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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance gave negative results in the Ames test with Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and Escherichia coli strain WP2 uvrA, with and without metabolic activation. Negative results were also observed in the chromosome aberration test with human lymphocytes in the presence and absence of metabolic activation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
- Preliminary toxicity assay: 1, 3, 6, 8, 11, 21 and 37 mmoles/L (24,500; 73,400; 147,000; 196,000, 269,000; 513,000 and 905,000 ppm, respectively)
- Mutagenicity and confirmatory assay: 3, 6, 8, 11, 21 and 37 mmoles/L (73,400; 147,000; 196,000, 269,000; 513,000 and 905,000 ppm, respectively).
- Confirmatory retest: 0.7, 1, 3, 6, 8, 11, 21 and 37 mmoles/L (17,200; 24,500; 73,400; 147,000; 196,000, 269,000; 513,000 and 905,000 ppm, respectively)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: test system was exposed to the test article via the desiccator methodology, a modification of the plate incorporation methodology originally described by Ames et al. (1975) and updated by Maron and Ames (1983). This test system has been shown to detect a wide range of classes of chemical mutagens (McCann et al., 1975; McCann and Ames, 1976). The desiccator methodology has been shown to be an effective method for detecting the genotoxic activity of volatile and gaseous test articles (Wagner et al., 1992). Briefly, once agar had solidified, the plates were inverted in 9 liter dessicators and an appropriate quantity of test material was introduced into each dessicator by withdrawing the appropriate amount of air and replacing with test material.

DURATION
- Original assay: following 24 hour exposure to test article, the plates were removed from the dessicators and allowed to inclubate at 37 °C for an additional 24 hours.
- Confirmatory assay: exposure duration 48 hours in dessicator.

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; number of revertants
Evaluation criteria:
- Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value.
- Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value.
- An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited.
- A response will be evaluated as negative, if it is neither positive nor equivocal.
Key result
Species / strain:
other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
PRELIMINARY TOXICITY ASSAY
- No background lawn toxicity was observed however a reduction in revertant counts was observed beginning at 11, 21 or at 37 mmoles/L with some test conditions.

MUTAGENICITY ASSAY: No positive mutagenic responses were observed with any of the tester strains in the presence or absence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 11, 21 or at 37 mmoles/L.

CONFIRMATORY MUTAGENICITY ASSAY
No positive mutagenic response was observed with tester strains TA100, TA1535 and WP2 uvrA in the absence of S9 activation and with tester strains TA100, TA1535 and TA1537 in the presence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 21 or 37 mmoles/L. Tester strain TA98 in the presence and absence of S9 was not evaluated due to confluent growth but was retested. Due to excessive toxicity, as a result of a drop in the revertant counts beginning at 8 mmoles/L, tester strain TA 1537 in the absence of S9 was retested. Due to an unacceptable vehicle control value, tester strain WP2 uvrA in the absence of S9 activation was not evaluated but was retested. In the retest of the confirmatory mutagenicity assay, no positive responses were observed with tester strains TA98 and TA1537 in the absence of S9 activation and with tester strains TA98 and WP2 uvrA in the presence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 21 or 37 mmoles/L.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.










































































































































































































































































































































































































 



 



-S9


 


 

+S9


 


 

Strain



Test conc


(mmol/L)



Average reverants original



Average revertants – confirmatory



Average reverants – original



Average revertants – confirmatory



 



 



 



 



 



 



TA98



Vehicle



35



30



24



12



 



0.7



NA



31



NA



9



 



1



NA



34



NA



9



 



3



27



29



15



10



 



6



28



24



25



9



 



8



22



27



18



11



 



11



19



22



13



10



 



21



4



15



3



10



 



37



0



0



0



0



 



Positive control



129



351



442



408



TA100



Vehicle



179



96



209



106



 



3



214



108



216



88



 



6



179



113



203



83



 



8



184



130



236



72



 



11



216



88



214



63



 



21



205



40



267



40



 



37



0



0



0



0



 



Positive control



 



772



 



407



TA1535



Vehicle



10



11



7



9



 



3



10



8



7



10



 



6



9



9



6



5



 



8



11



11



10



12



 



11



8



7



8



7



 



21



6



11



5



7



 



37



0



0



0



0



 



Positive control



293



666



73



140



TA1537



Vehicle



7



10



7



7



 



0.7



NA



6



NA



NA



 



1



NA



6



NA



NA



 



3



3



10



4



6



 



6



3



11



3



6



 



8



5



7



4



4



 



11



0



6



7



5



 



21



1



4



0



5



 



