:
- In experiment No. 1 with and without S-9 mix some values are above the mutation rate of 15 x 10^-6 set as the limit. However, there is no dose-response relationship. Since cloning efficiency did not indicate a toxic effect at the highest dose, two of the test acceptance criteria were not fulfilled. Therefore a second experiment was started.
In experiment No. 2 without S-9 mix one value is above the limit of 15 x 10^-6 , however, there is no dose-response relationship. Cytotoxicity was found to be high enough with S-9 mix but not without S-9 mix. Therefore, a third experiment was started.
In experiment No. 3 without S-9 mix one value is above the limit of 15 x 10^-6 while S-9 mix data indicate some mutagenicity. But this is in clear contrast with respect to the doses to experiments No. 1 and 2. In addition, cytotoxicity shows that 2.5 mg/mL is the highest acceptable
dose level.
Although experiments No. 1 and 2 do have some deficiencies, data from all three experiments together provide with respect to reproducibility and dose response no clear evidence for mutagenicity. Therefore, it is assumed that the test material is not mutagenic under the test conditions employed. - Remarks on result:
- other: Not mutagenic under the test conditions.
- Conclusions:
- Under the conditions the test material is assumed to be non-mutagenic.
- Executive summary:
The mutagenicity of the test material was assessed according to OECD Test Guideline 476 and in compliance with GLP.
The treatment period was 4 hours in hypoxanthin-free medium after a 24 hours attachment period. In a range finding toxicity assay (detachment assay: 16 hours exposure) reduced cloning efficiency was found with S-9 mix at 2.5 mg/mL and without S-9 mix at 2.5 mg/mL. In the attachment assays during the mutagenicity tests (18 - 20 hours after the 4 hours exposure time) reduced cloning efficiency was found with S-9 mix at 2.5 - 5 mg/mL and without S-9 mix at 2.5 - 5 mg/mL.
Under the conditions the test material is assumed to be non-mutagenic.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- 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
- Justification for type of information:
- An in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from in vivo cytogenicity tests are available.
Referenceopen allclose all
Study Results
+/- S9 Mix | Concentration (µg/plate) | Mean number of colonies/plate | ||||
TA1535 | TA1537 | TA1538 | TA98 | TA100 | ||
+ | PC Vehicle 20 100 500 2500 5000 | 376 20 16 15 14 11 12 | 105 12 13 11 10 4 B | 1408 27 26 22 25 20 11 | 1623 40 40 38 36 31 23 | 2175 103 117 112 117 113 111 |
- | PC Vehicle 20 100 500 2500 5000 | 2263 16 16 14 13 13 14 | 503 7 8 9 6 5 B | 264 14 15 15 11 12 10 | 790 20 19 19 19 17 14 | 2250 117 122 124 136 122 117 |
Mean number of revertant colonies/4 replicate plates
PC = Positive control
B= reduced his- background growth
Experiment 3
With Metabolising System (S-9 mix) |
|||||||||||||
Concentration (mg/mL) |
Mutagenicity |
Mutation Rate Not Corrected for Cytotoxicity |
Mutation Rate Corrected for Cytotoxicity |
Cytotoxicity |
Cytotoxicity |
||||||||
No. of Cells 7 Days After Seeding ca. 300 000 cells per Flask (80 cm^2) into Selection Medium |
Cloning Efficiency of Cells 18 – 20 h Post Exposure |
% |
Cloning Efficiency of Cells at the End of Expression Period |
% |
|||||||||
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
210 |
209 |
104.75 |
163 |
175 |
84.50 |
0.025 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
191 |
190 |
95.25 |
134 |
110 |
61.00 |
0.050 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
189 |
182 |
92.75 |
89 |
97 |
46.50 |
0.1 |
2 |
2 |
1 |
4 |
1 |
6.67 |
11.16 |
156 |
185 |
85.25 |
118 |
121 |
59.75 |
0.25 |
2 |
2 |
2 |
2 |
1 |
6.00 |
10.08 |
137 |
129 |
66.50 |
108 |
130 |
59.50 |
0.50 |
0 |
0 |
2 |
0 |
1 |
2.00 |
3.17 |
176 |
151 |
81.75 |
140 |
112 |
63.00 |
1.0 |
5 |
4 |
4 |
7 |
2 |
14.67 |
20.66 |
156 |
173 |
82.25 |
140 |
144 |
71.00 |
2.5 |
5 |
6 |
6 |
8 |
3 |
18.47 |
32.75 |
104 |
77 |
45.25 |
102 |
126 |
57.00 |
5.0 |
- |
- |
- |
- |
-* |
-* |
-* |
1 |
2 |
0.75 |
- |
- |
0 |
Positive Control: 0.01 MCA |
31 |
35 |
33 |
32 |
35 |
110.67 |
434.00 |
82 |
130 |
53.00 |
48 |
54 |
25.50 |
Cloning Efficiency: No. f colonies and % ratio of mean value to no. of seeded sells (approx. 200).
Mutation rate: Number of mutants per 10^6 cells; correction for cytotoxicity applies to the cloning efficiency at the end of expression period.
*: No cells survived.
Without Metabolising System |
|||||||||||||
Concentration (mg/mL) |
Mutagenicity |
Mutation Rate Not Corrected for Cytotoxicity |
Mutation Rate Corrected for Cytotoxicity |
Cytotoxicity |
Cytotoxicity |
||||||||
No. of Cells 7 Days After Seeding ca. 300 000 cells per Flask (80 cm^2) into Selection Medium |
Cloning Efficiency of Cells 18 – 20 h Post Exposure |
% |
Cloning Efficiency of Cells at the End of Expression Period |
% |
|||||||||
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
180 |
194 |
93.50 |
106 |
109 |
53.75 |
0.025 |
1 |
0 |
1 |
2 |
1 |
3.33 |
8.82 |
184 |
191 |
93.75 |
76 |
75 |
37.75 |
0.050 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
169 |
176 |
86.25 |
135 |
117 |
63.00 |
0.1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
171 |
163 |
83.50 |
119 |
154 |
68.25 |
0.25 |
3 |
3 |
4 |
7 |
6 |
15.33 |
26.32 |
141 |
153 |
73.50 |
109 |
124 |
58.25 |
0.50 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
145 |
183 |
82.00 |
113 |
114 |
56.75 |
1.0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
180 |
191 |
92.75 |
83 |
97 |
45.00 |
2.5 |
1 |
0 |
0 |
0 |
1 |
1.33 |
2.97 |
95 |
93 |
47.00 |
89 |
90 |
44.75 |
Positive control: 0.3 EMS |
77 |
72 |
77 |
86 |
78 |
260.00 |
650.00 |
100 |
102 |
50.50 |
70 |
90 |
40.00 |
Cloning efficiency: No. of colonies and % ratio of mean value to no. of seeded cells (approx. 200).
Mutation Rate: Number of mutants per 10^6 cells; correction for cytotoxicity applies to the cloning efficiency at the end of expression period.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Genetic Toxicity In Vivo
In Vivo Mammalian Cell Study: DNA Damage and/or Repair - Sister Chromatid Exchange (SCE): Engelhardt (1985a)
Under the conditions of this study, the test material has a very weak SCE-inducing activity in vivo on bone marrow cells of Chinese hamsters. However, the weakly positive reaction was observed at dose levels only at which clear signs of toxicity could be observed. The dose which did not lead to any clinical signs or symptoms did not lead to any increase in the SCE rate either.
In Vivo Mammalian Somatic Cell Study: Cytogenicity/ Bone Marrow Chromosome Aberration: Engelhardt (1985b)
Under the conditions of this study, only in the highest dose group of 3 800 mg/kg body weight, at which clear signs of toxicity were found, was a slight, but statistically significant, increase in the number of aberrant metaphases including gaps and excluding gaps observed at the 6-hour sacrifice interval in comparison with the solvent control. A slight, but statistically non-significant increase in the aberration rate was also observed at the sacrifice interval of 24 hour. However, this increase is influenced by less than 50 % of the animals/group, the majority of the animals exhibiting an aberration rate which is in the range of that of the control. Therefore, the results of the cytogenetic study do not allow any final conclusions to be drawn regarding the clastogenic activity of the test material.
In Vivo Mammalian Somatic Cell study: Cytogenicity/ Erythrocyte Micronucleus: Durward (2000)
Under the conditions of this study, the test material was considered to be non-genotoxic.
In Vivo Mammalian Somatic Cell Study: Bone Marrow Chromosome Aberration: Durward (2000)
Under the conditions of this study the test material did not induce any significant increases in the frequency of chromosome aberrations in rat bone marrow under the conditions of the test. The test material was considered to be non-clastogenic to rat bone marrow cells in vivo.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 13 June 2000 to 10 August 2000
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.11 (Mutagenicity - In Vivo Mammalian Bone-Marrow Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: UKEMS Sub-Committee on Guidelines for Mutagenicity Testing, Report, Part 1 revised
- Version / remarks:
- Basic Mutagenicity Test: UKEMS recommended procedures, 1990
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: US, EPA, TSCA and FIFRA Guidelines
- Version / remarks:
- Not specified
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian bone marrow chromosome aberration test
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: Approximately eight to ten weeks old.
- Weight at study initiation: 225 to 270g
- Assigned to test groups randomly: Yes. The animals were selected at random and given a number unique within the study by ear punching and a number written on a colour coded cage card.
- Housing: The animals were housed in groups of up to seven by sex in solid-floor polypropylene cages with woodflakes bedding.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: Minimum acclimatisation period of seven days.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 30 to 70 %
- Air changes (per hr): approximately fifteen changes per hour.
- Photoperiod (hrs dark / hrs light): twelve hours light and twelve hours darkness.
- Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: Arachis oil
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was freshly prepared as required as a suspension at the appropriate concentration in arachis oil.
