Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From September 29th to October 26th, 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The test was conducted by means of Read Across approach. The reliability of the source study report is 1. Further information was attached at section 13

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted May 26, 1983
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
Salmonella typhimurium histidine (his) reversion system.
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell lines (if applicable):
- Supplier: bacterial strains were obtained from Dr. Heinz Träger, Knoll AG, D-6700 Ludwigshafen, F.R.G.
- Storage: the strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.
- Precultures: from the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. This nutrient medium contains per litre: 8 g Merck Nutrient Broth, 5 g NaCl.
- Incubation: the bacterial culture was incubated in a shaking water bath for 10 hours at 37° C.
- Regular checking: regular checking of the properties of the strains with regard to membrane permeability and ampicillin resistance as well as normal spontaneous mutation rates is performed in testing laboratory.
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix (Rat liver)
Test concentrations with justification for top dose:
Pre-experiment for toxicity: 10.0, 33.3, 100.0, 333.3, 1000.0, 2500.0 and 5000.0 µg/ml
Main study (two independent experiments): 33.3, 100.0, 333.3, 1000.0, 2500.0 and 5000.0 µg/ml
Vehicle:
- Vehicle test substance was dissolved in aqua bidest.
- Justification for choice of vehicle: the solvent was chosen because of its solubility properties.
Controls
Negative controls:
yes
Remarks:
concurrent untreated
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine // 2-aminoanthracene
Details on test system and conditions:
METHOD OF APPLICATION
EXPERIMENT I: PLATE INCORPORATION TEST
The following materials were mixed in a test tube and poured onto the selective agar plates: 100 µl test solution at each dose level, solvent control, negative control, or reference mutagen solution (positive control); 500 µl S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation); 100 µl bacteria suspension (cf. test system, pre-culture of the strains); 2000 µl overlay agar.

EXPERIMENT II: PRE-INCUBATION TEST
In the pre-incubation assay 100 µl test solution, 500 µl S9 mix /S9 mix substitution buffer and 100 µl bacteria suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2.0 ml overlay agar was added to each tube. The mixture was poured on minimal agar plates.

After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

NUMBER OF REPLICATIONS: each concentration, including the controls, was tested in triplicate.

DATA RECORDING
The colonies were counted using the AUTOCOUNT. The counter was connected to a compatible PC with printer which printed out the individual values and the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. If precipitation of the test article precluded automatic counting the revertant colonies were counted by hand.

PRE-EXPERIMENT FOR TOXICITY
- Strains: to evaluate the toxicity of the test article a prestudy was performed with strains TA 98 and TA 100.
- Concentrations: 10.0, 33.3, 100.0, 333.3, 1000.0, 2500.0 and 5000.0 µg/ml
- Replicates: 3 plates per concentration.
- Experimental conditions: the same as described for the experiment I (plate incorporation test).
- Evaluation: toxicity of the test article may be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.

MAIN TEST COCNENTRATIONS
- Concentrations: the maximum concentration was 5000.0 µg/plate; the concentration range included two logarithmic decades.
- Spacing factor: six adequately spaced concentrations were tested.

MAMMALIAN MICROSOMAL FRACTION S9 MIX
- S9 Preparation: the S9 liver microsomal fraction was obtained from the liver of 8-12 weeks old male Wistar rats, strain WU (weight approx. 150 - 200 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 in olive oil 5 days previously. After cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate, diluted 1+3 in KCl was centrifuged cold at 9000 g for 10 minutes.
- Storage of stock solution: stock of the supernatant containing the microsomes was frozen in ampoules of 2, 3 or 5 ml and stored at -70 °C. Small numbers of the ampoules are kept at -20 °C for only several weeks before use. The protein concentration in the S9 preparation was 33.6 mg/ml.
- S9 Mix: before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution in a ratio 1.5:8.5. The composition of the cofactor solution was concentrated to yield the following concentrations in the S9 mix: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 5 mM NADP, in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
- Storage: during the experiment the S9 mix was stored in an ice bath.
- Preparation: the S9 mix preparation was performed according to Ames et al.

