Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not reported.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: A non-GLP study performed to sound scientific principles, with a sufficient level of reporting to assess the reliability of the submitted data.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1980
Report date:
1980

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
Five S. typhimurium strains were assessed for evidence of mutagenicity as a result of exposure to the test material in an Ames test. Strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were exposed to concentrations of the test material dissolved in DMSO ranging from 0.0001 to 10 µL/plate in both the presence and absence of metabolic activation. The mean number of revertant colonies together with the individual plate counts were recorded for evaluation. Solvent controls, positive controls, sterility and viability tests were run concurrently.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-phenoxyethyl isobutyrate
EC Number:
203-127-1
EC Name:
2-phenoxyethyl isobutyrate
Cas Number:
103-60-6
Molecular formula:
C12H16O3
IUPAC Name:
2-phenoxyethyl 2-methylpropanoate
Test material form:
other: liquid (undefined)
Details on test material:
- Name of test material (as cited in study report): phenoxyethyl isobutyrate (PEIB)
- Physical state: Clear liquid.

Method

Target gene:
Histidine operon.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
> Bacterial Growth Inhibition Test:
10, 1, 0.1, 0.01 and 0 µL/plate with all strains.
> Mutation Test:
10, 1, 0.1, 0.01 and 0 µL/plate with strains TA 1535, TA 1537, TA 1538 and TA 98.
> First Repeat Test:
0.1, 0.01, 0.001, 0.0001 and 0 µL/plate, with strain TA 100
0.001, 0.0001 and 0 µL/plate with strains TA 1535 TA 1537, TA 1538 and TA 98.
> Second Repeat Test:
0.05, 0.02, 0.01, 0.005 µL/plate with TA 100.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
sodium azide
other: 4-nitro-o-phenylene diamine, 2-amino-anthracence and neutral red.
Details on test system and experimental conditions:
CONTROLS
Sterility and viability controls were also performed.
Evaluation criteria:
Substantial increases in revertant colony numbers was the criteria used to determine whether or not the test material was mutagenic.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Bacterial Growth Inhibition and Mutation Test: The test material proved to be toxic to bacteria at the two highest concentrations, 10 and 1 µL/plate, resulting in either absence or incomplete formation of a bacterial lawn. According to these findings the repeat tests were performed at lower test concentrations. See Table 1 for growth inhibition results and Table 2 for the mutation test results.

First Repeat Test: In this repeat test a slight increase in revertant colony numbers was observed with tester strain TA 100, in the absence of activation at the 0.01 µL/plate dose level only. As this increase was slight and restricted to one dose level a second repeat test was performed over a narrower dose range. No substantial increase in the revertant colony numbers were observed for any of the remaining four strains, regardless of the dose level or the presence/absence of metabolic activation. Results can be seen in Table 3. Positive and sterility control results from concurrently run tests can be seen in Table 4. Viability results are in Table 5.

Second Repeat Test: The test material failed to produce any substantial increase in revertant colony numbers with tester strain TA 100. The results of the first repeat were slight and unrepeatable and therefore not considered to be significant. Results can be seen in Table 6. Concurrently run positive and sterility control results can be seen in Table 7. Viability results are in Table 8.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Bacterial Growth Inhibition Test

Strain Conc. of Test Material (µL/well) Zone of Inhibition on his- medium well (mm)
TA 1535 10 18
1 10
0.1 10
0.01 10
0 10
TA 1537 10 20
1 10
0.1 10
0.01 10
0 10
TA 1538 10 19
1 10
0.1 10
0.01 10
0 10
TA 98 10 17
1 10
0.1 10
0.01 10
0 10
TA 100 10 20
1 10
0.1 10
0.01 10
0 10

Table 2: Mutation Test

Strain Conc. of Test Material (µL/plate) Metabolic Activation Mean Revertant Colony Counts
TA 1535 10 N NL
1 N NL
0.1 N 13
0.01 N 16
0 N 16
10 Y IL
1 Y 15
0.1 Y 16
0.01 Y 14
0 Y 15
TA 1537 10 N NL
1 N NL
0.1 N 6
0.01 N 7
0 N 7
10 Y IL
1 Y 7
0.1 Y 7
0.01 Y 6
0 Y 7
TA 1538 10 N NL
1 N NL
0.1 N 16
0.01 N 18
0 N 15
10 Y IL
1 Y IL
0.1 Y 16
0.01 Y 16
0 Y 15
TA 98 10 N NL
1 N NL
0.1 N 34
0.01 N 35
0 N 34
10 Y IL
1 Y IL
0.1 Y 31
0.01 Y 40
0 Y