- Dose volume: 10 mL/kg
- Concentration:
125 mg/L group: 12.5 mg/mL
250 mg/L group: 25 mg/mL
500 mg/L group: 50 mg/mL - Duration of treatment / exposure:
- Single oral administration
- Frequency of treatment:
- Once
- Post exposure period:
- The animals were sacrificed between 24 - 48 hours after test material administration.
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- Vehicle control
- Dose / conc.:
- 125 mg/kg bw/day (nominal)
- Remarks:
- Low dose
- Dose / conc.:
- 250 mg/kg bw/day (nominal)
- Remarks:
- Mid dose
- Dose / conc.:
- 500 mg/kg bw/day (nominal)
- Remarks:
- High dose
- No. of animals per sex per dose:
- Vehicle control: 14 males total (Kill Time 48 and 24 hours)
Positive control: 5 males
Test material low and mid dose: 7 males
Test material high dose: 14 males (Kill time 48 and 24 hours) - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Cyclophosphamide:
- Doses / concentrations: 25 mg/kg. The positive control material was freshly prepared as required as a solution at the appropriate concentration in distilled water. - Tissues and cell types examined:
- 100 metaphase cells of adequate quality were scored, if possible, from the slides prepared from each animal for both numerical and structural chromosome aberrations.
- Details of tissue and slide preparation:
- DOSE SELECTION RATIONALE:
A range-finding toxicity study was performed to determine a suitable dose level and route of administration for the chromosome aberration study. The dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2 000 mg/kg. The range-finding toxicity study was also used to determine if the main study was to be performed using both sexes or males only. Using toxicity data supplied by the sponsor it was considered unnecessary to investigate the intraperitoneal route. The supplied data also showed no marked differences between the sexes and therefore only male rats were used to determine the maximum tolerated dose level for use in the main study.
All animals were dosed once only at the appropriate dose level by gavage using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to its bodyweight at the time of dosing.
Animals were observed one hour after dosing and subsequently once daily for two days. Any deaths and evidence of overt toxicity were recorded at each observation. No necropsies were performed.
In the main study, groups, each of seven rats, were dosed once only via the oral route with the test material at 125, 250 or 500 mg/kg. One group of rats from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed at 500 mg/kg was killed after 48 hours. In addition, three further groups of rats were included in the study; two groups (seven rats) were dosed via the oral route with the vehicle alone (arachis oil) and a third group (five rats) was dosed orally with cyclophosphamide, a positive control material known to produce chromosome aberrations under the conditions of the test. The vehicle controls were killed 24 and 48 hours following treatment and positive control group animals were killed 24 hours following treatment.
All animals were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable and immediately prior to termination.
TREATMENT AND SAMPLING TIMES:
Animals were injected i.p. with a solution of colchicine at 4 mg/kg approximately 2 to 3 hours prior to bone marrow harvest. At the scheduled time, animals were killed by cervical dislocation and one femur was extracted from each animal and cleaned of muscle and connective tissue. The bone marrow was aspirated into 5 mL of Hank's balanced salt solution (HBSS) and spun down in a centrifuge. The supernatant was removed and the cell pellet resuspended in 0.075 M KCl at room temperature for 15 minutes (including five minutes centrifugation). The cells were recentrifuged and all but 1 mL of the supernatant removed. After resuspension of the cell pellet, the cells were fixed by the addition of freshly prepared fixative (methanol:glacial acetic acid, 3:1). The fixative was changed several times and the cells stored at 4 °C for at least 4 hours.
DETAILS OF SLIDE PREPARATION:
After storage the cell suspensions were recentrifuged and the fixative removed to leave a sufficient amount to give a milky suspension on resuspension of the cell pellet. A few drops of each cell suspension was dropped onto clean, wet slides and air-dried. When completely dry the slides were stained in 5 % Giemsa for 10 minutes and rinsed in tap water and distilled water. When dry the slides were coverslipped using a mounting medium.
Groups, each of seven rats, were dosed once only via the oral route with the test material at 125, 250 or 500 mg/kg. One group of rats from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed at 500 mg/kg was killed after 48 hours. In addition, three further groups of rats were included in the study; two groups (seven rats) were dosed via the oral route with the vehicle alone (arachis oil) and a third group (five rats) was dosed orally with cyclophosphamide, a positive control material known to produce chromosome aberrations under the conditions of the test. The vehicle controls were killed 24 and 48 hours following treatment and positive control group animals were killed 24 hours following treatment. - Evaluation criteria:
- SLIDE EVALUATION
The stained slides were coded and examined 'blind' using light microscopy at x 100 and x 1 000 magnifications.
If the cell had 40 to 44 or more chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1990 UKEMS Guidelines for Mutagenicity Testing. Aberrations recorded by the slide scorers were checked by a Senior Cytogeneticist. - Statistics:
- Comparisons were made between the negative control groups and each corresponding treatment dose group, with a chi-squared test, using observed numbers of cells with aberrations.
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
In animals dosed with test material via the oral route, clinical signs were observed at and above 500 mg/kg as follows: hunched posture, ataxia, lethargy, loss of righting reflex, decreased and laboured respiration, ptosis, pile-erection and splayed gait. When observations were made approximately 24 hours after dosing there was some concern with the condition of the animals dosed with 750 mg/kg. These animals were observed again at approximately 26 hours and, due to the severity of the toxicity of the test material, they were killed in extremis.
Adequate evidence of test material toxicity was demonstrated via the oral route of administration, therefore, this was selected for use in the main study. A maximum tolerated dose (MTD) of the test material, 500 mg/kg, was selected for use in the main study, with 125 and 250 mg/kg as the lower dose levels.
RESULTS OF DEFINITIVE STUDY
Evaluation of bone marrow slides:
- There were no clear statistically significant reductions in the mean mitotic index in any of the test material treatment groups when compared to their concurrent vehicle control groups. The mean mitotic index of the positive control group was sharply depressed compared to that of the 24-hour vehicle control group.
- All the negative control animals gave values of chromosome aberrations within the expected range. The mean frequency of cells with aberrations was consistent between the two vehicle control groups, the highest frequency (0.3 % cells with aberrations excluding gaps) being seen in the 24 hour group.
- The positive control group animals showed highly significant increases in the frequency of cells with aberrations indicating that the test method itself was operating as expected.
- The test material was seen to induce no significant, dose-related increases in the frequency of cells with aberrations in any of the treatment groups.
- The test material did not induce a significant increase in the numbers of polyploid cells in any of the treatment groups.
Historical Aberration Ranges for Vehicle and Untreated Control Cultures
Experiments with rat bone marrow cells have established a range of aberration frequencies acceptable for control animals, these are commonly in the range of 0 to 3 % cells with structural aberrations.
A positive response was recorded for a particular treatment if the % cells with aberrations markedly exceeded that seen in the concurrent control. For modest increases in aberration frequency, appropriate statistical tests may be applied in order to record a positive response. - Conclusions:
- Under the conditions of this study the test material did not induce any significant increases in the frequency of chromosome aberrations in rat bone marrow under the conditions of the test. The test material was considered to be non-clastogenic to rat bone marrow cells in vivo.
- Executive summary:
The potential of the test material to produce damage to chromosomes or the mitotic apparatus when administered to rats was assessed according to OECD Test Guideline 475 and EU Method B.11, in compliance with GLP.
A range-finding study was performed to find suitable dose levels of the test material. Adequate oral toxicity data in rats was supplied by the sponsor which showed that there was no marked differences in test material toxicity between the sexes. Therefore, the main study was performed using only male rats. The chromosome aberration study was conducted using the oral route in groups of seven rats (males) at the maximum tolerated dose (MTD) 500 mg/kg with 250 and 150 mg/kg as the lower dose levels. Further groups of rats were given a single oral dose of arachis oil (7 rats) or dosed orally with cyclophosphamide (5 rats), to serve as vehicle and positive controls respectively. Animals were killed 24 or 48 hours later, the bone marrow extracted and slide preparations made and stained. Bone marrow cells were scored for the presence of chromosome aberrations.
No statistically significant decreases in the mitotic index mean values were observed in the 24 or 48 hour test material groups when compared to their concurrent vehicle control group. However, the observation of clinical signs indicated that systemic absorption, and consequently, exposure to the target tissue, had occurred.
There was no evidence of a statistically significant increase in the incidence of chromosome aberrations in animals dosed with the test material when compared to the concurrent vehicle control groups. The positive control material produced a marked increase in the frequency of chromosome aberrations.
Under the conditions of this study the test material did not induce any significant increases in the frequency of chromosome aberrations in rat bone marrow under the conditions of the test. The test material was considered to be non-clastogenic to rat bone marrow cells in vivo.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 22 May 2000 to 13 July 2000
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian erythrocyte micronucleus test
- Species:
- mouse
- Strain:
- CD-1
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: Approximately five to eight weeks old
- Weight at study initiation: 24 to 30 g
- Assigned to test groups randomly: Yes
- Housing: The animals were housed in groups of up to seven in solid-floor polypropylene edges with wood flakes bedding.
- Diet: Ad libitum
- Water: Ad libitum mains drinking water.
- Acclimation period: A minimum acclimatisation period of seven days.
ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25 °C
- Humidity: Relative humidity 30 to 70 %
- Air changes: Approximately fifteen changes per hour.
- Photoperiod: Twelve hours light and twelve hours darkness. - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle used: Arachis oil
- Concentration of test material in vehicle: 187.5, 375 or 750 mg/kg
- Amount of vehicle: 10 mL/kg
- Lot/batch no: T36 - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
- The test material was freshly prepared as required as a suspension at the appropriate concentration in arachis oil. The concentration, homogeneity and stability of the test material preparations were not determined. - Duration of treatment / exposure:
- Single oral administration
- Frequency of treatment:
- Once
- Post exposure period:
- One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed at 750 mg/kg was killed at 48 hours.
