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Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
( Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
Sponsor's identification: Carbohydrazide CAS 497-18-7
CAS number : 497-18-7
Identifier : NALCO TIS I0352
Description : White solid
Purity : 99.974%
Batch number : OJ24-21M
Date received : 4 April 2011
Expiry date : 24 January 2013
Storage conditions: Room temperature in the dark over silica gel

Method

Target gene:
Histidine for Salmonella.
Tryptophan for E.coli

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta­naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment one (Range-finding Test - Salmonella strains): 50, 150, 500, 1500 and 5000 µg/plate
Experiment one (Range-finding Test - E.coli strain): 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment two - Not performed as a clear positive result was obtained in the first experiment
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile distilled water.
- Justification for choice of solvent/vehicle: The test material was fully soluble in sterile distilled water at 50 mg/ml in solubility checks performed in-house and was therefore selected as the vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
(Spontaneous mutation rates of TA100, TA1535, WP2uvrA, TA98, TA1537)
Negative solvent / vehicle controls:
yes
Remarks:
(Sterile distilled water)
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
N-ethyl-N-nitro-N-nitrosoguanidine: Without S9 mix. 2 µg/plate for WP2uvrA; 3 µg/plate for TA 100; 5 µg/plate for TA 1535.
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
9-aminoacridine: Without S9 mix. 80 µg/plate for TA 1537.
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
4-nitroquinoline-1-oxide: Without S9 mix. 0.2 µg/plate for TA 98.
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
1 µg/plate for TA 100; 2 µg/plate for TA 1535 and TA 1537; 10 µg/plate for WP2uvrA
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
5 µg/plate for TA98
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) for Experiment 1.
- Exposure duration: Approximately 48 hours

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.




Evaluation criteria:
Acceptance Criteria:

The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks.
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
All tester strain cultures should be in the range of 0.9 to 9 x 10E9 bacteria per ml.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
There should be a minimum of four non-toxic dose levels.
There should be no evidence of excessive contamination.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS.
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgement about test item activity. Results of this type will be reported as equivocal.
Statistics:
Standard deviation
Statistical analysis of data as determined by UKEMS

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The test item caused a visible reduction in the growth of the bacterial background lawn at 5000 µg/plate to all strains in the absence of S9 but only in WP2uvrA in the presence of S9 mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
There were dose related and statistically significant increases in the frequency of TA 100 and TA 1535 revertant colonies at sub-toxic test item dose levels, in both the absence and presence of S9-mix.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The test item caused a visible reduction in the growth of the bacterial background lawn at 5000 µg/plate to all strains in the absence of S9 but only TA 1537 in the presence of S9 mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was fully soluble in sterile distilled water at 50 mg/ml in solubility checks performed in-house.
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:

The test item was toxic at 5000 µg/plate to the E.coli strain (WP2uvrA) of bacteria only. No toxicity was observed in the Salmonella strain, TA 100. The test item formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: None


Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

RANGE-FINDING/SCREENING STUDIES:

Preliminary Toxicity Test:

The test item was toxic at 5000 µg/plate to the E.coli strain (WP2uvrA) of bacteria only. No toxicity was observed in the Salmonella strain, TA 100. The test item formulation and S9-mix used in this experiment were both shown to be sterile. Large increases in revertant colony frequency were observed at 5000 µg/plate in TA 100 in the absence of S9-mix only.

The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

136

113

102

113

119

115

95

140

219

290

433

+

TA100

152

120

122

117

106

137

85

109

132

147

136

-

WP2uvrA

31

34

29

35

27

26

40

33

41

42

22 T

+

WP2uvrA

37

49

46

34

57

40

51

33

55

55

40T

 

T Partial absence of bacterial background lawn

Mutation Test

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 (see below) and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, reference item and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for the Main experiment. The results are also expressed graphically in Figures 1 and 2. (see attached background material for Tables and Figures).

A history profile of untreated/vehicle and positive control (reference items) is presented in attached background material (historical profiles).

The test item caused a visible reduction in the growth of the bacterial background lawn at 5000 µg/plate to all of the strains in the absence of S9 but only in WP2uvrA and TA1537 in the presence of S9-mix. A substantial reduction in revertant colony frequncy was also observed at 5000 µg/plate in TA98 (with S9 mix only). Therefore the test item was tested up to the maximum recommended dose level of 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were dose related and statistically significant increases in the frequency of TA100 and TA1535 revertant colonies at sub-toxic test item dose levels, in both the absence and presence of S9-mix. The increases observed for TA1535 were particularly large and in excess of the in-house historical control ranges with increases greater than 12 fold over the concurrent vehicle control. Therefore, the test item was considered to be mutagenic under the conditions of the test. The OECD 471 test guideline and the study plan permits

non-repetition of the experiment when a clear, positive response is obtained in the first experiment, therefore a second confirmatory experiment was not required.

All of the reference items (positive control chemicals) used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table 1 Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1 (Range-finding test)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA 100

TA 1535

WP2uvrA

TA98

TA1537

103

13

19

10

7

120 (101)

12 (15)

28 (24)

19 (16)

6 (6)

79

19

24

20

4

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive

There were dose related and statistically significant increases in the frequency of TA100 and TA1535 revertant colonies at sub-toxic test item dose levels, in both the absence and presence of S9-mix. The increases observed for TA1535 were particularly large and in excess of the in-house historical control ranges with increases greater than 12 fold over the concurrent vehicle control. Therefore, the test item was considered to be mutagenic under the conditions of the
test. The OECD 471 test guideline and the study plan permits non-repetition of the experiment when a clear, positive response is obtained in the first experiment, therefore a second confirmatory experiment was not required.

The test item, Carbohydrazide was considered to be mutagenic under the conditions of this test.
Executive summary:

Introduction.

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with Carbohydrazide using the Ames plate incorporation method at up to six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors) The dose range for Experiment 1 (range-finding test) was determined in a preliminary toxicity assay and ranged between 15 and 5000 µg/plate, depending on strain type. The experiment was not repeated on a separate day due to the results of the main experiment.

An additional dose level (15 µg/plate) was included and an expanded dose range was selected in order to achieve four non-toxic dose levels and the toxic limit of the test item.

Results

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic

activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item caused a visible reduction in the growth of the bacterial background lawn at 5000 µg/plate to all of the strains in the absence of S9 but only in WP2uvrA and TA1537 in the presence of S9-mix. A substantial reduction in revertant colony

frequncy was also observed at 5000 µg/plate in TA98 (with S9 mix only). Therefore the test item was tested up to the maximum recommended dose level of 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either

the presence or absence of S9-mix.

There were dose related and statistically significant increases in the frequency of TA100 and TA1535 revertant colonies at sub-toxic test item dose levels, in both the absence and presence of S9-mix. The increases observed for TA1535 were particularly

large and in excess of the in-house historical control ranges with increases greater than 12 fold over the concurrent vehicle control. Therefore, the test item was considered to be mutagenic under the conditions of the test. The OECD 471 test guideline and

the study plan permits non-repetition of the experiment when a clear, positive response is obtained in the first experiment, therefore a second confirmatory experiment was not required.

Conclusion

The test item, Carbohydrazide was considered to be mutagenic under the conditions of this test.