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Toxicological information

Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
TK
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
A continuous cell log was kept to record growth, doubling times, and subculture operations. Laboratory cultures were periodically tested for mycoplasma contamination and karyotype. To reduce the frequency of spontaneous TK-‘. mutants prior to use in the mutation assay, cell cultures were exposed to conditions that selected against the TK +/- phenotype (exposure to methotrexate).
The medium used for this study was RPMI 1640 supplemented with Pluronic F68, L-glutamine, sodium pyruvate, antibiotics, and heat-inactivated horse serum (10% by volume). Treatment medium was Fischers medium with the same media supplements used in the culture medium except that the horse serum concentration was reduced to 5% by volume. Cloning medium consisted of the preceding growth medium with up to 20% horse serum, without Pluronic” F68 and with the addition of BBL purified agar at a final concentration of 0.24% to achieve a semisolid state. Selection medium was cloning medium that contained 3 ug/ml of TFT.
Metabolic activation:
with and without
Metabolic activation system:
rat liver enzymes (S9 fraction, Aroclor induced) and an energy producing system (CORE) comprised of nicotinamide adenine dinucleotide phosphate (NADP., sodium salt) and isocitrate.
Test concentrations with justification for top dose:
5 - 80 µg/ml
Vehicle / solvent:
- insoluble in dimethylsulfoxide (DMSO) at 50 mg/ml and 100 mg/ml
- formed a clear, yellow solution in ethanol at 100 mg/ml. Ethanol was therefore chos’en as the vehicle. The test article was prepared in ethanol at 100x times the highest desired concentration. Lower 100x stocks were prepared by serial dilution with ethanol
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h
- Expression time (cells in growth medium): 2 days

NUMBER OF REPLICATIONS: two independent experiments, every sample in triplicate

NUMBER OF CELLS EVALUATED: all colonies are counted

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER EXAMINATIONS:
- pH
- osmolarity
- solubility
- cell morphology
Evaluation criteria:
- A dose-related or toxicity-related increase in mutant frequency should be observed. It is desirable to obtain this relation for at least three doses, but this depends upon the concentrat,ion steps chosen for the assay and the toxicity at which mutagenic activity appears.

- If the mutant frequency obtained for a single dose at or near the highest, testable toxicity is about two or more times the minimum criterion, the test article will be considered mutagenic in a single trial. Smaller increases at a single dose near the highest testable toxicity will require confirmation by a repeat assay.

- For some test articles, the correla,tion between toxicity and applied concentration is poor. The proportion of the applied test article that effectively interacts with the cells to cause genetic alterations is not always repeatable or under control. Conversely, measurable changes in frequency of induced mutants may occur with concentration changes that cause only small changes in observed toxicity. Therefore, either parameter, applied concentration or toxicity (perceni. relative growth), can be used to establish whether the increase in mutant frequency is related to an increase in effective treatment.

- Treatments that induce less than 10% relative growth are included in the assay, but are not used as primary evidence for mutagenicity as it relates to risk assessment.

A test article is evaluated as non-mutagenic in a single assay only if the minimum increase in mutant frequency is not observed for a range of applied concentrations
that extends to toxicity causing 10% to 20% relative growth or, in the case of relatively nontoxic materials, a range of applied concentrations extending to the maximum of 5000 pglml or to at least twice the solubility limit in medium.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
cytotoxicity was observed from 15 ug/ml onward
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Dose Range Finding Assay

The test article was tested in a preliminary dose range finding assay both with and without S9 metabolic activation. Ten dose levels were used in each case that ranged from 1.95 μg/ml to 1000 μg/ml; vehicle and untreated controls were included under each activation condition (Table 1). The test article was weakly to non-cytotoxic with and without metabolic activation from 1.95 μg/ml to 31.3 μg/ml and treatments from 62.5 μg/ml to 1000 μg/ml were very highly cytotoxic or lethal under both activation conditions. The mutation assays were initiated with treatments based on the results of the cytotoxicity assay.