- Dose / conc.:
- 750 mg/kg bw (total dose)
- Dose / conc.:
- 375 mg/kg bw (total dose)
- Dose / conc.:
- 187.5 mg/kg bw (total dose)
- No. of animals per sex per dose:
- Seven males per dose per kill time.
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Cyclophosphamide
- Route of administration: Oral
- Doses / concentrations: 50 mg/kg freshly prepared as a solution at the approriate concentration in distilled water. The concentration homogeneity and stability of the positive control material and its preparation were not determined by analysis. - Tissues and cell types examined:
- The incidence of micronucleated cells per 2 000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1 000 erythrocytes were counted; these cells were also scored for incidence of micronuclei.
The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations. - Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
- A range-finding toxicity study was performed to determine a suitable dose level and route of administration for the micronucleus study. The dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of cytotoxicity up to a maximum recommended dose of 2 000 mg/kg. The range-finding toxicity study was also used to determine if the main study was to be performed using both sexes or males only. Using existing toxicology data it was considered to be unnecessary to investigate the intraperitoneal route of administration.
- All animals were dosed once only at the appropriate dose level by gavage using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to its bodyweight at the time of dosing. Animals were observed one hour after dosing and subsequently once daily for up to two days. Any deaths and evidence of overt toxicity were recorded at each observation. No necropsies were performed.
- In animals dosed with the test material via the oral route, premature deaths occurred at and above 1 000 mg/kg, and clinical signs were observed at and above 500 mg/kg as follows: hunched posture, lethargy, ataxia, splayed gait, decreased respiratory rate, laboured respiration, occasional body tremors and ptosis. The severity of the observations increased with increased dose concentration. The test material showed no marked difference in its toxicity to male or female mice, it was therefore considered to be acceptable to use males only for the main study. Adequate evidence of test material toxicity was demonstrated via the oral route of administration, therefore, this was selected for use in the main study. The maximum tolerated dose (MTD) of the test material, 750 mg/kg, was selected for use in the main study, with 187.5 and 375 mg/kg as the lower dose levels.
TREATMENT AND SAMPLING TIMES
- Groups, each of seven mice, were dosed once only via the oral route with the test material at 187.5, 375 or 750 mg/kg. One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed at 750 mg/kg was killed at 48 hours. In addition, three further groups of mice were included in the study; two groups (seven mice) were dosed via the oral route with the vehicle alone (arachis oil) and a third group (five mice) was dosed orally with cyclophosphamide, a positive control material known to produce micronuclei under the conditions of the test. The vehicle controls were killed 24 or 48 hours following dosing and positive control group animals were killed 24 hours following dosing. All animals were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable and immediately prior to termination.
DETAILS OF SLIDE PREPARATION:
- Immediately following termination (i.e. 24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with foetal calf scrum and bone marrow smears prepared following centrifugation and re- suspension. The smears were air-dried, fixed in absolute methanol and stained in May-Grilnwald/Giemsa, allowed to air-dry and coverslipped using mounting medium.
METHOD OF ANALYSIS:
- Slide Evaluation: Stained bone marrow smears were coded and examined blind using light microscopy at x 1 000 magnification. The incidence of micronucleated cells per 2 000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1 000 erythrocytes were counted; these cells were also scored for incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations. - Evaluation criteria:
- A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test material groups and the number occurring in the corresponding vehicle control group.
A positive mutagenic response was demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to their corresponding control group.
If these criteria were not demonstrated, then the test material was considered to be non-genotoxic under the conditions of the test.
A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group. - Statistics:
- All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a √(x + 1) transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Evaluation of Bone Marrow Slides
- There were no statistically significant decreases in the PCE/NCE ratio in the 24 or 48-hour test material groups when compared to their concurrent vehicle control groups. However, a marked reduction in the PCE/NCE ratio was observed in the 48-hour 750 mg/kg test material group indicating a cytotoxic response in the target tissue, bone marrow. This, accompanied by the premature deaths and clinical observations indicated that systemic absorption had occurred and that exposure to the target tissue had been achieved.
- There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test material dose groups when compared to their concurrent vehicle control groups. However, a small increase in the frequency of micronucleated PCEs which was outside the normal historical range was observed in the 750 mg/kg 48-hour test material dose group. It was considered that the toxic response seen with the test material in the bone marrow induced an increased rate of erythropoiesis. It has been reported that increased erythropoiesis may cause some cells to cycle more quickly than in control animals, thereby reducing the time available for repair of spontaneously-occurring DNA damage before final mitosis and enucleation. In such circumstances, micronucleus frequencies are observed to increase. It was therefore considered that the modest increases in micronucleated cells were not due any genotoxic action in the test material.
- The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.
- The test material was found not to produce a significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test. - Conclusions:
- Under the conditions of this study, the test material was considered to be non-genotoxic.
- Executive summary:
The potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice was investigated in accordance with the standardised guidelines OECD 474, EU Method B12, OPPTS 870.5395, under GLP conditions.
A range-finding study was performed to find suitable dose levels of the test material and investigate if there was a marked difference in toxic response between the sexes. There was no marked difference in test material toxicity between the sexes, therefore the main study was performed using only male mice. The micronucleus study was conducted using the oral route in groups of seven mice (males) at the maximum tolerated dose (MTD) 750 mg/kg with 375 and 187.5 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) were scored for the presence of micronuclei. Further groups of mice were given a single oral dose of arachis oil (7 mice) or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively.
No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48-hour test material dose groups when compared to their concurrent vehicle control groups. However, the presence of premature deaths and clinical signs indicated that systemic absorption had occurred and it was considered that target tissue exposure had been achieved. There was no evidence of a statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups.
The positive control material produced a marked increase in the frequency of micronucleated polychromatic erythrocytes.
Under the conditions of this study, the test material was considered to be non-genotoxic.
- Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- January 1984
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The aim of the study was to test the test material for its possible induction of sister chromatid exchanges (SCEs) in chromosomes of Chinese hamsters after a single oral administration. For this purpose, a cytogenetic examination was carried out in somatic cells (bone marrow).
- GLP compliance:
- not specified
- Type of assay:
- sister chromatid exchange assay
- Species:
- hamster, Chinese
- Strain:
- not specified
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Weight at study initiation: 27 to 31 g
- Assigned to test groups randomly: Yes according to a randomisation plant compiled by the Computer Centre, BASF.
- Housing: For the duration of about one week the animals were housed in Makrolon cages, type M III, in groups of 5 and separately according to sex. One day before the beginning of the experiment the animals were transferred to Makrolon cages, type M I, and housed individually under the same experimental conditions until the end of the study.
- Diet: Standardised pelleted feed was available ad libitum
- Water: Drinking water from bottles was available ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 24 °C
- Humidity: relative humidity 30 to 70 %
- Photoperiod: The day/night rhythm was 12 hours (12 hours light from 6.00 - 18.00 hours and 12 hours darkness from 18.00 – 6.00 hours). - Route of administration:
- oral: gavage
- Vehicle:
- Aqueous 0.5 % carboxymethyl cellulose (CMC).
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
- The test material was suspended in an aqueous 0.5 % CMC formulation
- All test material formulations were prepared immediately before administration.
- Administration was 10 mL/kg bodyweight as a suspension with the following concentrations:
3 800 mg test material/ kg body weight group: 38 g/100 mL
470 mg test material/ kg body weight group: 4.7 g/100 mL
60 mg test material/ kg body weight group: 0.6 g/100 mL
- The amount of test material or volume to be administered was related to the specific weight of the individual animals on the day of the experiment. - Duration of treatment / exposure:
- Single oral administration
- Frequency of treatment:
- Once
- Post exposure period:
- The animals were sacrificed 24 hours after the administration of the test material.
- Dose / conc.:
- 3 800 mg/kg bw (total dose)
- Dose / conc.:
- 470 mg/kg bw (total dose)
- Dose / conc.:
- 60 mg/kg bw (total dose)
- No. of animals per sex per dose:
- Five animals per sex per dose
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Cyclophosphamide:
- Doses / concentrations: 20 mg/kg body weight dissoled in aqua bidest., 10 mL/kg bodyweight. - Tissues and cell types examined:
- Analysis was carried out with coded preparations. As a rule, 30 differentially stained metaphases using 2 or 3 preparations were evaluated from each male and female animal of the solvent control group and of the two dose groups. In the positive control group, the evaluation was limited to only 10 metaphases per animal.
One animal of the 470 mg/kg group and one hamster of the 60 mg/kg group could not be evaluated because only a few differentially stained metaphases were found. - Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
- In the determination of the acute toxicity of the test material after a single oral administration, deaths were observed down to a dose of 4 640 mg/kg body weight. At the dose level of 3 830 mg/kg body weight, all animals survived, but distinct clinical signs of toxicity, such as dyspnea, apathy, tremors and twitching, piloerection and squatting posture were observed about 15 minutes after test material administration. After 2 hours a narcotic-like state and staggering were detected, which lasted about 3 hours. On the 2nd and 3rd day after test material administration no abnormalities were detected any longer in any of the animals. Therefore a dose of 3 800 mg/kg body weight was selected as the maximally tolerated dose in the present cytogenetic investigations. 470 mg/kg body weight is to be administered as the intermediate dose. At a similar amount of 464 mg/kg body weight used for the determination of the acute toxicity the same clinical signs except for staggering and a narcotic like state were observed after about 15 minutes as described for 3 830 mg/kg, but they were less pronounced. 60 mg/kg body weight was selected as the lowest dose and was tolerated by all animals without any signs or symptoms.
TREATMENT
- After shaving the animals' necks and icing them with chloroethyl 26 hours before the animals were sacrificed, a cut measuring about 5 mm in length was made in the skin of the neck using a scalpel, a 50 mg BrdU tablet was implanted, and the cut was covered with Histoacryl adhesive. Two hours later the animals received a single oral administration of the test material suspended in an aqueous 0.5 % CMC formulation (carboxy methyl cellulose) in doses of 3 800 mg/kg, 470 mg/kg and 60 mg/kg body weight. The volume of administration was 10 mL/kg bodyweight. The hamsters were sacrificed 24 hours after the administration of the test material.