37



0



0



0



0



 



Positive control



992



633



36



473



WP2 uvrA



Vehicle



14



45



19



42



 



0.7



NA



NA



NA



39



 



1.0



NA



NA



NA



42



 



3



13



56



23



43



 



6



12



55



16



28



 



8



11



39



13



35



 



11



4



33



6



32



 



21



2



15



5



34



 



37



0



8



0



4



 



Positive control



168



372



181



198


Conclusions:
The results of the bacterial reverse mutation test using gas-phase exposure indicate that, under the conditions of this study, the test article did not cause a positive mutagenic response in either the presence or absence of Aroclor induced rat liver S9.
Executive summary:

In a reverse mutation assay, performed according to OECD Guideline 471 and GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test material using the desiccator methodology, a modification of the Ames plate incorporation methodology, in the presence and absence of S9 -mix. The assay was performed in three phases: a preliminary toxicity assay, a mutagenicity assay and a confirmatory mutagenicity assay. Based on the findings of the preliminary toxicity assay, the maximum dose plated in the confirmatory mutagenicity assay was 37 mmoles/L (905,000 ppm). In the mutagenicity and confirmation assay, no precipitation was observed and toxicity was observed beginning at 11, 21 or at 37 mmoles/L. In the mutagenicity and confirmation assay, no positive mutagenic response was observed. In conclusion, under the conditions of this study, the test article did not cause a positive mutagenic response in either the presence or absence of Aroclor-induced rat liver S9.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Qualifier:
according to guideline
Guideline:
other: ICH (1996) Guideline S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals
Qualifier:
according to guideline
Guideline:
other: ICH (1998) Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals.
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human
Cytokinesis block (if used):
Colcemid
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction obtained from phenobarbital and 5,6-benzoflavone induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
- First test: 0, 60, 89, 101.48, 169.13, 281.88, 469.80, 783, and 1305 ug/mL in the absence and presence of S9.
- Second test: 0, 169.13, 281.88, 469.80, 783, and 1305 ug/mL in the absence and presence of S9.
Vehicle / solvent:
Ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION
- Pre-incubation

DURATION (first test)
- Pre-incubation period: 48 hours
- Exposure duration: 3 hours with and without S9 mix
- Expression time (cells in growth medium): 18 hours recovery
- Fixation time (start of exposure up to fixation or harvest of cells): 21 hours

DURATION (second test)
- Pre-incubation period: 48 hours
- Exposure duration: 3 hours with S9 mix and 21 hours continuous without S9 mix
- Expression time (cells in growth medium): 18 hours recovery (with S9 mix only)
- Fixation time (start of exposure up to fixation or harvest of cells): 21 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): 10 % Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 1000 cells per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Noted when observed
- Determination of endoreplication: Noted when observed

OTHER
- All cultures were treated in sealed glass bottles (with an internal volume of 160 mL) using a syringe and needle, inserted through the septum cap to prevent any loss of 1233zd within the test system. The glass bottles were then incubated on their sides at 37 °C in a roller apparatus, which rotates the bottles once every eight minutes approximately. The lymphocytes coat the inside of the bottles and were immersed in culture medium once every revolution and exposed directly to 1233zd for the rest of the revolution.
Evaluation criteria:
- An assay is considered to be acceptable if the negative and positive control values lie within the current historical control range.
- The test substance is considered to cause a positive response if the following conditions are met: Statistically significant increases (P<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentration. The increases exceed the negative control range of this laboratory, taken at the 99 % confidence limit. The increases are reproducible between replicate cultures. The increases are not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity. Evidence of a concentration-related response is considered to support the conclusion.
- A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any concentration.
Statistics:
The number of aberrant metaphase cells in each test substance group was compared with the solvent control value using the one-tailed Fisher exact test. A Cochran-Armitage test for trend was applied to the control and all test substance groups. If significance was found at the 1 % level, the test was reiterated excluding the highest concentration group - this process continued until the trend test was no longer significant.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
GENOTOXICITY:
- First test: In both the absence and the presence of S9 mix, the test substance caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any concentration, when compared with the solvent control. All mean values for the solvent control (ethanol), and all 1233zd treatment concentrations were within the historical control range for this laboratory, when taken at the 99 % confidence limit.
- Second test: Metaphase analysis: In both the absence and the presence of S9 mix, the test substance caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any concentration, when compared with the solvent control. In the absence of S9 mix, the mean values for the solvent control (ethanol) were on the limit of the laboratory historical control range (including gaps), and outside the laboratory historical range (excluding gaps). Although increased values were also observed at 1233zd treatment concentrations, as no statistical significance was observed at any treatment level with no associated concentration-related response, the result was considered negative. In the presence of S9 mix, all mean values for the solvent control (ethanol), and all 1233zd treatment concentrations were within the laboratory historical control range, when taken at the 99% confidence limit.
- Positive controls: In both tests, both positive control compounds, Mitomycin C and Cyclophosphamide, caused statistically significant increases (p<0.001) in the proportion of aberrant cells. This demonstrated the efficacy of the S9 mix and the sensitivity of the test system.
- Polyploid analysis: No statistically significant increases in polyploid metaphases were observed during metaphase analysis in both tests.