TABLE 1: Cytotoxicity Assay

  Without S9 Activation With S9 Activation
Applied concentration (µg/ml) cell density/ml (x105) % vehicle control cell density/ml (x105) % vehicle control
negative (media) control 10.5 147.9 13.3 133.0
vehicle control (1% ethanol) 7.1 100.0 10.0 100.0
1.95 NTC -- NTC --
3.91 NTC -- NTC --
7.81 NTC -- NTC --
15.6 12.8 180.3 12.9 129.0
31.3 4.5 63.4 8.3 83.0
62.5 0.0 0.0 0.3 3.0
125 0.0 0.0 0.0 0.0
250 0.0 0.0 0.0 0.0
500 0.0 0.0 0.0 0.0
1000 0.0 0.0 0.0 0.0

NTC = not counted

Mutation Assay

Three non-activation mutation assays were performed with the test article but Trial 2 was unacceptable because of high vehicle control mutant frequencies. In Trial 1 (Table 2), nine dose levels were initiated at 5.00, 10.0, 15.0, 20.0, 30.0, 40.0, 50.0, 60.0 and 80.0 μg/ml. Treatments from 20.0 μg/ml to 80.0 μg/ml were terminated because of excessive cytotoxicity. The remaining three treatments induced little or no cytotoxicity (90.4% to 62.5% relative growths), and a small increase in concentration from 15.0 μg/ml to 20.0 μg/ml was excessively cytotoxic.

In order for a culture to be evaluated as mutagenic in Trial 1 of the non-activation assay, a mutant frequency greater than 92.6 x 10-6 was required. This threshold value was equal to twice the average mutant frequency of the concurrent vehicle controls. None of the analyzed test item treatments induced a mutant frequency that exceeded the minimum criterion and the test article was considered non-mutagenic in this trial. A confirmatory assay was initiated.

TABLE 2: Mutation assay without activation (Trial 1)

   Daily cell counts (cells/ml, 105 units)   Suspension growth   Total mutant colonies  Total viable colonies  Cloning efficiency   Relative growth (%)   Mutant Frequencies (10-6 units) 
1 2
 Vehicle control  17.8 14.6 28.9 97 355 59.2 100.0 54.6
 Vehicle control  16.2 14.6 26.3 87 383 63.8 100.0 45.4
 Vehicle control  17.4 16.0 30.9 75 386 64.3 100.0 38.9
 Vehicle average      28.7     62.4    
 MMS 5 nl/ml  12.8 16.8 23.9 291 417 69.5 92.8 139.6#
 MMS 10 nl/ml  9.2 12.9 13.2 393 187 31.2 23.0 420.3#
 MMS 15 nl/ml  9.0 12.3 12.3 317 113 18.8 12.9 561.1#
      relative to vehicle control (%)     relative to vehicle control (%)    
 Test article 5 µg/ml  15.6 15.3 92.4 82 366 97.8 90.4 44.8
 Test article 10 µg/ml  13.0 15.3 77.0 97 404 107.9 83.1 48.0
 Test article 15 µg/ml  12.2 12.2 56.7 115 413 110.3 62.5 55.7

Suspension Growth = (Day 1 count / 3) x (Day 2 count) / (3 or Day 1 count if not split back)

Cloning Efficiency = total viable colony count / number of cells seeded x 100

Relative Growth = (Relative suspension growth x relative cloning efficiency) / 100

Mutant Frequency = (total mutant colonies/total viable colonies) x 2x10-4 (decimal is moved to express the frequency in units of 10-6)

Vehicle control = 1% ethanol; MMS = Methyl methansulfonate positive control

# mutagenicity exceeds minimum criterion of 92.6 x 10-6

In Trial 3 of the non-activation assay (Table 3), ten treatments at 5.00, 7.50, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0 and 50.0 μg/ml were initiated and treatments above 25.0 μg/ml were terminated because of excessive cytotoxicity. However, there was a calculation error and the 25.0 μg/ml treatment was actually 20.0 μg/ml and is indicated as such in Table 3. The six analyzed treatments induced a wide range of toxic action (97.5% to 14.8% relative growths). The minimum criterion for a positive response in Trial 3 of the nonactivation assay was 247.1 x 10-6. None of the assayed treatments induced this level of mutant action and no dose related trend was observed. The test article was therefore considered non-mutagenic without activation in this assay. The background mutant frequency (average vehicle control mutant frequency) was 123.6 x 10-6, which is slightly above the usual range of 30 x 10-6 to 120 x 10-6. The range was meant as a guide and all other acceptance criteria were met for this assay. Higher backgrounds are occasionally observed when especially good recovery of mutants occur, therefore, in this cast. the small increase above the usual range was not considered indicative of an unacceptable assay.