DETAILS OF SLIDE PREPARATION:
- Preparation of the bone marrow: Two hours prior to sacrifice the animals were intraperitoneally injected with 3.3 mg Colcemid/kg body weight in order to arrest mitosis in the metaphase. The two femora were prepared from the animals sacrificed by cervical dislocation and all soft parts were removed.
- After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube in reciprocal directions using a cannula filled with Hank's solution which was at 7 °C (about 2 mL/femur). The suspension was thoroughly mixed with a pipette and subsequently centrifuged at 1 500 rpm for 5 minutes, the supernatant was pipetted off except for a few drops and the precipitate was resuspended.
- For hypotonic treatment about 5 mL of a 1 % sodium citrate solution which was at 7 °C was added, and subsequently the suspension was kept in a water bath at 7 °C for 20 minutes while thoroughly mixing it every 4 or 5 minutes with a pipette.
- After re-centrifugation at 1 500 rpm for 5 minutes, the supernatant was pipetted off except for one drop, and about 2 mL of fixative (methanol:glacial acetic acid/ 3:1) was added to the sediment dropwise while constantly shaking. After 30 minutes the fixative was replaced, the centrifuge tube was closed with Parafilm, and this suspension was kept overnight at 4 °C.
- After re-centrifugation at 1 500 rpm the supernatant was pipetted off except for one drop and a suspension was prepared with fresh fixative.
- 3 - 5 drops of this suspension were dropped onto clean microscopic slides stored in aqua dest. at 4 °C using a Pasteur pipette and were then rapidly passed through a Bunsen burner flame. The preparations were dried in the air and subsequently stained.
- After being stained in Hoechst 33 258 (10 minutes) and rinsed twice in buffer (pH 6.8), the preparations were exposed to UV light (254 nm) for a period of 25 minutes. Then the preparations were stored in a 2xSSC solution in a water bath at 60 °C for 90 minutes. After the preparations had cooled, they were stained in Giemsa. After being rinsed twice in aqua dest. and clarified in xylene; the preparations were embedded in Entellan. - Evaluation criteria:
- The means of the SCEs/ cell are considered to be characteristic of a group. Accordingly, these values of the dose group were compared with the values of the control group.
- Statistics:
- In order to clarify the question of whether there are significant differences regarding the SCE rate between the negative control group and the dose groups, the many-one rank test according to Steel (nonparametric, one-sided test; significance levels of 5 % and 1 %; multiple location comparison between independent random samples) was applied.
The means of the SCEs/cell are considered to be characteristic of a group. Accordingly, these values of the dose group were compared with the values of the control group.
The statistical evaluation for the positive control group was omitted. - Key result
- Sex:
- male/female
- Genotoxicity:
- positive
- Toxicity:
- yes
- Remarks:
- clinical signs observed
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Determination of the SCE rate
- After a single oral administration of the test material in doses of 470 mg/kg and 3 800 mg/kg body weight an only slight but statistically significant, dose-dependent increase in the sister chromatid exchange rate was observed. When compared with 3.06 SCEs/cell in the solvent control, the test material led to 3.77 SCEs/cell in the 470 mg/kg group (statistical significance of 95 %) and to 5.31 SCEs/ cell in the 3 800 mg/kg group (statistical significance of 99 %).
- After a single oral administration of 60 mg/kg body weight, however, the number of SCEs/cell (3.19) was in the range of that of the control (3.06).
- With 25.12 SCEs/cell the positive control substance cyclophosphamide at a dose level of 20 mg/kg body weight, as expected, led to a very clear increase in the sister chromatid exchange rate. - Conclusions:
- Under the conditions of this study, the test material has a very weak SCE-inducing activity in vivo on bone marrow cells of Chinese hamsters. However, the weakly positive reaction was observed at dose levels only at which clear signs of toxicity could be observed. The dose which did not lead to any clinical signs or symptoms did not lead to any increase in the SCE rate either.
- Executive summary:
The genetic toxicity of the test material was investigated in Chinese hamsters using the sister chromatid exchange method.
The test material suspended in an aqueous 0.5 % CMC formulation, was administered once orally to male and female animals at dose levels of 3 800, 470 and 60 mg/kg body weight in a volume of 10 mL/kg body weight. For control purposes, male and female hamsters were administered merely the 0.5 % CMC formulation by the same route. As a positive control, 20 mg of cyclophosphamide/kg body weight, dissolved in aqua dest., was administered once orally to male and female animals in a volume of 10 mL/kg body weight. In general 5 male and 5 female animals per test group were analysed for SCEs.
The single oral administration both of the CMC formulation and or the positive control substance cyclophosphamide was tolerated by all animals without any clinical signs of toxicity.
The highest dose of 3 800 mg/kg body weight led to clinical signs, such as apathy, piloerection, irregular respiration and squatting posture, trembling and twitching which were detected after about 15 minutes. After 60 minutes staggering was additionally observed. With the exception of staggering the signs were still observed on the day after the administration.
About 15 minutes after the administration of 470 mg/kg body weight piloerection, apathy and, in some cases, atony, irregular respiration, twitching and squatting posture were detected. On the day after treatment irregular respiration, apathy and piloerection were found.
The lowest dose of 60 mg/kg body weight did not lead to any signs or symptoms.
The gross-pathological examination of the internal organs of the male and female animals of all test groups sacrificed at the end of the study did not reveal any pathological changes that could be attributed to the test material.
Two hours before the administration of the test material or before the administration of the positive and negative control substances cyclophosphamide and CMC formulation, a 5-bromodeoxyuridine tablet was implanted subcutaneously in the nape of the animals' necks in order to obtain chromosomes with sister chromatids differently substituted with BrdU. After an appropriate differential staining sister chromatid exchanges (SCEs) could thus be detected. The animals were sacrificed and the bone marrow of the two femora as prepared 24 and 26 hours after the administration of the test material and after the implantation of the BrdU tablet respectively. About two hours before the animals were sacrificed, they were intraperitoneally injected with 3.3 mg of Colcemid/kg body weight in order to arrest mitosis in the metaphase. After preparation of the bone marrow and combined staining of the resulting preparations with Hoechst 33258 and Giemsa, 30 differentially stained metaphases were evaluated per animal of the solvent group, and of the three dose groups, and the sister chromatid exchange rate was determined. In the case of the positive control group, the evaluation was limited to 10 cells/animal.
When compared with the solvent control showing 3.06 SCEs/cell, the single oral administration of the test material led to a slight but statistically significant, dose-dependent increase in the SCE rate both in the 470 mg/kg group showing 3.77 SCEs/cell and in the 3 800 mg/kg group showing 5.31 SCEs/cell. The rate of 3.19 SCEs/cell in the lowest dose group of 60 mg/kg body weight, however, was in the range of that of the control. With 25.12 SCEs/cell the positive control substance cyclophosphamide at a dose level of 20 mg/kg body weight led to a very clear increase in the sister chromatid exchange rate.
Under the conditions of this study, the test material has a very weak SCE-inducing activity in vivo on bone marrow cells of Chinese hamsters. However, the weakly positive reaction was observed at dose levels only at which clear signs of toxicity could be observed. The dose which did not lead to any clinical signs or symptoms did not lead to any increase in the SCE rate either.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- April 1984
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The aim of the present study was to test the test material for possible clastogenicity in Chinese hamsters after a single oral administration. For this purpose, a chromosome analysis was carried out in somatic cells (bone marrow).
- GLP compliance:
- not specified
- Type of assay:
- mammalian bone marrow chromosome aberration test
- Species:
- hamster, Chinese
- Strain:
- not specified
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Weight at study initiation: 23.82 g
- Age at study initiation: 7 to 13 weeks
- Assigned to test groups randomly: Yes, animals were assigned to the test groups according to a randomisation plan compiled by the computer centre of the test facility.
- Housing: For the duration of about one week the animals were housed in Makrolon cages, type M III, in groups of 5 and separately according to sex. One day before the beginning of the experiment the animals were transferred to Makrolon cages, type M I, and housed individually under the same experimental conditions until the end of the study.
- Diet: Standardised pelleted feed was available ad libitum.
- Water: Drinking water from bottles was available ad libitum.
ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 24 °C
- Humidity: relative humidity 30 to 70 %
- Photoperiod: The day/night rhythm was 12 hours (12 hours light from 6.00 - 18.00 hours and 12 hours darkness from 18.00 – 6.00 hours). - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle used: Aqueous 0.5 % carboxymethylcellulose (CMC).
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
- The test material was suspended in an aqueous 0.5 % CMC formulation.
- All test material formulations were prepared immediately before administration.
- Administration was 10 mL/kg bodyweight as a suspension with the following concentrations:
3 800 mg test material/ kg body weight group: 38 g/100 mL
470 mg test material/ kg body weight group: 4.7 g/100 mL
60 mg test material/ kg body weight group: 0.6 g/100 mL
- The amount of test material or volume to be administered was related to the specific weight of the individual animals on the day of the experiment. - Duration of treatment / exposure:
- Single oral administration
- Frequency of treatment:
- Once
- Post exposure period:
- The animals were sacrificed 6, 24 and 48 hours after test material administration.
- Dose / conc.:
- 3 800 mg/kg bw (total dose)
- Dose / conc.:
- 470 mg/kg bw (total dose)
- Dose / conc.:
- 60 mg/kg bw (total dose)
- No. of animals per sex per dose:
- Five animals per sex, per dose, per sacrifice interval
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Cyclophosphamide
- Doses / concentrations: 60 mg/kg body weight, dissolved in aqua dest. administered once orally to male and female animals at at volume of 10 mL/kg bodyweight. - Tissues and cell types examined:
- Chromosome analysis was carried out with coded preparations. As a rule 100 metaphases using 3 or 4 slides were evaluated from each male and female. The mitotic index based on 1 500 cells is determined or all male and female animals of every test group.
