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Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
For the evaluation of genetic toxicity several experimental data with the test substance and a structural analogue (free acid) are available. The test substance was investigated for its ability to induce reverse mutations in S. typhimurium strains. The test substance caused a positive response in one of five tester strain in the absence of S9-mix. However, no increase of revertant colonies could be observed in a second Ames Test conducted with the test substance in the presence and absence of a metabolic activation system. The negative result of the second Ames test is supported by the results of three Ames Tests and two gene mutation assays on mammalian cells (MLA and UDS) with the read-across substance (free acid). Furthermore, no increase in micronuclei in bone marrow polychromatic erythrocytes was observed in an in vivo micronucleus test with the structural analogue. Therefore it is concluded that the test substance has no mutagenic and clastogenic potential.
Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1989-10-10 to 1989-12-19
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted under the regulations of GLP and the respective guideline. A read-across approach to the free acid of the test substance was used. For justification please refer to IUCLID section 13.
Qualifier:
according to
Guideline:
other: U.S. EPA FIFRA Guideline 84
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague-Dawley, Inc., Frederick, MD.
- Assigned to test groups randomly: yes, under following basis: body weight
- Weight at study initiation: 28.6 - 39.1 grams and 20.0 - 24.3 grams for the male and female animals
- Housing: group-housed by sex up to five per cage
- Diet: Purina Certified Laboratory Chow* #5002 ad libitum
- Water: tab water ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72±6
- Humidity(%): 50 ± 20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: corn oil
- Justification for choice of vehicle: Common vehicle
- Concentration of test material in vehicle: 400, 133.4, 40 mg/mL
- Amount of vehicle: 12.5 ml/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Based upon the solubility data from the dose limit test, the vehicle used to solubilize the test article for the bone marrow micronucleus assay was corn oil. The dosing solutions for the assay were prepared by making a 400 mg/mL stock for the high dose (5000 mg/kg). Dilutions of this stock were prepared for the 1667 and 500 mg/kg dose levels.
Duration of treatment / exposure:
single treatment
Frequency of treatment:
Once
Post exposure period:
24, 48, 72 hours
Remarks:
Doses / Concentrations:
5000, 1667, 500 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: orally by gavage
- Dose: 80 mg/kg bw
Tissues and cell types examined:
Bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The dose levels used in this assay were based upon the results of the previously conducted dose limit test in which there was no toxicity at 5000 mg/kg bw over a fourteen day observation period.

TREATMENT AND SAMPLING TIMES:
The test article dosed animals were euthanatized 24, 48 and 72 hours after administration of the test article. The positive and vehicle control animals were euthanatized 24 hours after the administration of the control articles.

DETAILS OF SLIDE PREPARATION:
At the appropriate harvest time, the animals were euthanatized with C02 and the adhering soft tissue and epiphyses of both tibiae were removed. The marrow was flushed into a centrifuge tube (one tube for each animal) with 3 mL fetal calf serum. Following centrifugation to pellet the tissue, most of the supernatant was drawn off, the cells were resuspended, and the suspension spread on slides and air-dried. The slides were then fixed in methanol, stained in May-Gruenwald solution followed by Giemsa, and rinsed in deionized water. After being air-dried, the slides were coverslipped using Depex mounting medium.

