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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October-November 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1977
Qualifier:
according to guideline
Guideline:
other: Standard Operation Procedure of Level Biotechnolocy Inc. (SOP: MP007-04)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Dipentaerythritol hexaesters of 3,5,5-trimethylhexanoic and n-heptanoic acids
EC Number:
945-883-1
Cas Number:
1379424-11-9
IUPAC Name:
Dipentaerythritol hexaesters of 3,5,5-trimethylhexanoic and n-heptanoic acids
Test material form:
liquid
Details on test material:
Colour: Gardner: 0.1

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Due to migration, the value was transferred to one of the current document's attachments
Test concentrations with justification for top dose:
The test article will be dissolved to the stock concentration of 100 mg/mL by ethanol. 5mg/plate of the test item will be used as the highest dose in "dose range finding test". In the groups with metabolic activation, 0.5 mL of metabolic mixture will be used instead of 0.2M phosphate buffer stated in 6.4B of the study report.
According to the result of the dose range finding test, 5 mg/plate was chosen as the highest dose and the other four doses (2.5, 1.25, 0.625, 0.313 mg/plate) were then determined for five Salmonella typhimurium strains.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The vehicle should not be suspended of chemical reaction with the test article and should be compatible with the survival of the bacteria and the S9 activity.
Controls
Negative solvent / vehicle controls:
yes
Remarks:
water/ethanol
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-aminoanthracene, SA
Details on test system and experimental conditions:
BACTERIAL SYSTEM:
- Salmonella typhimurium strain TA98, TA100, TA102, TA1535, TA1537
- Bacterial source: Moltox Inc., USA
- The genotype confirmation of bacterial strains was performed according to SOP MP007-04 before testing.

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : triplicate (all groups including positive, negative, vehicle and five dosage testing groups)
- Number of independent experiments : 1

METHOD OF TREATMENT/ EXPOSURE:

A. The doses used were:
without S9 mix:
TA 98: negative control (water), vehicle control (ethanol), test article 5, 2.5, 1.25, 0.625, 0.313 mg/plate, positive control (2-nitrofluorene, 1µg/plate)
TA100: negative control (water), vehicle control (ethanol), test article 5, 2.5, 1.25, 0.625, 0.313 mg/plate, positive control (sodium azide, 1µg/plate)
TA102: negative control (water), vehicle control (ethanol), test article 5, 2.5, 1.25, 0.625, 0.313 mg/plate, positive control (mitomycin C, 0.2 µg/plate)
TA1535: negative control (water), vehicle control (ethanol), test article 5, 2.5, 1.25, 0.625, 0.313 mg/plate, positive control (sodium azide, 1µg/plate)
TA1537: negative control (water), vehicle control (ethanol), test article 5, 2.5, 1.25, 0.625, 0.313 mg/plate, positive control (9-aminoacridine, 50 µg/plate)

without S9 mix:
TA 98: negative control (water), vehicle control (ethanol), test article 5, 2.5, 1.25, 0.625, 0.313 mg/plate, positive control (2-nitrofluorene, 1µg/plate)
TA100: negative control (water), vehicle control (ethanol), test article 5, 2.5, 1.25, 0.625, 0.313 mg/plate, positive control (benzo(a)pyrene, 1µg/plate)
TA102: negative control (water), vehicle control (ethanol), test article 5, 2.5, 1.25, 0.625, 0.313 mg/plate, positive control (2-aminoanthracene, 5 µg/plate)
TA1535: negative control (water), vehicle control (ethanol), test article 5, 2.5, 1.25, 0.625, 0.313 mg/plate, positive control (2-aminoanthracene, 5 µg/plate)
TA1537: negative control (water), vehicle control (ethanol), test article 5, 2.5, 1.25, 0.625, 0.313 mg/plate, positive control (2-aminoanthracene, 5 µg/plate)