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
- In the determination of the acute toxicity of the test material after a single oral administration deaths were observed down to a dose of 4 640 mg/kg body weight. At the dose level of 3 830 mg/kg body weight, all animals survived, but distinct clinical signs of toxicity, such as dyspnea, apathy, tremors and twitching, piloerection and squatting posture were observed about 15 minutes after test material administration. After 2 hours a narcotic-like state and staggering were detected, which lasted about 3 hours. On the 2nd or 3rd day after test material administration no abnormalities were detected any longer in any of the animals. Therefore a dose of 3 800 mg/kg body weight was selected as the maximally tolerated dose in the present cytogenetic investigations. 470 mg/kg body weight is to be administered as the intermediate dose. At a similar amount of 464 mg/kg body weight used for the determination of the acute toxicity the same clinical signs except for staggering and a narcotic-like state were observed after about 15 minutes as described for 3830 mg/kg, but they were less pronounced. 60 mg/kg body weight was selected as the lowest dose and was tolerated by all animals without any signs or symptoms.
TREATMENT AND SAMPLING TIMES:
- Male and female animals for each sacrifice interval were given the test material suspended in an aqueous 0.5 % CMC formulation (carboxymethyl cellulose) at dose levels of 3 800, 470 and 60 mg/kg body weight. Treatment consisted of a single oral administration. The volume of administration was 10 mL/kg body weight. For control purposes, male and female animals were given merely the 0.5 % CMC formulation by the same route. As a positive control, 60 mg cyclophosphamide/kg body weight, dissolved in aqua dest., was administered once orally to male and female animals in a volume of 10 mL/kg body weight.
DETAILS OF SLIDE PREPARATION:
- About two hours prior to sacrifice the animals were intraperitoneally injected with 3.3 mg Colcemid/kg body weight in order to arrest mitosis in the metaphase.
- The two femora were prepared from the animals sacrificed by cervical dislocation and all soft parts were removed. After cutting of the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube in reciprocal directions using a cannula filled with Hanks' solution which was at 37 °C (about 2 mL/femur). Subsequently the suspension was mixed thoroughly with a pipette, centrifuged at 1 500 rpm for 5 minutes, the supernatant was pipetted off except for a few drops, and the precipitate was resuspended.
- For hypotonic treatment about 5 mL of a 1 % sodium citrate solution which was at 37 °C was added, and subsequently the suspension was kept in a water bath at 37 °C for 20 minutes while thoroughly mixing it every 4 or 5 minutes with a pipette.
- After re-centrifugation at 1 500 rpm for 5 minutes, the supernatant was pipetted off except for one drop, and about 2 mL of fixative (methanol:glacial acetic acid/ 3:1) was added to the sediment dropwise while constantly shaking. After 30 minutes the fixative was replaced, the centrifuge tube was closed with Parafilm, and this suspension was kept overnight at 4 °C.
- After re-centrifugation at 1 500 rpm the supernatant was pipetted off, and a suspension was prepared with fresh fixative. 3 - 5 drops of this suspension were dropped onto clean microscopic slides stored in aqua dest. at 4 °C using a Pasteur pipette and were then rapidly passed through a Bunsen burner flame. The preparations were dried in the air, subsequently stained in a solution of Giemsa and Titrisol (10 mL Giemsa, 200 mL Titrisol, pH 7.2) for 10 minutes and rinsed in aqua dest. twice for 5 minutes. After being clarified in xylene the preparations were embedded in Entellan.
METHOD OF ANALYSIS:
- Chromosome analysis: As a rule, 100 metaphases from each of the male and female animals of every test group were analysed for the following chromosome aberrations:
a) Structural chromosome aberrations
- G' and G": Chromatid gap and isochromatid gap respectively, unstained regions (so-called achromatic lesions) without dislocation of the segment which appears to be separated.
- B' and B": Chromatid break and chromosome break respectively, visible discontinuity in chromatid or chromosomal structure with lateral or longitudinal dislocation of the fragment.
- F' and F": Chromatid fragment and chromosome fragment respectively, acentric chromosome segments which occur singly or in pairs.
- D’ and D": Chromatid deletion and chromosome deletion respectively, loss of a segment on the level of chromatids or chromosomes.
- m.A.: Multiple aberrations, metaphases with 5 or more aberrations; they are classified according to multiple aberrations with and without exchanges. If there are multiple aberrant metaphases, the number of chromosomes cannot generally be determined exactly; therefore, this number is not given.
- Disintegration of chromosomal structure (pulverisations): The chromosomes are present as irregular particles; a chromosomal structure can no longer be detected.
- Exchanges: These exchange aberrations are divided into intrachanges and interchanges:
Int' and Int": Intrachanges on the level of chromatids and chromosomes respectively the joining of broken ends capable of reuniting two or several chromatid regions within a chromosome (e.g., centric and acentric ring chromosomes, pericentric inversions).
I' and I": Intrachanges on the level of chromatids and chromosomes respectively, the joining of broken ends capable of reuniting two or several chromosomes (multiple interchanges). They are classified according to:
- Symmetric interchanges (e.g., reciprocal translocations between nonhomologous chromosomes, centric fusions, quadriradials).
- Asymmetric interchanges (e.g., dicentric and polycentric chromosomes, triradials and quadriradials).
b) Numerical chromosome aberrations (so-called heteroploidies)
- Aneuploidy: Metaphases with missing or additional chromosomes. Only metaphases with additional chromosomes, so-called hyperploidies, are recorded. Hypoploidies, i.e. metaphases with missing chromosomes, are not taken into account.
- Euploidy (polyploidy): Changes in the number of chromosomes by complete chromosome sets (3 n and more).
For analysis the preparations were coded. If only a few metaphases were found, a chromosome analysis was not carried out.
- Mitotic index: The mitotic index based on 1 500 cells is determined or all male and female animals of every test group. - Evaluation criteria:
- Chromosome analysis and mitotic index were evaluated.
- Statistics:
- Two statistical tests were used to answer the questions of whether there are significant differences between control group and dose group or between the individual dose groups concerning the rate of aberrant metaphases: First, the exact test according to FISHER, which was applied to register significant differences between the relative frequencies of a characteristic of two groups, and, second, the asymptotic U test according to MANN-WHITNEY (rank test modified according to WILCOXON). The relative frequencies of metaphases with aberrations per animal were used as a criterion of the rank determination for the U test. The two tests were calculated at the levels of 95 and 99 %. Significances were marked with *(95 %) and with **(99 %).
A sequential statistical test was used to answer the question of a possible change of the characteristic in terms of time within a dose group ("test for homogeneity in terms of time"). On the basis of the hypothesis that the relative frequencies of the characteristic are identical at all times, the relative frequencies which reject this hypothesis at the level of 95 or 99 % were determined successively. Significances were again marked with *(95 %) or with ** (99 %). - Sex:
- male/female
- Remarks on result:
- other: The results of the cytogenetic study do not allow any final conclusions to be drawn regarding the clastogenic activity of the test material.
- Additional information on results:
- Chromosome Analysis
- Analysis of the solvent control group: In 18 of a total of 20 animals which could be evaluated and which were administered the 0.5 % CMC formulation once orally 27 aberrant metaphases including gaps (1.35 %) or 7 aberrant metaphases excluding gaps (0.35 %) were found. Exchanges, multiple aberrant cells or disintegration of chromosomal structure were not observed. Furthermore 22 polyploid but no aneuploid cells were found.
- Analysis of the 3 800 mg/kg group: After the single administration of 3 800 mg/kg body weight 34 metaphases with aberrations including gaps (3.4 %) or 12 chromosomally damaged metaphases excluding gaps (1.2 %) could be observed in 9 of 10 animals 6 hours after the administration with a significance of 95 – 99 % depending on the statistical method. Exchanges or disintegration of chromosomal structure were not found. At the sacrifice interval of 24 hours 6 of 10 hamsters shoved a total 21 aberrant cells including gaps (2.1 %) and 8 metaphases with aberrations excluding gaps (0.8 %). Aberrations, such as exchanges or disintegration of chromosomal structure, were not detected. 48 hours after administration 7 chromosomally damaged cells including gaps (0.78 %) and 1 aberrant metaphase excluding gaps (0.11 %) were analysed in 7 of 9 animals which could be evaluated. Multiple aberrations or disintegration of chromosomal structure were not observed. No aneuploid cells were found at any of the three intervals. Polyploid cells were found at the 6-hour interval (9 cells), 24-hour interval (16 cells) and 48-hour interval (13 cells).
- Analysis of the 470 mg/kg group: At the sacrifice interval of 24 hours a total of 15 metaphases with aberrations including gaps (1.5 %) or 2 damaged cells excluding gaps (0.2 %) were analysed in 7 of 10 hamsters which received the test material in a dose of 470 mg/kg body weight. Exchanges and disintegration of chromosomal structure were not found. Regarding the numerical chromosome aberrations only 15 polyploid cells were detected.
- Analysis of the 60 mg/kg group: After the single administration of 60 mg/kg body weight 8 of a total of 10 animals which could be evaluated showed 15 aberrant metaphases including gaps (1.5 %) or 1 aberrant metaphase excluding gaps (0.1 %) 24 hours after administration. Exchanges, multiple aberrant cells or disintegration of chromosomal structure were not observed. Furthermore 11 polyploid cells but no aneuploid cells were observed.
- Analysis of the positive control group: After the administration of the positive control substance cyclophosphamide in a dose of 60 mg/kg body weight there was a very clear increase in the rate of structural chromosome aberrations with a total of 261 aberrant cells including gaps (26.1 %) and 222 aberrant metaphases excluding gaps (22.2 %), including 95 multiple aberrant metaphases, 125 cells with exchanges and 24 cells with disintegration of chromosomal structure. However, no increase in the number of numerical chromosomal aberrations {27 polyploid cells and 1 aneuploid cell) was observed.