METHOD OF ANALYSIS:
The coded slides were then scored for micronuclei and the polychromatic (PCE) to normochromatic (NCE) cell ratio. Standard forms were used to record these data. 1000 PCEs per animal were scored. The frequency of micronucleated cells as expressed as percent micronucleated cells based on the total PCEs present in the scored optic field. The normal frequency of micronuclei in this mouse strain is about 0.0-0.4%.
The frequency of PCEs versus NCEs was determined by scoring the number of NCEs observed in the optic fields while scoring the 1000 PCEs for micronuclei.
Evaluation criteria:
The criteria for the identification of micronuclei were those of Schmid (1976). Micronuclei were darkly stained and generally round, although almond and ring-shaped micronuclei occasionally occur. Micronuclei had sharp borders and were generally between 1/20 and 1/5 the size of the PCE. The unit of scoring was the micronucleated cell, not the micronucieus; thus the occasional cell with more than one micronucieus was counted as one micro-nucleated PCE, not two (or more) micronuclei. The staining procedure permitted the differentiation by color of PCEs and NCEs (bluish-grey and red, respectively).
The criteria for determining a positive response involved a statistically significant dose-related increase in micronucleated PCEs, or the detection of a reproducible and statistically significant positive response for at least one dose level. A test article that induced neither a statistically significant dose response nor a statistically significant and reproducible increase at one dose level was considered negative. In either case, the final decision was based on scientific judgment.
Statistics:
Data were summarized to include tables indicating the individual animal results and in tables with animal results summarized by sex and dose groups at the different time points. The analysis of the data was performed using an Analysis of Variance (p<0.05) on the square root arcsine transformation (Sokal and Rohlf, 1981) which was performed on the proportion of cells with micronuclei per animal (square root arcsine proportion). Once the Analysis of Variance had been performed, Tukey's Studentized range test (HSD) with adjustment for multiple comparisons (p<0.05) was used at each harvest time to determine which dose groups, if any, were significantly different from the vehicle control. Analyses were performed separately for each harvest time and sex combination, and also at each harvest time for the sexes combined.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: All animals appeared normal after dosing and remained healthy until the appropriate harvest times.
- Induction of micronuclei: The test article induced no significant increases in micronucleated polychromatic erythrocytes over the levels observed in the vehicle controls in either sex or at any of the harvest times. The positive control, CP, induced significant increases in micronucleated PCEs in both sexes, with means and standard errors of 2.48% ± 0.54% and 1.28% ± 0.69% for the males and females, respectively.
- Ratio of PCE/NCE:
Vehicle control: Males: 0.57 ± 0.06; Females: 0.71 ± 0.09
Positive control: Males: 0.76 ± 0.16; Females: 0.69 ± 0.03
500 mg/kg bw (48h): Males: 0.60 ± 0.06; Females: 0.83 ± 0.16
1667 mg/kg bw (48h): Males: 0.45 ± 0.07; Females: 0.58 ± 0.07
5000 mg/kg bw (48h): Males: 0.49 ± 0.08; Females: 0.65 ± 0.04
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

In vitro

Ames test

The mutagenic activity of the test substance was examined in the Salmonella/Mammalian-Microsome Reverse Mutation Assay (Ames Test, Sandoz 14562-0-401, 1991). This assay evaluates the test article and/or its metabolites for their ability to induce reverse mutations at the histidine locus in the genome of specific Salmonella typhimurium tester strains both in the presence and absence of an exogenous metabolic activation system of mammalian microsomal enzymes derived from Aroclor-induced rat liver (S9).

The doses tested in the mutagenicity assay were selected based on the results of a dose range-finding study using tester strain TA100 and ten dose levels of test article ranging from 5000 to 6.67 µg per plate, one plate per dose, both in the presence and absence of S9.

The tester strains used in this study were TA98, TA100, TA1535, TA1537 and TA1538. The assay was conducted using three plates per dose level both in the presence and absence of S9. Six dose levels of the test article were tested, from 100 to 5000 µg per plate in both the presence and absence of S9.

The results indicate that under the conditions of this study, the test article did not cause a positive increase in the number of histidine revertants per plate of any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor-induced rat liver (S9).

 

Ames test

The assay (Sandoz, 1988) evaluates the mutagenic potential of the test article (or its metabolites) for its ability to induce back mutations at selected loci of several strains of Salmonella typhimurium in the presence and absence of an exogenous metabolic activation system of microsomal enzymes derived from Aroclor induced rat liver. The tester strains used in this study were TA98, TA100, TA1535, TA1537 and TA1538.

The results indicate that under the conditions of this study, the test article did cause a positive response on tester strain TA1537 (12.5-fold) in the absence of microsomal enzymes.

In addition non-dose responsive increases in the number of revertants per plate were observed on tester strain TA98 (2.0-fold) in the absence of microsomal enzymes and on tester strain TA1537 (2.0-fold) in the presence of microsomal enzymes. However, non-dose responsive increases in the number of revertants per plate are not evaluated as positive. All criteria for a valid study were met as described in the protocol.

 

 

Ames test

Read-across

In a bacterial reverse mutation assay (Sandoz 1103-0-401, 1989) a structural analogue substance (free acid ) was tested for its mutagenic potential in 5 bacterial strains, Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA1538. Following concentrations were tested in a plate incorporation test using two independent experiments: 333, 557, 1000, 3330, 6670, 10000 µg/plate.

Each assay was conducted with and without metabolic activation (S9 Mix). The concentrations, including the respective positive and negative controls, were tested in triplicate.

No substantial increases or decreases were observed in revertant colony numbers of any of the five test strains following treatment with test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

Thus, it can be concluded that the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.

 

Ames test

Read-across

The test item (free acid of the registered substance) was assayed in tester strains TA-98, TA-100, TA-1535, TA-1537, and TA-1538 at concentrations of 667, 1000, 3333, 6667, and 10000 µg/plate in the absence or presence of an exogenous metabolic activation system (BASF 98/5030). There were no increases in the number of revertants/plate in any of the tester strains either in the presence or absence of metabolic activation, there was no cytotoxicity or reduction in the bacterial lawn, but on tester strain TA-1537, in the absence of microsomal enzymes, a 2-fold non-dose response increase was seen in the number of revertants/plate. This did not meet the criteria for a positive response. The results of this assay indicate that the test item did not cause a positive response in any of the tester strains with or without metabolic activation by induced rat liver microsomes.