CYTOTOXICITY
-In both tests In the absence and presence of S9 mix the test substance caused no reduction in the mitotic index at 1305 μg/mL, the highest tested concentration, compared to the solvent control value. The concentrations selected for metaphase analysis were 469.80, 783 and 1305 μg/mL.
Conclusions:
Under the experimental conditions described, the test substance did not show evidence of causing an increase in the frequency of structural chromosome aberrations.
Executive summary:

An in vitro chromosome aberration study according to OECD Guideline 473 and GLP was performed. Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin, and exposed to the test substance both in the absence and presence of S9 mix derived from rat livers. Solvent and positive control cultures were also prepared. Two hours before the end of the incubation period, cell division was arrested using Colcemid®, the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage. In the first test, cells were treated with 0, 60.89, 101.48, 169.13, 281.88, 469.80, 783, and 1305 ug/mL and in the second test with 0, 169.13, 281.88, 469.80, 783, and 1305 ug/mL. In the first test, cells were treated for three hours followed by 18 hour recovery. In the second test, cells were treated for three hours followed by 18 hour recovery in the presence of S9 mix, and in the absence of S9 mix cells were continuously treated for 21 hours. The dose levels selected for the metaphase analysis were 469.80, 783, and 1305 μg/mL. In both the absence and presence of S9 mix, the test substance caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any concentration, when compared with the solvent control, in either test. No statistically significant increases in the proportion of polyploid cells were observed during metaphase analysis, in either test. All positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix. It is concluded that the test substance has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test substance was negative in the following in vivo genotoxicity studies: micronucleus mouse (acute inhalation exposure) and rats (4-week inhalation exposure).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Deutschland, Sulzfeld, Germany
- Age at study initiation: approx. 7 weeks
- Weight at study initiation: 294.2 g
- Fasting period before study: No
- Housing: Macrolon cages with bedding of wood shavings (Lignocel, type 3/4, Rettenmaier, Rosenberg, Germany) and shreds of paper as environmental enrichment (Enviro-dri, Lillico, Betchworth, England)
- Diet: Rat and Mouse No. 3 Breeding Diet RM3 was available ad libitum, except during periods of exposure to test substance.
- Water: Tap water was available ad libitum via polypropylene bottles, except during periods of exposure to test substance.
- Acclimation period: 21 Days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 -70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The inhalation chamber consisted of a cylindrical polypropylene (groups 1 to 4) or stainless steel (group 5) column, surrounded by a transparent cylinder with a volume of ca. 50 litres. Each column included two rodent tube sections, each accommodated 20 ports for animal exposure. Additional ports were used for test atmosphere sampling, temperature and relative humidity. Only the nose of the rats protruded into the interior of the column. The animals were placed in the exposure units after stabilisation of the test atmosphere.
- Method of holding animals in test chamber: The animals were secured in plastic animal holders, positioned radially through the outer hood around the central column (males and females alternated). The remaining ports were closed.
- System of generating test atmospheres: The inhalation equipment was designed to expose rats to a continuous supply of fresh test atmosphere. Compressed dry nitrogen was used to pressurise the cylinder containing the test material. The test atmosphere was generated by mixing a controlled amount of liquid test material (using a peristaltic pump with a mass flow controlled amount of humidified air. For groups 4 and 5 also a mass flow controlled stream of oxygen was added. The test material was allowed to evaporate in a U-tube at 30ºC and was led to the entrance at the top of the exposure chambers. Each morning before the start of the exposure, the settings of the various mass flow controllers were recorded. The animals were placed in the exposure unit after stabilisation of the test atmosphere concentration. The period between the start of the generation and the start of the exposure was about 20 minutes.
- Temperature, humidity in air chamber: 22 ± 2 °C, 30 – 70 % relative humidity,