Mitotic Index
- There was a slight decrease in the mitotic index in the highest dose group at a sacrifice interval of 6 hours and in the positive control group. - Conclusions:
- Under the conditions of this study, only in the highest dose group of 3 800 mg/kg body weight, at which clear signs of toxicity were found, was a slight, but statistically significant, increase in the number of aberrant metaphases including gaps and excluding gaps observed at the 6-hour sacrifice interval in comparison with the solvent control. A slight, but statistically non-significant increase in the aberration rate was also observed at the sacrifice interval of 24 hour. However, this increase is influenced by less than 50 % of the animals/group, the majority of the animals exhibiting an aberration rate which is in the range of that of the control. Therefore, the results of the cytogenetic study do not allow any final conclusions to be drawn regarding the clastogenic activity of the test material.
- Executive summary:
The possible clastogenic activity of the test material in Chinese hamsters was investigated using the bone marrow method.
For this purpose, the test material suspended in an aqueous 0.5 % CMC formulation was administered once orally to male and female animals at dose levels of 3 800, 470 and 60 mg/kg body weight in a volume of 10 mL/kg body weight. For control purposes, male and female hamsters were administered merely the 0.5 % CMC formulation by the same route. As a positive control, 60 mg of cyolophosphamide/kg body weight, dissolved in aqua dest., was administered once orally to male and female animals in a volume of 10 mL/kg body weight.
The single oral administration both of the CMC formulation and of the positive control substance cyclophosphamide was tolerated by all animals without any clinical signs of toxicity.
The highest dose of 3 800 mg/kg body weight led to clinical signs such as apathy, piloerection, irregular respiration and, in some cases, to squatting posture, trembling and twitching, which were detected after about 15 minutes. After 60 minutes staggering was additionally detected. Some of these signs were still observed two days after the administration. One animal died on the 1st day after treatment. About 15 minutes after the administration 470 mg/kg body weight led to irregular respiration, piloerection, atony and, in some cases, to twitching and squatting posture. On the day after treatment irregular respiration, apathy and piloerection were found. The lowest dose of 60 mg/kg test material did not lead to any signs or symptoms. The gross-pathological examination of the internal organs of the male and female animals of all test groups sacrificed at the end of the study did not reveal any pathological changes that could be attributed to the test material administered.
The animals were sacrificed and the bone marrow of the two femora was prepared 6, 24 and 48 hours after administration in all three dose groups. However, only in the highest dose group were these three sacrifice intervals investigated; in the intermediate and lowest dose groups only the preparations of the 24 hour sacrifice interval were analysed. In the solvent control group and the positive control group only the 24-hour sacrifice interval was selected. About two hours before the animals were sacrificed, they were intraperitoneally injected with 3.3 mg Colcemid/kg in order to arrest mitosis in the metaphase. After preparation of the bone marrow and staining of the preparations with Giemsa, 100 metaphases were analysed per animal. The mitotic index based on 1 500 cells/animal was also determined.
Under the conditions of this study, only in the highest dose group of 3 800 mg/kg body weight, at which clear signs of toxicity were found, was a slight, but statistically significant, increase in the number of aberrant metaphases including gaps and excluding gaps observed at the 6-hour sacrifice interval in comparison with the solvent control. A slight, but statistically non-significant increase in the aberration rate was also observed at the sacrifice interval of 24 hour. However, this increase is influenced by less than 50 % of the animals/group, the majority of the animals exhibiting an aberration rate which is in the range of that of the control. Therefore, the results of the cytogenetic study do not allow any final conclusions to be drawn regarding the clastogenic activity of the test material.
Referenceopen allclose all
Mortality and Clinical Results
There were no premature deaths seen in any of the dose groups. Clinical
signs were observed in animals dosed with the test material at and above
125 mg/kg in both the 24 and 48-hour groups where applicable, these were
as follows: lethargy, hunched posture, ataxia, decreased and laboured
respiration, ptosis, loss of righting reflex, splayed gait, stained
urine and diuresis. The severity of the clinical signs increased with
the increase in dose of the test material.
Group Mean Results of Chromosome Aberration Assay
Treatment Group |
Animals |
Numbers of Cells Scored |
Polyploid Cells |
Total Gaps |
Chromatid |
Chromosome |
Others |
Total Aberrations (+ Gaps) |
Total Aberrations (- Gaps) |
Cells with Aberrations (+ Gaps) |
Cells with Aberrations (- Gaps) |
||
Breaks |
Exchanges |
Breaks |
Exchanges |
||||||||||
Vehicle Control 48 hours |
Males |
700 |
1 (0.1) |
3 (0.4) |
1 (0.1) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
4 (0.6) |
1 (0.1) |
3 (0.4) |
1 (0.1) |
Vehicle Control 24 hours |
Males |
700 |
1 (0.1) |
1 (0.1) |
1 (0.1) |
0 (0.0) |
1 (0.1) |
0 (0.0) |
0 (0.0) |
3 (0.4) |
2 (0.3) |
3 (0.4) |
2 (0.3) |
Positive Control |
Males |
500 |
0 (0.0) |
61 (12.2) |
71 (14.2) |
58 (11.6) |
20 (4.0) |
2 (0.4) |
16 (3.2) |
212 (42.4) |
151 (30.2) |
113 *** (22.6) |
94 *** (18.8) |
Test Material 500 mg/kg 48 Hours |
Males |
700 |
0 (0.0) |
2 (0.3) |
3 (0.4) |
0 (0.0) |
1 (0.1) |
0 (0.0) |
0 (0.0) |
6 (0.9) |
4 (0.6) |
5 (0.7) |
3 (0.4) |
Test Material 500 mg/kg 24 Hours |
Males |
700 |
0 (0.0) |
5 (0.7) |
3 (0.4) |
0 (0.0) |
5 (0.7) |
0 (0.0) |
0 (0.0) |
13 (1.9) |
8 (1.1) |
9 (1.3) |
5 (0.7) |
Test Material 250 mg/kg 24 Hours |
Males |
700 |
1 (0.1) |
1 (0.1) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (0.1) |
0 (0.0) |
1 (0.1) |
0 (0.0) |
Test Material 125 mg/kg 24 Hours |
Males |
700 |
1 (0.1) |
5 (0.7) |
2 (0.3) |
0 (0.0) |
1 (0.1) |
0 (0.0) |
0 (0.0) |
8 (1.1) |
3 (0.4) |
8 (1.1) |
3 (0.4) |
X = > 10 aberrations per cell (not included in total aberrations)
Figures in brackets = aberrations per 100 cells
*** = p < 0.001
Summary of Micronucleus Study Group Mean Data
Treatment Group |
Number of PCE with Micronuclei per 2 000 PCE |
PCE/NCE Ratio |
||
Group Mean |
SD |
Group Mean |
SD |
|
1. Vehicle Control 48-hour Sampling Time |
1.7 |
1.9 |
1.21 |
0.28 |
2. Vehicle Control 24-hour Sampling Time |
1.1 |
0.9 |
1.52 |
0.46 |
3. Positive Control 24-hour Sampling Time |
59.8*** |
18.4 |
1.12 |
0.49 |
4. Test Material 750 mg/kg a 48-hour Sampling Time |
4.4 |
2.9 |
0.89 |
0.47 |
5. Test Material 750 mg/kg 24-hour Sampling Time |
2.1 |
1.6 |
1.37 |
0.66 |
6. Test Material 375 mg/kg 24-hour Sampling Time |
1.0 |
1.4 |
1.39 |
0.67 |
7. Test Material 187.5 mg/kg 24-hour Sampling Time |
1.0 |
1.0 |
1.22 |
0.27 |
a = Data from five animals
PCE = Polychromatic erythrocytes
NCE = Normochromatic erythrocytes
SD = Standard deviation
*** = P<0.05
Historical Vehicle Control Data from 60 Studies (120 Groups)
Relative Group Frequency of Mean Micronuclei per 1 000 PCEs
Frequency of Micronuclei Per 1 000 PCEs |
Number of Study Groups Per MN/PCR Frequency |
||
24 h Groups |
48 h Groups |
Combined |
|
0.0 |
3 |
3 |
6 |
0.1 |
1 |
1 |
2 |
0.2 |
2 |
6 |
8 |
0.3 |
0 |
1 |
1 |
0.4 |
9 |
10 |
19 |
0.5 |
0 |
0 |
0 |
0.6 |
13 |
10 |
23 |
0.7 |
2 |
3 |
5 |
0.8 |
7 |
5 |
12 |
0.9 |
3 |
0 |
3 |
1.0 |
5 |
2 |
7 |
1.1 |
22 |
4 |
6 |
1.2 |
4 |
6 |
10 |
1.3 |
2 |
1 |
3 |
1.4 |
1 |
1 |
2 |
1.5 |
0 |
0 |
0 |
1.6 |
2 |
6 |
8 |
1.7 |
0 |
0 |
0 |
1.8 |
0 |
0 |
0 |
1.9 |
0 |
1 |
1 |
2.0 |
1 |
0 |
1 |
2.1 |
0 |
0 |
0 |
2.2 |
1 |
0 |
1 |
2.3 |
1 |
0 |
1 |
2.4 |
1 |
0 |
1 |
Total |
60 |
60 |
120 |
Relative Group Frequency Categories Micronuclei per 1 000 PCEs
48 h Control Group (60 Groups)
Frequency Categories |
Groups |
0.0 – 0.4 |
21 (35 %) |
0.5 – 0.9 |
18 (30 %) |
1.0 – 1.4 |
14 (23 %) |
1.5 – 2.0 |
7 (12 %) |
2.1 – 2.5 |
0 (0 %) |
24 h Control Group (60 Groups)
Frequency Categories |
Groups |
0.0 – 0.4 |
15 (25 %) |
0.5 – 0.9 |
25 (42 %) |
1.0 – 1.4 |
14 (23 %) |
1.5 – 2.0 |
3 (5 %) |
2.1 – 2.5 |
3 (5 %) |
Combined 24 and 48 h Groups (120 Groups)
Frequency Categories |
Groups |
0.0 – 0.4 |
36 (30 %) |
0.5 – 0.9 |
43 (36 %) |
1.0 – 1.4 |
28 (23 %) |
1.5 – 2.0 |
10 (8 %) |
2.1 – 2.5 |
3 (3 %) |
Mortality Data and Clinical Observations
-There was one premature death seen in the 48-hour 750 mg/kg test material dose group and one animal was killed in extremis. Clinical signs were observed in animals dosed with the test material at and above 187 .5 mg/kg in both the 24 and 48-hour groups where applicable, these were as follows: hunched posture, lethargy, ataxia, splayed gait, ptosis, increased lacrimation, decreased respiratory rate, laboured respiration, dehydration and piloerection. As was seen in the range-finding toxicity study, the severity of the clinical signs increased with dose concentration. It was considered that the loss of two animals did not affect the integrity of the study, because at least five analysable animals were available per group, as recommended in the OECD guidelines.