B. Plate incorporaton method was employed and performed as following:
(a) the components were added sequentially according to SOP MP007-04: (1) 0.5 mL of 0.2M Phosphate buffer, pH=7.4 (without S9 metabolic activation), (2) 0.05 mL of each testing concentration of test article, negative, vehicle, or positive control solution, 83) 0.1 mL of overnight culture of the Salmonella typhimurium strains (containing aproximately 1-2x10E9 cells/mL), (4) 2 mL of molten top agar (with 0.5 mM histidine/biotin)
(b) the contents of each tube were mixed and poured onto the surface of minimal glucose agar plates.
(c) when the top agar has been solidified, the plates were inverted and placed in a 37+-1ºC incubator for 48-72 hours. The colonies were then counted.

In the groups with metabolic activation, 0.5 ml of S9 metabolic mixture was used instead of 0.5 mL of 0.2M Phosphate buffer stated in B(a)

Evaluation criteria:
1. The raw data of revertant colony values were represented with Mean+-SD.
2. Cell toxicity determination: (A) A cytotoxic effect was concluded when a decrease in revertant colonies over the negative/vehicle control was lower than 0.5-fold, loss of bacterial lawn, or pin colony appeared. (B) Plates would be labeled and excluded from statistics while cytotoxic effect occured.
3. An increase in revertants over the negative control would be as the cut-off between a mutagenic and non-mutagenic response: (A) TA98, TA100 and TA102: more than two-fold increase in revertants over the negative/vehicle control, then the test article would be considered as a potential mutagen. (B) TA1535 and TA1537: more than three-fold increase in revertants over the negative/vehicle control, then the test article would be considered as a potential mutagen.
4. If the test aritlce was considered as a potential mutagen, the raw data would be further analyzed by ANOVA to evaluate the difference between negative/vehicle control group and the test article groups, and p<0.05 indicates the significant difference.
5. If the data showed statistically significant, then to evaluate the dose-related response in the numbers of revertant colonies on the test article group as compared with the vehicle control group. Once dose-related response was confirmed, the test articles would be considered as a mutagen.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Positive controls validity:
valid

Any other information on results incl. tables

Table 1. Result of genotyping of Salmonella typhimurium strains.

 

Salmonella typhimurium strains

   Histidine requirement  UV sensitivity  Crystal Violet sensitivity  Ampicilin resistance  Tetracycline resistance  Spontaneous revertants
 

 His+

Bio+

Plate

His-

Bio+

Plate 

 UV irradiated  Zone of growth inhibition*  Ampicilin plate  Ampicilin tetracycline pate  without S9
 TA98  +  -  -  +  +  -  32.7 +-3.8
 TA100  +  -  -  +  +  -  158.0 +-4.6
TA102   +  -  +  +  +  463.3 +-15.5
 TA1535  +  -  -  +  -  -  15.3 +-5.1
 TA1537  +  -  -  +  -  -  12.7 +-2.1

* remark: +: a clear zone of inhibition appeared around the disc

Table 2. Result of the dose range finding test (in TA100).

 dose (mg/plate)      number of revertants / plate (mean+-S.D., n=3)
   without S9 mixture
 negative control (sterile water)  151.3 +-5.7
positive control (sodium azide, 1µ/plate)  478.0 +-10.6*
vehicle control (ethanol)  161.3 +-4.5
 test article  5  156.7 +-3.1
  test article  2.5  152.3 +-3.1
  test article  1.25  157.7 +-14.0
  test article  0.625  172.3 +-8.4
  test article  0.313  157.0 +-5.0