ANALYSIS TEST ATMOSPHERE
- The actual concentration of the test material in the test atmosphere was monitored with a total carbon analyser. The responses of the analysers were recorded on a PC every minute using a CAN transmitter. test atmosphere samples were taken continuously from the exposure unit at the animals’ breathing zone and were passed to the total carbon analyser through a sample line. The mean response was calculated by averaging values read every minute.
Prior to the various exposures, the total carbon analysers were calibrated for a particular target concentration by sampling from three concentrations in a range including the target concentration in duplicate. The concentrations were prepared by injecting a known amount of test substance in a sample bag containing a mixture of air and oxygen.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
5 days/week (i.e., 20 or 21 exposure days over a 28-day study period
Dose / conc.:
2 000 ppm
Remarks:
Group 2: Low dose. Corresponding with an analytical concentration of 1994 ppm
Dose / conc.:
4 500 ppm
Remarks:
Group 3: Low-mid dose. Corresponding with an analytical concentration of 4485 ppm
Dose / conc.:
7 500 ppm
Remarks:
Group 4: High-mid dose. Corresponding with an analytical concentration of 7492 ppm
Dose / conc.:
10 000 ppm
Remarks:
Group 5: High dose. Corresponding with an analytical concentration of 9966 ppm
No. of animals per sex per dose:
5 males per dose
Control animals:
yes
Positive control(s):
- Positive control: Mitomycin C
- Justification for choice of positive control(s): The positive control substance is a mutagen known to induce damage to chromosomes and/or micronuclei in the bone marrow cells of male rats.
- Route of administration: intraperitoneal injection
- Doses / concentrations: 10 mL/kg body weight; 0.15 mg/mL in saline). The concentration of the positive control in the test solution was not determined analytically; the dose level quoted is therefore a nominal one.
Tissues and cell types examined:
bone marrow smears, 2 smears per animal were prepared, one slide per animal was examined

Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Doses were selected based on results of a sub-acute (14-day) inhalation toxcity study of this test substance in rats.

DETAILS OF SLIDE PREPARATION:
Bone marrow cells of one femur from each animal were collected into foetal calf serum and processed into glass drawn smears. Two bone marrow smears per animal were made, air-dried and fixed in methanol. One fixed smear was stained for analysis with a May-Grünwald Giemsa solution. The other unstained fixed smear was kept in reserve and discarded after completion of analysis.

METHOD OF ANALYSIS:
The slides were randomly coded by a person not involved in the scoring of slides. The slides (one slide per animal) were read by moving from the beginning of the smear (label end) to the leading edge in horizontal lines taking care that areas selected for evaluation were evenly distributed over the whole smear. The numbers of polychromatic and normochromatic erythrocytes (PE and NE, respectively) were recorded in a total of 200 erythrocytes per animal; if micronuclei were observed, these were recorded as micronucleated polychromatic erythrocytes (MPE) or micronucleated normochromatic erythrocytes (MNE). Once a total number of 200 erythrocytes (PE + NE) had been scored, an additional number of PE was scored for the presence of micronuclei until a total number of 2000 PE had been scored. Thus the incidence of MPE was recorded in a total of 2000 PE per animal and the number of MNE was recorded in the number of NE.
Evaluation criteria:
The assay was considered valid if the positive controls showed a statistically significant increase in the mean number of MPE/2000 PE and if the negative controls were within the historical range.
A test substance was considered to cause chromosomal damage and/or damage to the mitotic apparatus, if it showed a positive response, namely: the mean number of MPE/2000 PE was statistically significantly higher compared to the negative control group and was at least 6 MPE/2000 PE.
A test substance was considered to be negative if it produces no positive response at any of the dose levels analysed.
The test substance or its metabolites were considered to be cytotoxic to the bone marrow via general circulation, if the test substance statistically significantly reduced the mean number of PE/erythrocyte.
Both statistical significance and biological relevance were considered together in the evaluation.
Statistics:
Data on MPE and PE were analysed using pooled error variance t-tests or, if variances were not homogeneous, separate variance t-tests. These t-tests were applied to the negative control group versus treatment groups. All statistical tests were performed using SAS V9.1 statistical software Copyright (c) 2002-2003 by SAS Institute Inc., Cary, NC, USA.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Effects were observed on the haematological and clinical chemistry parameters in the high concentration group (10000 ppm) that were considered mild and reversible in a 14 day recovery period.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: Equivalent to 12932 mg/kg bw/day
Additional information on results:
- The highest dose level of 10000 ppm (53.9 g/m3) corresponds to about 3880 mg/rat per day, taking into account a minute ventilation of 200 mL/min, a 6-hour exposure period, and assuming 100 % absorption. The dose of 3880 mg per rat per day corresponds to about 12932 mg/kg-bw/day for a rat of 300 g, which is far above the limit concentration of 1000 mg/kg-bw/day for an exposure duration longer than 14 days.
Micronucleated polychromatic erythrocytes (MPE)
- 2000, 4500, 7500 and 10000 ppm: there were no statistically significant increases in the mean numbers of MPE between all test substance groups compared with the negative control group.
- 0 ppm: The mean number of MPE/2000 PE in the negative control group was within the historical range.
- Positive control: there was a statistically significant (p<0.001) increase in the mean number of MPE in the positive control group compared with the negative control group.
Polychromatic erythrocytes (PE)
- 2000, 4500, 7500 and 10000 ppm: there were marginal statistically significant (p<0.05) decreases in the mean number of PE in group 3 (4500 ppm) and group 5 (10000 ppm), when compared to the mean number of PE found in the negative control group. However, a clear treatment-related cytotoxicity to the bone marrow cells could not be demonstrated because 1) the effect of the treatment, at increasing dose levels, was not consistent, 2) the standard deviation fluctuated at increasing dose levels, and 3) a dose-related decrease in the mean number of PE could not be demonstrated. In group 2 (2000 ppm) and group 4 (7500 ppm) there were no statistically differences in the mean number of PE, when compared to the mean number of PE found in the negative control group.
- Positive control group: The mean number of PE in the positive control group was statistically significantly lower (p< 0.01) than the mean number in the negative control group. This indicated that the positive control substance Mitomycin C induced cytotoxicity to the bone marrow.
Conclusions:
It was concluded that repeated inhalation exposure to the test substance at concentrations up to 10000 ppm, resulting in a dose of 12932 mg/kg bw/day, which was far in excess of the limit dose of 1000 mg/kg bw/day, did not induce damage to the chromosomes and/or mitotic spindle apparatus (micronuclei) in the bone marrow cells of male rats under the conditions of this study.
Executive summary:

In a GLP-compliant erythrocyte micronucleus test, performed according to OECD guideline 474, five groups of Sprague-Dawley rats were exposed to the test substance at concentrations of 0, 2000, 4500, 7500, 10000 ppm via inhalation 6 hours/day on five days/week for four weeks. The group mean numbers of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) in the positive control group (Mitomycin C; 1.5 mg/kg bw, administered once intraperitoneally) showed a statistically significant increase compared to the negative control group. The mean numbers of MPE per 2000 PE in the negative control group were within the historical control range. Therefore, the study was considered valid. The results showed no statistically significant differences in the number of MPE per 2000 PE between the test substance treated groups and the negative control group (clean air). The number of PE per 200 erythrocytes in the positive control group was statistically significantly lower than that in the negative control group, indicating that the positive control substance induced cytotoxicity to the bone marrow. Though the mean numbers of PE per 200 erythrocytes in the groups exposed to the test substance showed some marginal decreases compared to negative controls (at 4500 and 10000 ppm), a clear treatment-related cytotoxicity to the bone marrow cells could not be demonstrated. It was concluded that, under the conditions of this study, the test substance at exposure levels up to 10000 ppm, resulting in a dose of 12932 mg/kg bw/day, which far exceeded the limit dose of 1000 mg/kg-bw/day, did not induce damage to the chromosomes and/or mitotic spindle apparatus (micronuclei) in the bone marrow target cells of male rats.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
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
- Source: Charles River Deutschland, Sultzfeld, Germany
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: Mean weight: Negative control: 31.55 g; Treated: 31.45 g; Postive control: 31.20 g
- Fasting period before study: none
- Housing: Sterilsed macrolon cages with bedding of wood shavings (Lignocel, type 3/4, Rettenmaier, Rosenberg, Germany) with strips of paper (Enviro-dri, Lillico, Betchworty, England)
- Diet: ad libitum Rat & Mouse No.3 Breeding Diet, RM3 (SDS Special Diets Services, Witham, England)
- Water: tap water, ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 40 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/2
Route of administration:
inhalation: gas
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The inhalation chamber consisted of a cylindrical stainless steel column, surrounded by a transparent cylinder with a volume of ca. 50 litres. The column consisted of 2 mixing chambers, a rodent tube section underneath, and the exhaust section at the bottom. The rodent tube section had 20 ports for animal exposure. Additional ports were used for test atmosphere sampling, and measurement of oxygen, temperature and relative humidity. Only the nose of the rats protruded into the interior of the column.
- Method of holding animals in test chamber: The animals were secured in plastic animal holders, positioned radially through the outer hood around the central column. The remaining ports were closed.
- System of generating test atmosphere: The inhalation equipment was designed to expose mice to a continuous supply of fresh test atmosphere. Liquid test material was evaporated in a temperature-controlled brass tube. Compressed dry nitrogen was used to pressurize the cylinder containing the test material. Before entering the top of the inhalation chamber, the test substance was mixed with humidified compressed air. Oxygen was added to ensure an oxygen concentration of at least 19.5%. The animals were placed in the exposure unit after stabilization of the test atmosphere concentration. The period between the start of the generation and the start of the exposure was about 103 minutes. The exposure unit for the control animals was supplied with a rotameter-controlled stream of dry compressed air only.
- Temperature, humidity, oxygen in air chamber: 21.3-23.1°C, 49.5-55.4% relative humidity, 20.6 - 20.7% oxygen
- Air flow rate: 15.6 L/min