 

Mouse Lymphoma Test

Read across

The objective of this in vitro assay (Sandoz 110360-0-431, 1996) was to evaluate the ability of a structural analogue (free acid of the registered substance) to induce forward mutations at the thymidine kinase (TK) locus in the L5178Y mouse lymphoma cell line. A preliminary range finding test was conducted which showed that the test item was soluble in dimethyl sulfoxide and remained in solution in culture medium up to a concentration of 0.5 mg/mL. Cell toxicity was observed starting at a test item concentration of 1.0 mg/mL.

The mutagenic potential of test item was evaluated in two independent experiments both with and without metabolic activation (S9 mix) using single cultures per dose level.

The following test item concentrations were used:

Experiment 1: 0.05, 0.1, 0.5, 0.8, 1.0, 1.5, 2.0, 3.0 mg/mL

Experiment 2: 0.05, 0.1, 0.5, 0.6, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0 mg/mL

The test article produced dose-related increases in toxicity starting at 1.0 mg/mL in both trials. In both experiments, significant mutant frequencies were obtained only at toxic, insoluble test article concentrations. Because increased mutant frequencies were observed only at highly toxic and insoluble concentrations, the result of this test was regarded as negative.

 

UDS Test

Read-across

In the in vitro rat primary hepatocyte unscheduled DNA synthesis (UDS) assay (Sandoz 11030-0-447, 1990) a structural analogue substance (free acid of the registered substance) did not induce significant increases in UDS. In the assay, freshly prepared rat hepatocytes were exposed to the test item at 15 concentrations ranging from 0.025 mg/mL to 1000 mg/mL in the presence of 10 µCi/mL 3HTdR (42 Ci/mmole). The test material was soluble in media at all concentrations tested with a slightly acidic pH shift over the range of 100 mg/mL to 1000 mg/mL. The highest treatments of 1000 mg/mL and 500 mg/mL were not analyzed for nuclear labeling due to excessive toxicity. Treatments from 250 mg/mL to 5.00 mg/mL covered a good range of toxicity (50.8% to 98.8% survival) and were selected for analysis of nuclear labeling. None of the criteria used to indicate UDS were approached by any of the analyzed treatments and no dose-related response was observed. The test item was therefore evaluated as inactive in the in vitro rat primary hepatocyte UDS Assay. The test material did not induce significant changes in the nuclear labeling of rat primary hepatocytes for an applied concentration range of 250 mg/mL to 5.00 mg/mL. The test item was therefore evaluated as inactive in the in vitro rat primary hepatocyte UDS assay

 

In vivo

Micronucleus Assay

Read-across

The objective of the in vivo micronucleus assay (Sandoz 11030-0-445, 1990) was to evaluate the ability of a structural analogue substance (free acid of the registered product) to induce micronuclei in bone marrow polychromatic erythrocytes of ICR mice. The test article was suspended in corn oil and dosed by oral gavage at 500, 1667, and 5000 mg/kg bw based upon the results of a previously conducted dose limit test, in which a dose of 5000 mg/kg was evaluated. Ten animals (five males and five females) were randomly assigned to each dose/harvest time group. Vehicle and positive control groups euthanatized 24 hours after dosing were included in the assay. The animals were dosed with the test article and were euthanatized 24, 48 and 72 hours after dosing for extraction of the bone marrow. The test material did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse bone marrow micronucleus test.

 

Conclusion

For the evaluation of genetic toxicity there are several experimental data with the test substance and a structural analogue (free acid) available. The test substance was investigated for its ability to induce reverse mutations in S. typhimurium strains. The test substance caused a positive response in one of five tester strain in the absence of S9-mix. However, no increase of revertant colonies could be observed in a second Ames Test conducted with the test substance in the presence and absence of a metabolic activation system. The negative result of the second Ames test is supported by the results of three Ames Tests and two gene mutation assays on mammalian cells (MLA and UDS) with the read-across substance (free acid). Furthermore, no increase in micronuclei in bone marrow polychromatic erythrocytes was observed in an in vivo micronucleus test with the structural analogue.

Therefore it is concluded that the test substance has no mutagenic and clastogenic potential.

 


Justification for selection of genetic toxicity endpoint
One in vivo micronucleus assay on mice is available and is considered acceptable for the assessment of the genetic toxicity potential of the test item. There are further in vitro studies with the test substance and a structural analogue available.

Justification for classification or non-classification

Based on the results obtained in the in vitro and in vivo studies the test item is not considered to be genotoxic/mutagenic or clastogenic and thus has not to be classified according to Directive 67/548/EEC and Regulation (EC) No 1272/2008.