ACCEPTANCE CRITERIA
The generally accepted conditions for the evaluation of the results are:
- Corresponding background growth on both negative control and test plates
- Normal range of spontaneous reversion rates.
Range of spontaneous reversion frequencies (the following values refer to the negative control without metabolic activation (+) The range of strain TA 102 was determined from testing laboratory historical control datas)
TA 1535: 3 - 37
TA 1537: 4 - 31
TA 1538: 12 - 37
TA 98: 15 - 60
TA 100: 75 - 200
TA 102 (+): 120 - 300
Evaluation criteria:
A test article is considered as positive if either a dose related and reproducible increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a dose related and reproducible increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

A significant response is described as follows: a test article is considered as mutagenic if in strain TA 100 and TA 102 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate; also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.
Statistics:
No evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at the test performance time.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
No toxic effects occurred in the test groups with and without metabolic activation.
The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used.
No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with test item at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of significance.

A slight increase in revertant colony numbers (factor 1.5) was observed in strain TA 100 with metabolic activation at the highest investigated dose in experiment II (pre-incubation test). This effect is considered not to be biologically relevant. It is due to the relatively low level of the corresponding solvent control. Additionally the factor of 2.0 which is recommended for a mutagenic response in strain TA 100 could not be reached.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced revertant colonies.

PRE-EXPERIMENT FOR TOXICITY
The pre-study was performed with strains TA 98 and TA 100.
The plates with the test article showed normal background growth up to 5000.0 µg/plate in strain TA 98 and TA 100, respectively.

Any other information on results incl. tables

SUMMARY OF RESULTS

Revertants/plate mean from three plates, without S9 mix

Dose µg/plate TA 1535 TA 1537 TA 98 TA 100 TA 102
I II I II I II I II I II
Neg. contr. 11 24 8 12 17 33 91 74 144 206
Solv. contr. 12 25 6 12 26 28 90 77 177 208
33.3 8 29 9 13 15 28 100 68 190 208
100.0 9 30 8 13 15 29 98 72 188 175
333.3 9 23 9 11 20 25 82 72 152 201
1000.0 9 29 8 13 15 28 89 79 202 225
2500.0 9 28 8 14 26 29 85 76 167 207
5000.0 10 27 8 12 19 29 108 81 184 199
Positive controls

Sodium azide (10 µg/plate)

856

655

1018

1105

4-Nitro-o-phenylene-diamine (50 µg/plate)

647

302

1972

2280

1116

1233

Revertants/plate mean from three plates, with S9 mix

Dose µg/plate TA 1535 TA 1537 TA 98 TA 100 TA 102
I II I II I II I II I II
Neg. contr. 12 22 11 19 21 46 98 95 225 279
Solv. contr. 13 25 8 24 22 49 104 85 296 310
33.3 10 23 9 23 29 49 93 75 278 263
100.0 14 22 9 24 22 47 112 90 280 273
333.3 11 26 9 21 19 49 115 96 265 315
1000.0 14 24 8 22 21 45 113 100 332 366
2500.0 12 23 9 23 19 47 116 112 328 331
5000.0 10 27 9 22 24 44 135 124 320 381
Positive control
2-Aminoanthracene (2.5 µg/plate) 416 422 355 351 937 668 898 789 1515 966

PRE-EXPERIMENT FOR TOXICITY

Test groups

Concentration per plate µg

Revertants per plate

-

+

-

+

Negative control

-

17

21

91

98

Solvent control

-

26

22

90

104

4-NOPD

50

1972

/

/

/

Sodium azide

10

/

/

1018

/

2-aminoanthracene

2.5

/

937

/

898

Test article

3.3

14

25

93

107

10

22

18

88

124

33.3

15

29

100

93

100

15

22

98 112
333.3 20 19 82 115
1000 15 21 89 113
2500 26 19 85 116
5000 19 24 108 135

* -: without S9 mix; +: with S9 mix

/ not performed

Applicant's summary and conclusion

Conclusions:
Not mutagenic
Executive summary:

Method

The study was performed to investigate the potential of test material to induce gene mutations, according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 TA 100 and. TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 33.3, 100.0, 333.3, 1000.0, 2500.0 and 5000.0 µg/plate.

Results

No toxic effects occurred in the test groups with and without metabolic activation.

The plates incubated with the test article showed normal background growth up to 5000.0 ug/plate with and without S9 mix in all strains used.

No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with test item at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of significance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.