Test Material Analysis
- For checking the stability of the test material in the carrier throughout the study period the samples were kept at room temperature until the treatment of the last animal (approximately 1 h) and then deep frozen until they were determined analytically.
- The homogeneity of the test material in the vehicle CMC was guaranteed by constant stirring during the removal and administration of the test material formulation and by analytical determination of two individual samples of each concentration. Depending on the dosage about 85 – 92 % of the theoretical values could be determined analytically. Experience has shown that for CMC suspensions the theoretical and analytically determined concentrations differ from each other and that generally 65 – 95 % of the theoretical values are found. Therefore, the differences between the analytically determined and the theoretical values are acceptable.
Test Group |
Theoretical Values |
Values Determined Analytically |
|
Individual Values (mg/mL) |
Mean (mg/mL) |
||
2 |
380 mg/L |
332 |
350 |
369 |
|||
3 |
47 mg/L |
10.5 |
39.85 |
39.2 |
|||
4 |
6 mg/L |
5.10 |
5.29 |
5.47 |
|||
Feed Analysis
- The feed used in the study was assayed for contaminants. On the basis of duration of use and the analytical findings, the feed was found to be suitable.
Water Analysis
- On the basis of the analytical findings, the drinking water was found to be suitable.
Clinical Examinations
- Clinical signs: The single oral administration of the 0.5 % CMC formulation in a volume of 10 mL/kg body weight was tolerated by all animals without any signs or symptoms.
The dose of 3 800 mg/kg body Weight led to apathy, piloerection, irregular respiration and, in some cases, to squatting posture, or trembling and twitching, which were observed after about 15 minutes. After 60 minutes staggering was additionally detected. With the exception of staggering the signs were still observed on the day after treatment, and the general state of the animals was poor.
About 15 minutes after test material administration 470 mg/kg body weight led to piloerection and apathy; in some cases atony, irregular respiration, twitching and squatting posture were observed. On the day after treatment irregular respiration, apathy and piloerection were still found.
Amounts of 60 mg/kg body weight did not lead to any evident signs of toxicity when administered once orally.
The single administration of the positive. control substance cyclophosphamide was also tolerated by all animals without any clinical signs or symptoms.
- Necropsy: The gross-pathological examination of the animals sacrificed at the end of the study did not reveal any changes of the internal organs which could be attributed to the test material administered.
Summary of Results
Group |
No. of animals (m/f) |
Metaphases examined/ animal |
G-SCEs/ metaphase |
Standard deviation |
P ≤ 0.05 |
P ≤ 0.01 |
Solvent control (0.5 % CMC) |
5/5 |
30 |
3.06 |
0.562 |
|
|
Test material: 3 800 mg/kg bw |
5/5 |
30 |
5.31 |
0.992 |
X |
x |
Test material: 470 mg/kg bw |
4/5 (1+) |
30 |
3.77 |
0.482 |
X |
- |
Test material: 60 mg/kg bw |
4/5 (1+) |
30 |
3.19 |
0.580 |
- |
- |
Positive control (20 mg/kg bw cyclophosphamide) |
5/5 |
10 |
25.12 |
5.481 |
|
|
G-SCEs/ metaphase: Mean of the SCEs per metaphase for the group
P: P-value ≤ 0.05/0.01, yes = x, no = -
+: Animal not evaluable
Summary of the Results of All Test Groups at the 24-Hour Sacrifice Interval
|
Group 1 (Solvent Control) |
Group 2 (3 800 mg/kg Test Material) |
Group 3 (470 mg/kg Test Material) |
Group 4 (60 mg/kg Test Material) |
Group 5 (60 mg/kg Cyclophosphamide) |
Animal Total |
20 |
10 |
10 |
10 |
10 |
Analysed Metaphases |
2 000 |
1 000 |
1 000 |
1 000 |
1 000 |
Number of Animals with Aberrant Metaphases |
18 |
6 |
7 |
8 |
10 |
Metaphases with Aberrations Including Gaps /100 |
27/1.35 |
21/2.10 |
15/1.58 |
15/1.5 |
261/26.1 XX ++ |
Metaphases with Aberrations Excluding Gaps /100 |
7/0.35 |
8/0.80 |
2/0.20 |
1/0.10 |
222/22.20 XX ++ |
Exchanges /100 |
0/0.0 |
0/0.0 |
0/0.0 |
0/0.0 |
125/12.50 XX ++ |
Mult. Aberr. Met |
0 |
1 |
0 |
0 |
95 |
Pulverisations |
0 |
0 |
0 |
0 |
24 |
Aneuploidy |
0/0.0 |
0/0.0 |
0/0.0 |
0/0.0 |
1/0.10 |
Polyploidy |
22/1.10 |
16/1.60 |
15/1.50 |
11/1.10 |
27/2.70 |
XX: 99 % significance Fisher-Yates Test
++: 99 % significance U Test
Mean Mitotic Index Values
Test Groups |
Mitotic Index (%) |
||
6 h |
24 h |
48 h |
|
Solvent control (0.5 % CMC) |
- |
5.80 |
5.32 |
3 800 mg/kg Test Material |
2.53 |
6.03 |
- |
470 mg/kg Test Material |
- |
6.74 |
- |
60 mg/kg Test Material |
- |
5.39 |
- |
Positive Control (Cyclophosphamide) |
- |
2.89 |
- |
Test Material Analysis
- For checking the stability of hte test material in the carrier throughout the study period the samples were kept at room temperature until the treatment of the last animal (approximately 1 h) and then deep frozen until they were determined analytically.
- The homogeneity of the test material in the carrier CMC was guaranteed by constant stirring during the removal and administration of the test material formulation and by analytical determination of two individual samples of each concentration.
Test Group |
Theoretical Values |
Values Determined Analytically |
|
Individual Values (mg/mL) |
Mean (mg/mL) |
||
2 |
380 mg/L |
323 |
318.5 |
314 |
|||
3 |
47 mg/L |
38.7 |
38.7 |
38.7 |
|||
4 |
6 mg/L |
4.51 |
4.17 |
3.83 |
|||
- Depending on the dosage, about 70 – 84 % of the theoretical values could be determined analytically. Experience has shown that for CMC suspensions the theoretical and analytically determined concentrations differ from each other and that generally 65 – 95 % of the theoretical values are found. Therefore, the differences between the analytically determined and theoretical values are acceptable.
Clinical Examinations
- Clinical signs: The single oral administration of the 0.5 % CMC formulation in a volume of 10 ml/kg body weight was tolerated by all animals without any signs or symptoms. The dose of 3800 mg/kg body weight led to apathy, piloerection, irregular respiration and, in some. cases, to squatting posture, trembling and twitching, which were observed after about 15 minutes. After 60 minutes staggering was additionally detected. Some of these signs were still observed two days after treatment, and the general state of the animals was poor. One animal died on the 1st day after administration. About 15 minutes after test material administration 470 mg/kg body weight led to irregular respiration, piloerection, atony and, in some cases, to twitching and squatting posture. On the day after treatment irregular respiration, apathy and piloerection were found. Amounts of 60 mg/kg body weight did not lead to any evident signs of toxicity when administered once orally. The single administration of the positive control substance cyclophosphamide was also tolerated by all animals without any clinical signs or symptoms.
- Necropsy: The gross-pathological examination of the animals sacrificed at the end of the study did not reveal any changes of the internal organs which could be attributed to the test material administered.
Additional information
Genetic Toxicity In Vitro
In Vitro Gene Mutation Study in Bacteria (Ames Test): Engelhardt (1981)
The genotoxicity of the test material was investigated, according to a similar method described in OECD Test Guideline 471. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
The test material was tested for mutagenicity in the Ames test in a dose range of 20 µg - 5 000 µg/plate using the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100. The test was carried out directly and in the presence of a mammalian metabolising system (9 000 g supernatant of liver homegenate from rats treated with Aroclor 1254, the so-called S-9 mix) in order to register possible mutagenic metabolites. Under all these experimental conditions no mutagenicity was detected for the test material. The number of his+ revertants was always in the range of that of the control, both using the base pair strains TA 1535 and TA 100 and using the frameshift strains TA 1537, TA 1538 and TA 98. The highest dose of 5 000 µg/plate was slightly toxic for some strains and, in some cases, led to a reduced his- background growth or to a slight decrease in the number of mutant colonies.
Under the conditions of this study, the test material was not mutagenic.
In Vitro Gene Mutation Study in Mammalian Cells (CHO): Engelhardt (1988)
The mutagenicity of the test material was assessed according to OECD Test Guideline 476 and in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).