TABLE 3: Mutation assay without activation (Trial 3, confirmatory assay)

   Daily cell counts (cells/ml, 105 units)   Suspension growth   Total mutant colonies  Total viable colonies  Cloning efficiency   Relative growth (%)   Mutant Frequencies (10-6 units) 
1 2
 Vehicle control  9.5 12.3 13.0 205 444 74.0 100.0 92.3
 Vehicle control  12.0 10.7 14.3 302 465 77.5 100.0 129.9
 Vehicle control  7.8 14.0 12.1 288 388 64.7 100.0 148.5
 Vehicle average      13.1   72.1  
 MMS 5 nl/ml  7.2 10.8 8.6 973 305 50.8 46.3 638.0#
 MMS 10 nl/ml  8.8 6.3 6.2 697 199 33.2 21.8 700.5#
 MMS 15 nl/ml  6.0 4.4 2.9 255 48 8.0 2.5 1062.5#
      relative to vehicle control (%)     relative to vehicle control (%)    
 Test article 5 µg/ml  6.7 13.8 78.4 235 446 103.1 80.3 105.4
 Test article 7.5 µg/ml  6.7 11.9 67.6 194 309 71.4 48.3 125.6
 Test article 10 µg/ml  6.9 8.3 48.6 240 453 104.7 50.9 106.0
 Test article 15 µg/ml  11.1 10.8 101.7 204** 394** 95.9 97.5 103.6
 Test article 20 µg/ml  1.6* 5.8 14.8 256 432 99.9 14.8 118.5
 Test article 20 µg/ml  3.0* 8.2 20.9 261 571 132.0 27.6 91.4

Suspension Growth = (Day 1 count / 3) x (Day 2 count) / (3 or Day 1 count if not split back)

Cloning Efficiency = total viable colony count / number of cells seeded x 100

Relative Growth = (Relative suspension growth x relative cloning efficiency) / 100

Mutant Frequency = (total mutant colonies/total viable colonies) x 2x10-4(decimal is moved to express the frequency in units of 10-6)

Vehicle control = 1% ethanol; MMS = Methyl methansulfonate positive control

# mutagenicity exceeds minimum criterion of 247.1 x 10-6

* not split back

** dilution error: actually plated 570 cells instead of 600 for total viable colonies and 2.85 x 106 instead of 3 x 106 for total mutant colonies.

Three trials of the activation assay were also initiated but Trial 2 was unacceptable because of excessively high vehicle control mutant frequencies. The results from Trials 1 and 3 are shown in Tables 4 and 5. In Trial 1 (Table 4), nine cultures were dosed with the test article at 5.00, 10.0, 15.0, 20.0, 30.0, 40.0, 50.0, 60.0 and 80.0 μg/ml but doses from 50.0 μg/ml to 80.0 μg/ml were terminated because of excessive cytotoxicity. The remaining six doses were non-cytotoxic to highly cytotoxic (89.4% to 20.1% relative growth). In order for a culture to be evaluated as mutagenic in this assay, a mutant frequency of greater than 102.4 x 10-6 was required. None of the analyzed doses induced a mutant frequency that exceeded the minimum criterion. The test article was considered non-mutagenic in this trial. Another trial was initiated to confirm these results.