ANALYSIS OF TEST ATMOSPHERE
The actual concentration of the test material in the test atmosphere was monitored with a total carbon analyser. The responses of the analysers were recorded on a PC every minute using a CAN transmitter. Test atmosphere samples were taken continuously from the exposure unit at the animals’ breathing zone and were passed to the total carbon analyser through a sample line. The mean response was calculated by averaging values read every minute. Prior to the actual study, the total carbon analyser was calibrated for the target concentration by sampling from 4 concentrations in a range including the target concentration. The concentrations were prepared by mixing mass flow controlled amounts of air, oxygen and test material.
Duration of treatment / exposure:
4 hrs
Frequency of treatment:
once
Post exposure period:
24 and 48 hrs
Dose / conc.:
50 000 ppm
Remarks:
Corresponding with an analytical concentration of 48719 ppm.
No. of animals per sex per dose:
5 males/time point (negative control, test material), 5 positive control males
Control animals:
yes, sham-exposed
Positive control(s):
- Positive contol: mitomycin C
- Justification for choice of positive control(s): positive in this assay
- Route of administration: ip injection
- Doses / concentrations: 0.75 mg/kg bw
Tissues and cell types examined:
bone marrow smears, 4 smears per animal were prepared, at least one slide per animal was examined
Details of tissue and slide preparation:
- Sacrifice, bone marrow sampling and processing: At 24 hours after the end of the exposure, 5 negative control, 5 test animals and 5 positive control animals were sacrificed by cervical dislocation. At 48 hours after the end of the exposure, 5 negative control and 5 test animals were sacrificed. Immediately following sacrifice, the bone marrow cells of both femurs were collected into foetal calf serum and processed into glass drawn smears. Four smears per animal were prepared air dried and fixed in methanol. All smears were stained with a May-Grunwald/Giemsa solution. One or more slides per animal were used for analysis.
- Microscopic examination of bone marrow smears: The slides were randomly coded by a person not involved in the scoring of slides. The slides were read by moving from the beginning of the smear (label end) to the leading edge in horizontal lines, taking care that areas selected for evaluation were evenly distributed over the whole smear. The following criteria were used for the scoring of cells:
Polychromatic erythrocyte (PE) is an immature erythrocyte that contains ribosomes. Normochromatic erythrocyte (NE) is a mature erythrocyter that lacks ribosomes. A micronucleus is a small, normally round nucleus with a diameter of approx. 1/20 to 1/5 of an erythrocyte, distinguished from the cytoplasm by a dark blue stain.
The numbers of PE and NE were recorded in a total of 200 erythrocytes (E) per animal. If micronuclei were observed, these were recorded as micronucleated polychromatic erythrocytes (MPE) or micronucleated normochromatic erythrocytes (MNE). Once a total of 200 E (PE +NE) had been scored, an additional number of PE was scored for the presence of micronuclei until a total number of 2000 PE had been scored. Thus the incidence of MPE was recorded in a total of 2000 PE per animal and the number of MNE was recorded in the number NE.
Evaluation criteria:
- The study was considered valid if positive controls showed a statistically significant increase in the mean number of MPE/2000 PE and if the negative controls were within the historical range.
- A test substance was considered to cause chromosomal damage and/or damage to the mitotic apparatus, if it showed a positive response, namely: the mean number of MPE/2000 PE was statistically significantly higher compared to the negative control and was at least 6 MPE/2000 PE.
- A test substance was considered to be negative if it produced no positive response.
- The test substance or its metabolites were considered to be cytotoxic to the bone marrow via general circulation, if the test substance statistically significantly reduced the mean number of PE.
- Both statistical significance and biological relevance were considered together in the evaluation.
Statistics:
Initially a one-way ANOVA was preformed. If it yielded a positive result (p<0.05) it was followed by pooled error variance t-tests or, if variences were not homogenious, separate variance t-tests.
All statistical tests were preformed using SAS V9.1 statistical software.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- Behaviour, clinical signs and mortality: Clear restless (graded as slight) was seen in all animals at every observation point during exposure. No abnormalities were observed in mice exposed to the control atmosphere. In a preliminary experiment, one male mouse was exposed for 4 hours to a target concentration of 50000 ppm. Besides slight restlessness and breathing at an increased rate (graded as slight), no signs of toxicity were observed and mortality did not occur.
- Statistical analysis of micronucleus results: At time point 24 and 48 hours after treatment there was no statistically significant difference in the mean number of MPE/2000E between the test substance group and the negative control group. The mean number of MPE/2000 PE in the negative control group was within the historical range. The mean number of MPE/2000E in the positive control was statistically significantly (p<0.0001) increased compared with the negative control group. The mean number of PE in the test substance group did not differ statistically significantly from that in the negative control group. The mean number of PE in the positive control group was statistically significantly lower (p = 0.0015) than that in the negative control group. This indicated that the positive control substance Mitomycin C induced cytotoxicity to the bone marrow.
Conclusions:
It was concluded that inhalation exposures to the test substance at a concentration of 50000 ppm for 4 hours, resulting in a dose of about 65800 mg/kg body weight, which was far in excess of the limit dose of 2000 mg/kg body weight, did not induce damage to the chromosomes and/or mitotic spindle apparatus (micronuclei) in the bone marrow cells of male mice under the conditions of this study.
Executive summary:

In a GLP-compliant erythrocyte micronucleus test, performed according to OECD guideline 474, two groups of 10 male CD-1 mice were exposed to the test substance at concentrations of 0 or 50000 ppm via inhalation for 4 hours. 24 and 48 hours after exposure 5 mice per group were sacrificed. A positive control group consisted of 5 males treated with a single intraperitoneal dose of mitomycin C (0.75 mg/kg bw) and sacrificed 24 hours after injection. The group mean numbers of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) in the positive control group showed a statistically significant increase compared to the negative control group. The mean number of MPE per 2000 PE in the negative control group was within the historical control range. Therefore, the study was considered valid. Both at 24 and 48 hours after inhalation exposure, no statistically significant differences between the test substance and the negative control group were found with respect to the mean number of PE per 200 erythrocytes (E) and the mean number of MPE per 2000 PE. It was therefore concluded that, under the conditions of this study, inhalation exposure to the test substance for 4 hours at a concentration of 50000 ppm, resulting in a dose of about 65800 mg/kg body weight, which far exceeded the limit dose of 2000 mg/kg body weight, did not induce damage to the chromosomes and/or mitotic spindle apparatus (micronuclei) in the bone marrow target cells of male mice.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro: Reverse mutation assay


In a reverse mutation assay, performed according to OECD Guideline 471 and GLP, salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test material using the desiccator methodology, a modification of the Ames plate incorporation methodology, in the presence and absence of S9 -mix (BioReliance 2011). The assay was performed in three phases: a preliminary toxicity assay, a mutagenicity assay and a confirmatory mutagenicity assay. Based on the findings of the preliminary toxicity assay, the maximum dose plated in the confirmatory mutagenicity assay was 37 mmoles/L (905,000 ppm). In the mutagenicity and confirmation assay, no precipitation was observed and toxicity was observed beginning at 11, 21 or at 37 mmoles/L. In the mutagenicity and confirmation assay, no positive mutagenic response was observed. In conclusion, under the conditions of this study, the test article did not cause a positive mutagenic response in either the presence or absence of Aroclor-induced rat liver S9.


 


In vitro: Chromosome aberration test


An in vitro chromosome aberration study according to OECD Guideline 473 and GLP was performed (Huntington Life Sciences, 2011). Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin, and exposed to the test substance both in the absence and presence of S9 mix derived from rat livers. Solvent and positive control cultures were also prepared. Two hours before the end of the incubation period, cell division was arrested using Colcemid®, the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage. In the first test, cells were treated with 0, 60.89, 101.48, 169.13, 281.88, 469.80, 783, and 1305 ug/mL and in the second test with 0, 169.13, 281.88, 469.80, 783, and 1305 ug/mL. In the first test, cells were treated for three hours followed by 18 hour recovery. In the second test, cells were treated for three hours followed by 18 hour recovery in the presence of S9 mix, and in the absence of S9 mix cells were continuously treated for 21 hours.The dose levels selected for the metaphase analysis were 469.80, 783, and 1305 μg/mL. In both the absence and presence of S9 mix, the test substance caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any concentration, when compared with the solvent control, in either test. No statistically significant increases in the proportion of polyploid cells were observed during metaphase analysis, in either test. All positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix. It is concluded that the test substance has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.