The treatment period was 4 hours in hypoxanthin-free medium after a 24 hours attachment period. In a range finding toxicity assay (detachment assay: 16 hours exposure) reduced cloing efficiency was found with S-9 mix at 2.5 mg/mL and without S-9 mix at 2.5 mg/mL. In the attachment assays during the mutagenicity tests (18 - 20 hours after the 4 hours exposure time) reduced cloning efficiency was found with S-9 mix at 2.5 - 5 mg/mL and without S-9 mix at 2.5 - 5 mg/mL.
Under the conditions the test material is assumed to be non-mutagenic.
Genetic Toxicity In Vivo
In Vivo Mammalian Cell Study: DNA Damage and/or Repair -Sister Chromatid Exchange (SCE): Engelhardt (1985a)
The genetic toxicity of the test material was investigated in Chinese hamsters using the sister chromatid exchange method. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
The test material suspended in an aqueous 0.5 % CMC formulation, was administered once orally to male and female animals at dose levels of 3 800, 470 and 60 mg/kg body weight in a volume of 10 mL/kg body weight. For control purposes, male and female hamsters were administered merely the 0.5 % CMC formulation by the same route. As a positive control, 20 mg of cyclophosphamide/kg body weight, dissolved in aqua dest., was administered once orally to male and female animals in a volume of 10 mL/kg body weight. In general 5 male and 5 female animals per test group were analysed for SCEs.
The single oral administration both of the CMC formulation and or the positive control substance cyclophosphamide was tolerated by all animals without any clinical signs of toxicity.
The highest dose of 3 800 mg/kg body weight led to clinical signs, such as apathy, piloerection, irregular respiration and squatting posture, trembling and twitching which were detected after about 15 minutes. After 60 minutes staggering was additionally observed. With the exception of staggering the signs were still observed on the day after the administration.
About 15 minutes after the administration of 470 mg/kg body weight piloerection, apathy and, in some cases, atony, irregular respiration, twitching and squatting posture were detected. On the day after treatment irregular respiration, apathy and piloerection were found.
The lowest dose of 60 mg/kg body weight did not lead to any signs or symptoms.
The gross-pathological examination of the internal organs of the male and female animals of all test groups sacrificed at the end of the study did not reveal any pathological changes that could be attributed to the test material.
Two hours before the administration of the test material or before the administration of the positive and negative control substances cyclophosphamide and CMC formulation, a 5-bromodeoxyuridine tablet was implanted subcutaneously in the nape of the animals' necks in order to obtain chromosomes with sister chromatids differently substituted with BrdU. After an appropriate differential staining sister chromatid exchanges (SCEs) could thus be detected. The animals were sacrificed and the bone marrow of the two femora as prepared 24 and 26 hours after the administration of the test material and after the implantation of the BrdU tablet respectively. About two hours before the animals were sacrificed, they were intraperitoneally injected with 3.3 mg of Colcemid/kg body weight in order to arrest mitosis in the metaphase. After preparation of the bone marrow and combined staining of the resulting preparations with Hoechst 33258 and Giemsa, 30 differentially stained metaphases were evaluated per animal of the solvent group, and of the three dose groups, and the sister chromatid exchange rate was determined. In the case of the positive control group, the evaluation was limited to 10 cells/animal.
When compared with the solvent control showing 3.06 SCEs/cell, the single oral administration of the test material led to a slight but statistically significant, dose-dependent increase in the SCE rate both in the 470 mg/kg group showing 3.77 SCEs/cell and in the 3 800 mg/kg group showing 5.31 SCEs/cell. The rate of 3.19 SCEs/cell in the lowest dose group of 60 mg/kg body weight, however, was in the range of that of the control. With 25.12 SCEs/cell the positive control substance cyclophosphamide at a dose level of 20 mg/kg body weight led to a very clear increase in the sister chromatid exchange rate.
Under the conditions of this study, the test material has a very weak SCE-inducing activity in vivo on bone marrow cells of Chinese hamsters. However, the weakly positive reaction was observed at dose levels only at which clear signs of toxicity could be observed. The dose which did not lead to any clinical signs or symptoms did not lead to any increase in the SCE rate either.
.
In Vivo Mammalian Somatic Cell Study: Cytogenicity / Bone Marrow Chromosome Aberration: Engelhardt (1985b)
The possible clastogenic activity of the test material in Chinese hamsters was investigated using the bone marrow method. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
For this purpose, the test material suspended in an aqueous 0.5 % CMC formulation was administered once orally to male and female animals at dose levels of 3 800, 470 and 60 mg/kg body weight in a volume of 10 mL/kg body weight. For control purposes, male and female hamsters were administered merely the 0.5 % CMC formulation by the same route. As a positive control, 60 mg of cyolophosphamide/kg body weight, dissolved in aqua dest., was administered once orally to male and female animals in a volume of 10 mL/kg body weight.
The single oral administration both of the CMC formulation and of the positive control substance cyclophosphamide was tolerated by all animals without any clinical signs of toxicity.
The highest dose of 3 800 mg/kg body weight led to clinical signs such as apathy, piloerection, irregular respiration and, in some cases, to squatting posture, trembling and twitching, which were detected after about 15 minutes. After 60 minutes staggering was additionally detected. Some of these signs were still observed two days after the administration. One animal died on the 1st day after treatment. About 15 minutes after the administration 470 mg/kg body weight led to irregular respiration, piloerection, atony and, in some cases, to twitching and squatting posture. On the day after treatment irregular respiration, apathy and piloerection were found. The lowest dose of 60 mg/kg test material did not lead to any signs or symptoms. The gross-pathological examination of the internal organs of the male and female animals of all test groups sacrificed at the end of the study did not reveal any pathological changes that could be attributed to the test material administered.
The animals were sacrificed and the bone marrow of the two femora was prepared 6, 24 and 48 hours after administration in all three dose groups. However, only in the highest dose group were these three sacrifice intervals investigated; in the intermediate and lowest dose groups only the preparations of the 24 hour sacrifice interval were analysed. In the solvent control group and the positive control group only the 24-hour sacrifice interval was selected. About two hours before the animals were sacrificed, they were intraperitoneally injected with 3.3 mg Colcemid/kg in order to arrest mitosis in the metaphase. After preparation of the bone marrow and staining of the preparations with Giemsa, 100 metaphases were analysed per animal. The mitotic index based on 1 500 cells/animal was also determined.
Under the conditions of this study, only in the highest dose group of 3 800 mg/kg body weight, at which clear signs of toxicity were found, was a slight, but statistically significant, increase in the number of aberrant metaphases including gaps and excluding gaps observed at the 6-hour sacrifice interval in comparison with the solvent control. A slight, but statistically non-significant increase in the aberration rate was also observed at the sacrifice interval of 24 hour. However, this increase is influenced by less than 50 % of the animals/group, the majority of the animals exhibiting an aberration rate which is in the range of that of the control. Therefore, the results of the cytogenetic study do not allow any final conclusions to be drawn regarding the clastogenic activity of the test material.
In Vivo Mammalian Somatic Cell study: Cytogenicity/ Erythrocyte Micronucleus: Durwood (2000)
The potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice was investigated in accordance with the standardised guidelines OECD 474, EU Method B12, OPPTS 870.5395, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).
A range-finding study was performed to find suitable dose levels of the test material and investigate if there was a marked difference in toxic response between the sexes. There was no marked difference in test material toxicity between the sexes, therefore the main study was performed using only male mice. The micronucleus study was conducted using the oral route in groups of seven mice (males) at the maximum tolerated dose (MTD) 750 mg/kg with 375 and 187.5 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) were scored for the presence of micronuclei. Further groups of mice were given a single oral dose of arachis oil (7 mice) or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively.
No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48-hour test material dose groups when compared to their concurrent vehicle control groups. However, the presence of premature deaths and clinical signs indicated that systemic absorption had occurred and it was considered that target tissue exposure had been achieved. There was no evidence of a statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups.
The positive control material produced a marked increase in the frequency of micronucleated polychromatic erythrocytes.
Under the conditions of this study, the test material was considered to be non-genotoxic.
In Vivo Mammalian Somatic Cell Study: Bone Marrow Chromosome Aberration: Durward (2000)
The potential of the test material to produce damage to chromosomes or the mitotic apparatus when administered to rats was assessed according to OECD Test Guideline 475 and EU Method B.11, in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).
A range-finding study was performed to find suitable dose levels of the test material. Adequate oral toxicity data in rats was supplied by the sponsor which showed that there was no marked differences in test material toxicity between the sexes. Therefore, the main study was performed using only male rats. The chromosome aberration study was conducted using the oral route in groups of seven rats (males) at the maximum tolerated dose (MTD) 500 mg/kg with 250 and 150 mg/kg as the lower dose levels. Further groups of rats were given a single oral dose of arachis oil (7 rats) or dosed orally with cyclophosphamide (5 rats), to serve as vehicle and positive controls respectively. Animals were killed 24 or 48 hours later, the bone marrow extracted and slide preparations made and stained. Bone marrow cells were scored for the presence of chromosome aberrations.
No statistically significant decreases in the mitotic index mean values were observed in the 24 or 48 hour test material groups when compared to their concurrent vehicle control group. However, the observation of clinical signs indicated that systemic absorption, and consequently, exposure to the target tissue, had occurred. There was no evidence of a statistically significant increase in the incidence of chromosome aberrations in animals dosed with the test material when compared to the concurrent vehicle control groups. The positive control material produced a marked increase in the frequency of chromosome aberrations.
Under the conditions of this study the test material did not induce any significant increases in the frequency of chromosome aberrations in rat bone marrow under the conditions of the test. The test material was considered to be non-clastogenic to rat bone marrow cells in vivo.
Justification for classification or non-classification
In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the material does not require classification with respect to genetic toxicity.
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