TABLE 4: Mutation assay with activation (Trial 1)

   Daily cell counts (cells/ml, 105 units)   Suspension growth   Total mutant colonies  Total viable colonies  Cloning efficiency   Relative growth (%)   Mutant Frequencies (10-6 units) 
1 2
 Vehicle control  13.2 22.1 32.4 119 526 87.7 100.0 45.2
 Vehicle control  14.0 18.6 28.9 124 442 73.7 100.0 56.1
 Vehicle control  10.7 18.9 22.5 137 524 87.3 100.0 52.3
 Vehicle average      27.9   82.9    
 MCA 2 µg/ml  10.0 15.4 17.1 502 402 67.0 49.5 249.8#
 MCA 4 µg/ml 5.8 15.7 10.1 557 393 65.5 28.6 283.5#
      relative to vehicle control (%)     relative to vehicle control (%)    
 Test article 5 µg/ml  10.2 18.3 74.3 147 492 98.9 73.5 59.8
 Test article 10 µg/ml  10.0 24.6 98.0 82 435 87.5 85.8 37.7
 Test article 15 µg/ml  11.1 17.3 76.5 98 581 116.8 89.4 33.7
 Test article 20 µg/ml  5.5 18.4 40.3 155** 667** 134.1 54.0 46.5
 Test article 30 µg/ml  7.8 19.7 61.2 117 424 85.2 52.1 55.2
 Test article 40 µg/ml  3.5* 21.8 26.0 81 385 77.4 20.1 42.1

Suspension Growth = (Day 1 count / 3) x (Day 2 count) / (3 or Day 1 count if not split back)

Cloning Efficiency = total viable colony count / number of cells seeded x 100

Relative Growth = (Relative suspension growth x relative cloning efficiency) / 100

Mutant Frequency = (total mutant colonies/total viable colonies) x 2x10-4(decimal is moved to express the frequency in units of 10-6)

Vehicle control = 1% ethanol; MCA = Methylchloranthrene positive control

# mutagenicity exceeds minimum criterion of 102.4 x 10-6

* not split back

** dilution error: actually plated 1010 cells instead of 600 for total viable colonies and 5.05 x 106instead of 3 x 106for total mutant colonies.

In the third activation assay (Table 5), ten doses, at 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 50.0, 60.0 and 70.0 μg/ml were initiated and six doses from 15.0 μg/ml to 40.0 μg/ml were chosen for mutant analysis. However, there was a calculation error and the 25.0 μg/ml treatment was actually 20.0 μg/ml and is indicated as such in Table 5. Treatments above 40.0 μg/ml were excessively cytotoxic, and the 10.0 μg/ml treatment was terminated because a sufficient number of noncytotoxic doses were available for analysis. The selected doses induced a wide range of cytotoxicity (102.9% to 22.2% relative growth). In order for a dose to be considered positive in this assay, a mutant frequency exceeding 260.2 x 10-6 was required. None of the analyzed test article treatments induced a mutant frequency that exceeded the minimum criterion and no dose-response was observed. These results confirmed the lack of activity observed in Trial 1. The results of the two activation mutation trials were therefore evaluated as negative for inducing forward mutation at the TK locus in mouse lymphoma cells. As was observed with Trial 3 of the nonactivation assay, the background mutant frequency (130.1 x 10-6) was slightly above the usual range. Since the increase was small and other criteria for an acceptable assay were met, the trial was considered acceptable.

TABLE 5: Mutation assay with activation (Trial 3, confirmatory assay)

   Daily cell counts (cells/ml, 105 units)   Suspension growth   Total mutant colonies  Total viable colonies  Cloning efficiency   Relative growth (%)   Mutant Frequencies (10-6 units) 
1 2
 Vehicle control  8.9 16.4 16.2 329 418 69.7 100.0 157.4
 Vehicle control  9.9 16.5 18.2 261 407 67.8 100.0 128.3
 Vehicle control  10.5 14.3 16.7 250 478 79.7 100.0 104.6
 Vehicle average      17.0   72.4    
 MCA 2 µg/ml  2.0* 8.6 2.9 828 226 37.7 8.9 732.7#
 MCA 4 µg/ml  4.9 13.6 7.4 924 260 43.3 26.0 710.8#
      relative to vehicle control (%)     relative to vehicle control (%)    
 Test article 15 µg/ml  10.6 15.6 108.1 C 325 74.8 80.9 -----
 Test article 20 µg/ml  9.2 18.3 110.0 176 307 70.7 77.8 114.7
 Test article 20 µg/ml  11.3 15.4 113.7 264 ++ 393 90.5 102.9 134.4
 Test article 30 µg/ml  7.9 16.3 84.2 280 400 92.1 77.5 140.0
 Test article 35 µg/ml  5.8 14.5 55.0 323 427 98.3 54.1 151.3
 Test article 40 µg/ml  2.1* 13.1 25.7 294 375 86.3 22.2 156.8