36

N = absence

Y = presence

NL = no bacterial lawn

IL = incomplete bacterial lawn

Table 3: Revertant Colony Counts Obtained per Plate, First Repeat Test

Strain Conc. Test Material (µL/plate) Metabolic Activation Mean Revertant Colony Counts Individual Revertant Colony Counts
TA 1535 0.001 N 17 17, 15, 18
0.0001 N 18 19, 17, 17
0 N 16 18, 14, 15
0.001 Y 10 11, 19, 9
0.0001 Y 9 9, 8, 11
0 Y 12 10, 11, 15 
TA 1537 0.001 N 9 5, C, 13
0.0001 N 8 9, 6, 9
0 N 8 10, 8, 6
0.001 Y 10 8, 11, 12
0.0001 Y 7 8, 7, 6
0 Y 10 12, 8, 10
TA 1538 0.001 N 14 17, 14, 12
0.0001 N 10 13, 8, 10
0 N 15 17, 18, 11
0.001 Y 14 15, 13, 13
0.0001 Y 12 12, 12, 13
0 Y 13 16, 10, 13
TA 98 0.001 N 39 31, 47, C
0.0001 N 39 40, 38, C
0 N 39 40, C, 37
0.001 Y 32 29, 35, 33
0.0001 Y 30 27, 32, 30
0 Y 31 30, 32, 31
TA 100 0.1 N IL IL
0.01 N 111 109, 97, 127
0.001 N 76 83, 80, 64
0.0001 N 85 97, 84, 74
0 N 86 86, 93, 80
0.1 Y 60 59, 64, 58
0.01 Y 62 59, 57, 71
0.001 Y 61 56, 67, 61
0.0001 Y 55 C, 57, 53
0 Y 68 64, 69, 70

N = absence

Y = presence

C = contaminated

IL = incomplete bacterial lawn

Table 4: Mutability and Sterility Tests (Positive Controls)

Strain Compound Conc Compound (µL/plate) Metabolic Activation Mean Revertant Colony Counts Individual Revertant Colony Counts
TA 1535 sodium azide 5 N 926 1016, 865, 897
TA 1537 4-nitro-o-phenylene diamine 500 N 136 158, 128, 122
TA 1538 N 1158 1132, 1044, 1298
TA 98 N 1600 1546, 1621, 1632
TA 100 sodium azide 5 N 644 785, 558, 589
TA 1535 2-amino-anthracene 2 Y 178 178, C, C
TA 1537 neutral red 10 Y 65 65, C, C
TA 1538 2-acetyl-amnofluorene 20 Y 183 C, C, 183
TA 98 2-amino-anthracene 2 Y C C, C, C
TA 100 2-amino-anthracene 2 Y C C, C, C
S-9 mix 500 µL   0 0
test material 0.1 µL N 0 0

N = absence

Y = presence

C = contaminated

Table 5: Viability Test

Dilution** Strain
TA 1535 TA 1537 TA 1538 TA 98 TA 100
10E-1 D 24 8 9 18 82
10E-6 R * * * * *
10E-7 R 245 205 217 228 205
10E-8 R 22 26 15 24 16

D = plated onto histidine deficient agar

R = plated onto histidine rich agar

* = too many colonies for accurate counting

** = 3 mL of each dilution per plate

Table 6: Revertant Colony Counts Obtained per Plate using strain TA 100, Second Repeat Test

Strain Conc. of Test Material (µL/plate) Metabolic Activation Mean Revertant Colony Counts Individual Revertant Colony Counts
TA 100 0.05 N 58 47, 76, 50
0.02 N 66 61, 72, 64
0.01 N 60 57, 55, 69
0.005 N 61 65, 56, 63
0 N 61 56, 68, 58

N = absence

Y = presence

Table 7: Mutability and Sterility Tests with Strain TA 100

Strain Compound Conc. of Compound (µL/plate) Metabolic Activation Mean Revertant Colony Counts Individual Revertant Colony Counts
TA 100 sodium azide 5 N 1673 1661, 1657, 1700
S-9 mix 500 µL   0 0
test material 50 µg/plate   0 0

N = absence

Table 8: Viability Test

Dilution** Strain TA 100
10E-1 D 88
10E-6 R *
10E-7 R 156
10E-8 R 26

D = plated onto histidine deficient agar

R = plated onto histidine rich agar

* = too many colonies for accurate counting

** = 3 mL of each dilution per plate

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of the study the test material gave a negative (i.e. non-mutagenic) response in Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 in the presence and absence of metabolic activation.
Executive summary:

The potential for the test material to cause gene mutation in S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100 was determined in an Ames test conducted in the presence and absence of metabolic activation. Strains were exposed to the test material at concentrations ranging from 0.0001 to 10 µL/plate dissolved in DMSO. The mean number of revertant colonies together with the individual plate counts were recorded for evaluation. Solvent controls, positive controls, sterility and viability tests were run concurrently.

During the bacterial growth inhibition and mutation tests (0, 0.01, 0.1, 0.1, 1.0 and 10.0 µL/plate), the test material proved to be toxic to bacteria at the two highest concentrations, 10 and 1 µL/well, resulting in either absence or incomplete formation of a bacterial lawn. According to these findings repeat tests were performed at lower test concentrations. The first repeat test was conducted at concentrations of 0, 0.0001, 0.001, 0.01 and 0.1 µL/plate for TA 100, for all other strains at concentrations of 0, 0.0001 and 0.001 µL/plate. A slight increase in revertant colony numbers was observed with tester strain TA 100, in the absence of metabolic activation at the 0.01 µL/plate dose level only. As this increase was slight and restricted to one dose level a second repeat test was performed over a narrower dose range. No substantial increase in the revertant colony numbers was observed for any of the remaining four strains, regardless of the dose level or the presence/absence of metabolic activation. The second repeat test was performed to verify the TA 100 results at concentrations of 0, 0.005, 0.1, 0.02 and 0.05 µL/plate. The test material failed to produce any substantial increase in revertant colony numbers. As the results of the first repeat were slight and unrepeatable the result was not considered to be significant. Concurrently run viability test and positive, vehicle and sterility control confirm the viability of the test system.

Under the conditions of the test, the test material was determined to be non-mutagenic giving a negative result.