* remark: more than two-fold increase in revertants over the negative control

Table 3. Result of Bacterial Reverse Mutation Test (5 strains)

    group (mg/plate)              Number of revertants / plate (without S9 activator, Mean +- S.D., n=3)
    TA98  TA100  TA102  TA1535  TA1537
    negative control  34.3 +-4.0  153.0 +-13.0  294.7 +-3.1  12.0 +-2.6  11.0 +-2.0
    positive control  246.0 +-14.4  486.7 +-31.9  2832.0 +-57.7  498.0 +-31.0  192.3 +-64.5
    vehicle control  31.3 +-9.0  138.0 +-13.2  263.3 +-20.0  11.7 +-1.5  12.3 +-4.0
 test article  5  28.7 +-1.5  128.7 +-12.7  276.0 +-12.2  10.0 +-1.0  16.0 +-2.6
   test article  2.5  37.7 +-2.5  135.3 +-6.5  277.3 +-20.0  11.0 +-2.0   18.3 +-4.5
   test article  1.25  37.7 +-2.1  126.7 +-17.0  302.7 +-17.5  10.7 +-3.1  12.7 +-2.5
   test article  0.625  40.7 +-7.2  124.3 +-2.5  298.0 +-28.2  9.0 +-1.0  13.3 +-4.2
   test article  0.313  43.0 +-4.0  153.7 +-12.9  312.7 +-10.1  9.7 +-2.3  14.7 +-1.5
    group (mg/plate)               Number of revertants / plate (with S9 activator, Mean +- S.D., n=3)
    negative control  38.0 +-2.6  111.3 +-6.0  323.0 +-43.8a  13.0 +-1.0  12.0 +-2.6
    positive control  806.0 +-24.6*  291.3 +-33.0*  670.7 +-7.0*  213.0 +-67.8*  337.3 +-36.9*
    vehicle control  41.7 +-1.2  129.7 +-2.1  283.3 +-34.9  16.0 +-1.0  8.7 +-2.9
  test article  5  42.7 +-5.0  130.0 +-13.7  296.0 +-19.1  11.3 +-1.5  12.3 +-4.2
  test article  2.5  39.3 +-10.3  110.0 +-5.6  283.0 +-15.6a  13.7 +-2.5  13.0 +-1.0
  test article  1.25  35.7 +-7.6

113.7 +-13.6 

288.7 +-37.2  13.0 +-1.0   10.3 +-2.5
  test article  0.625  34.0 +-9.5  121.0 +-19.2  273.3 +-20.1  11.3 +-1.2  13.0 +-3.5
  test article  0.313  41.0 +-8.2 120.3 +-5.8  301.3 +-14.2   9.7 +-1.5  12.0 +-3.5

Remark:

1. The use of positive control substance for each strain is listed above

2. *: more than two or three-fold increase in revertants over the negative control

3. a: n=2

Applicant's summary and conclusion

Conclusions:
According to the numbers of revertant colonies at all the tesing conditions of the five strains used in this study (TA98, TA100, TA102, TA1535, TA1537), the test article did not present genotoxic effect.
Executive summary:

"Bacterial Reverse Mutation Test" was used in this study to evaluate the genotoxicity of the substance in accordance with OECD Guideline for the testing of chemicals #471 (1997): Bacterial reverse mutation test, and the operation was executed according to the standard operation procedure of Level Biotechnology Inc. (SOP: MP007 -04).

Salmonella typhimurium TA100 was chosen and 5 mg/plate of the test article was used as highest dose in "dose range finding test" to determine the testing dose for "Bacterial Reverse Mutation Test". The result of the "dose range finding test" indicated that the test article showed non-cytotoxic and non-mutagenic effects in Salmonella typhimurium TA100.

According to the result of the "dose range finding test", 5 mg/plate was chosen as the highest dose and the other four doses (2.5, 1.25, 0.625, 0.313 mg/plate) were then determined for five Salmonella typhimurium strains, "Bacterial Reverse Mutation Test". Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 were used, and plate incorporation method in the presence and absence of S9 metabolic mixture were applied in this test.

Based on the results of this test, no significant increase in the number of revertant colonies was observed. The test article did not present genotoxic effect at al concentrations of testing strains in the condition of both presence and absence of S9 metabolic mixture. In conclusion, the test article was non-genotoxic in the testing system applied in this study.