Suspension Growth = (Day 1 count / 3) x (Day 2 count) / (3 or Day 1 count if not split back)

Cloning Efficiency = total viable colony count / number of cells seeded x 100

Relative Growth = (Relative suspension growth x relative cloning efficiency) / 100

Mutant Frequency = (total mutant colonies/total viable colonies) x 2x10-4(decimal is moved to express the frequency in units of 10-6)

Vehicle control = 1% ethanol; MCA = Methylchloranthrene positive control

# mutagenicity exceeds minimum criterion of 260.2 x 10-6

* not split back

C two or more plates contaminated

++ one plate contaminated: value determined by weight proportion

The average cloning efficiencies for the vehicle controls varied from 62.4% and 72.1% without activation to 82.9% and 72.4% with S9 metabolic activation which demonstrated acceptable cloning conditions for the assays. The positive control cultures, MMS (nonactivation) and MCA (activation) induced large increases in mutant frequency that were greatly in excess of the minimum criteria.

In conclusion, the test material was negative in the mouse lymphoma forward mutation assay both with and without S9 metabolic activation under the conditions of testing.

Applicant's summary and conclusion

Conclusions:
The test material was evaluated as negative in the mouse lymphoma forward mutation assay both with and without S9 metabolic activation over a wide range of cytotoxicity.
Executive summary:

The objective of this in vitro assay was to evaluate the ability of the test article to induce forward mutations at the thymidine kinase (TK) locus in the mouse lymphoma L5178Y cell line. The test article was soluble in ethanol at 100 mg/ml and soluble in medium up to 40 µg/ml. Higher concentrations of the test item in medium contained a white precipitate. In the preliminary dose range finding assay, cells were exposed to the test article for about four hours in the presence and absence of rat liver S9 metabolic activation. Treatment from 1.95 µg/ml to 1000 µg/ml were initiated. The test article was weakly to non-cytotoxic with and without metabolic activation from 1.95 µg/ml to 31.3 µg/ml and treatments from 62.5 µg/ml to 1000 µg/ml were very highly cytotoxic or lethal. The mutation assays were initiated with treatments based on the results of the cytotoxicity assay. Two non-activation mutation assays were used in the evaluation. In the first non-activation trial, the test article was lethal or excessively toxic above 15.0 µg/ml. The remaining three treatments from 5.00 µg/ml to 15.0 µg/ml induced weak cytotoxicity. None of the three analyzed treatments induced a mutant frequency that exceeded the minimum criterion for a positive response and a small increase in concentration from 15.0 µg/ml to 20.0 µg/ml was excessively cytotoxic. An independent repeat assay was performed to confirm these results. In the independent repeat nonactivation assay, six treatments from 5.00 µg/ml to 20.0 µg/ml were analyzed for mutant induction and a wide range of cytotoxicity was induced. Even in the presence of high cytotoxicity, there was no evidence for a mutagenic response. Two activation mutation trials were also used in the evaluation. In the first activation trial, six treatments from 5.00 µg/ml to 40.0 µg/ml were evaluated for mutant induction. In the confirmatory trial, six treatments from 15.0 µg/ml to 40.0 µg/ml were analyzed. A wide range of cytotoxicity was induced in both trials and no evidence for a mutagenic response was observed. The test material was evaluated as negative in the mouse lymphoma forward mutation assay both with and without S9 metabolic activation over a wide range of cytotoxicity.