 


In vivo: Micronucleus assay (mouse)


In a GLP-compliant erythrocyte micronucleus test, performed according to OECD guideline 474, two groups of 10 male CD-1 mice were exposed to the test substance at concentrations of 0 or 50000 ppm via inhalation for 4 hours (TNO, 2009c). 24 and 48 hours after exposure 5 mice per group were sacrificed. A positive control group consisted of 5 males treated with a single intraperitoneal dose of mitomycin C (0.75 mg/kg bw) and sacrificed 24 hours after injection. The group mean numbers of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) in the positive control group showed a statistically significant increase compared to the negative control group. The mean number of MPE per 2000 PE in the negative control group was within the historical control range. Therefore, the study was considered valid. Both at 24 and 48 hours after inhalation exposure, no statistically significant differences between the test substance and the negative control group were found with respect to the mean number of PE per 200 erythrocytes (E) and the mean number of MPE per 2000 PE. It was therefore concluded that, under the conditions of this study, inhalation exposure to the test substance for 4 hours at a concentration of 50000 ppm, resulting in a dose of about 65800 mg/kg body weight, which far exceeded the limit dose of 2000 mg/kg body weight, did not induce damage to the chromosomes and/or mitotic spindle apparatus (micronuclei) in the bone marrow target cells of male mice.


 


In vivo: Micronucleus assay (rat)


In a GLP-compliant erythrocyte micronucleus test, performed according to OECD guideline 474, five groups of Sprague-Dawley rats were exposed to the test substance at concentrations of 0, 2000, 4500, 7500, 10000 ppm via inhalation 6 hours/day on five days/week for four weeks TNO, 2009b). The group mean numbers of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) in the positive control group (Mitomycin C; 1.5 mg/kg bw, administered once intraperitoneally) showed a statistically significant increase compared to the negative control group. The mean numbers of MPE per 2000 PE in the negative control group were within the historical control range. Therefore, the study was considered valid. The results showed no statistically significant differences in the number of MPE per 2000 PE between the test substance treated groups and the negative control group (clean air). The number of PE per 200 erythrocytes in the positive control group was statistically significantly lower than that in the negative control group, indicating that the positive control substance induced cytotoxicity to the bone marrow. Though the mean numbers of PE per 200 erythrocytes in the groups exposed to the test substance showed some marginal decreases compared to negative controls (at 4500 and 10000 ppm), a clear treatment-related cytotoxicity to the bone marrow cells could not be demonstrated. It was concluded that, under the conditions of this study, the test substance at exposure levels up to 10000 ppm, resulting in a dose of 12932 mg/kg-bw/day, which far exceeded the limit dose of 1000 mg/kg-bw/day, did not induce damage to the chromosomes and/or mitotic spindle apparatus (micronuclei) in the bone marrow target cells of male rats.


 


In vivo: Unscheduled DNA synthesis test


In a GLP compliant unscheduled DNA synthesis-test performed according to OECD Guideline 486, the test substance was examined for its potential to damage DNA as reflected by induced DNA synthesis to repair the damage using hepatocytes harvested from exposed male Sprague-Dawley rats (TNO, 2009b). Groups of five male rats were exposed by inhalation at concentrations of 0 (clean air, negative control), 7500 or 10000 ppm 6 hours/day on 5 days per week for four weeks. An additional group of the same size was treated once by oral gavage with the positive control substance 2-Acetylaminofluorene (2-AAF). The mean net nuclear grains (NNG) in the 7500 and 10000 ppm groups (-8.33 and -7.47, respectively) were comparable to the mean NNG in the negative control group (-8.30) and clearly lower than the mean NNG in the positive control group (-3.63). The percentage ‘cells in repair’ in the 7500 and 10000 ppm groups (1.00% and 0.60%) was comparable to that in the negative control group (1.80 % ‘cells in repair’) and clearly lower than that in the positive control group (24.51 %). It is concluded that under the conditions of this study the test substance did not induce unscheduled DNA synthesis (UDS) in hepatocytes of male rats.

Justification for classification or non-classification

Based on the available data classification for genetic toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.