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Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 January 2020 to 04 June 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
JMAFF 2-1-19-1, November 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-amino-5-chloro-N,3-dimethylbenzamide
Cas Number:
890707-28-5
Molecular formula:
C9H11ClN2O
IUPAC Name:
2-amino-5-chloro-N,3-dimethylbenzamide
Test material form:
solid
Specific details on test material used for the study:
Name: 2-Amino-5-Chloro-N,3-Dimethylbenzamide
Batch/Lot Number: 628-042-00
Test item content: 99.0%
Physical State: Beige Solid
Date of certificate of analysis: 09th March 2020
Date of Expiry: 17 October 2021
Storage Conditions:
Storage Temperature: Room temperature (15 to 30º C)
Storage Condition: Cool and dry conditions
Storage Container: Kept in original container as supplied by the Sponsor
Storage Location: Test Item Control Office, JRF

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
The S9 fraction is buffered and supplemented with the essential co-factors β-NADP and glucose-6-phosphate to form the “S9 mix”. This mix is added to the top agar in this activated assay. The S9 fractions procured from Molecular Toxicology Inc., U.S.A. (MOLTOX) (Lot N ° 4149)
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : The S9 fractions procured from Molecular Toxicology Inc., U.S.A. (MOLTOX) (Lot N ° 4149). Species: Rat, Sex: Male, Tissue: Liver
- method of preparation of S9 mix : The S9 fraction is buffered and supplemented with the essential co-factors β-NADP and glucose-6-phosphate to form the “S9 mix”. This mix is added to the top agar in this activated assay.
- concentration or volume of S9 mix and S9 in the final culture medium: A volume of 0.1 mL of S9 mix (5% v/v S9 mix for initial toxicity-mutation test and 10% v/v S9 mix for confirmatory mutation test) prepared for treatment was added aseptically to 2 mL of top agar, mixed thoroughly and this mixture was poured onto an MGA plate.
Test concentrations with justification for top dose:
- initial toxicity-mutation test, in the absence and presence of the metabolic activation (5% v/v S9 mix):
eight concentration levels (three plates/concentration): 1.5, 5.0, 15, 50, 1500, 5000 µg/plate
- confirmatory mutation test, in the absence and presence of the metabolic activation (10% v/v S9 mix):
six concentration levels (three plates/concentration): 156.25, 312.5, 625, 1250, 2500, 5000 µg/plate
Vehicle / solvent:
- solvent used: DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble in distilled water, (Stock A, 50000 µg/mL) while found to be soluble in dimethyl sulfoxide (Stock B, 50000 µg/mL). Hence, dimethyl sulfoxide (Stock B, 50000 µg/mL) was selected as the vehicle for treatment.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulfoxid (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-Aminoanthracene
Details on test system and experimental conditions:
Number of replications:
- Initial toxicity-mutation test: eight concentration levels (three plates/concentration) from 1.5 to 5000 µg/plate for the strains (TA1537, TA1535, TA98, TA100, and TA102), in the absence and presence of the metabolic activation (5% v/v S9 mix).
- Confirmatory mutation test: six concentration levels (three plates/concentration) from 156.25 to 5000 µg/plate for TA1537, TA1535, TA98, TA100, and TA102, in the absence and presence (10% v/v S9 mix) of the metabolic activation.
After 48 hours of incubation at 37 ± 1°C, revertant colonies were enumerated.

Solubility and Precipitation Test:
The test item was insoluble in distilled water, (Stock A, 50000 µg/mL) while found to be soluble in dimethyl sulfoxide (Stock B, 50000 µg/mL). Hence, dimethyl sulfoxide (Stock B, 50000 µg/mL) was selected as the vehicle for treatment. A volume of 100 µL from stock B was added to 2 mL of top agar and poured on a Minimal Glucose agar plate to assess the precipitation. Precipitation was not observed at the tested concentration of 5000 µg/plate. Hence, 5000 µg/plate was selected as the highest concentration to be tested for the initial toxicity-mutation test both in the absence and presence (5% v/v S9 mix) of metabolic activation.

Cell Viability Test:
The cell viability of the tester strains was determined prior to treatment. The optical density of the cultures was found to be in the acceptable range.

Genotype Confirmation Test:
The genotype of the tester strain was confirmed for all the strains regularly (once a month). The tester strains of Salmonella typhimurium were tested for histidine dependence, biotin dependence, histidine and biotin dependence, rfa mutation, uvrB mutation through sensitivity to ultraviolet light and the R-factor resistance for ampicillin and tetracycline.

Sterility Check for the Operating System:
A sterility check for the operating system was performed along with a mutagenicity test for the following:

Top Agar: Top agar prepared and used for the study (2 mL) was poured aseptically onto an MGA plate.

Solvent: A volume of 0.1 mL of dimethyl sulfoxide (DMSO), used as a vehicle was added aseptically to 2 mL of top agar, mixed well and this mixture was poured onto an MGA plate.

Test Item: A volume of 0.1 mL of the test item stock solution of the highest concentration was added aseptically to 2 mL of top agar, mixed well and this mixture was poured onto an MGA plate.

ONB Solution: A volume of 0.1 mL of the ONB solution was added aseptically to 2 mL of top agar, mixed well and the mixture was poured on to an MGA plate.

0.2 M Sodium Phosphate Buffer: A volume of 0.1 mL of 0.2 M of sodium phosphate buffer was added aseptically to 2 mL of top agar, mixed well and the mixture was poured on to an MGA plate.

Tester Strain Integrity:
All Salmonella typhimurium tester strain cultures exhibited sensitivity to crystal violet, demonstrating presence of the rfa wall mutation.
All the tester strains demonstrated the requirement of the biotin except strain TA102 which is biotin independent. All the tester strains showed sensitivity to UV exposure except TA102 which is wild type.
All the tester strains demonstrated requirement of histidine for their growth. Tester strains TA98, TA100 and TA102 exhibited resistance to ampicillin, demonstrating presence of the pKM101 plasmid. Tester strains TA102 exhibited resistance to tetracycline, demonstrating the presence of pAQ1 plasmid.

Characteristic Number of Spontaneous Revertants:
All negative control cultures exhibited their characteristic number of spontaneous revertants per plate, demonstrating the requirement for histidine (Salmonella typhimurium).

Tester Strain Culture Density:
Cell densities (OD at 660 nm) of all tester strains were within the required range to produce cultures with approximately 1–2 × 109 bacterial cells/mL, demonstrating that appropriate numbers of bacteria were plated.

Positive Control Values in the Absence of Metabolic activation System (-S9):
The mean value of the positive control for each tester strain exhibited clear increase over the mean value of the negative control for that strain, demonstrating that tester strains were capable of identifying a mutagen.

Positive Control Values in the Presence of Metabolic activation System (+S9):
The mean value of the positive control for each tester strain exhibited clear increase over the mean value of the negative control for that strain, demonstrating that the S9 mix was capable of metabolising a pro-mutagen to its mutagenic form(s). An acceptable positive control in the presence of S9 for a specific strain was evaluated as having demonstrated integrity of the S9 mix and the ability of the tester strain to detect a mutagen.
Evaluation criteria:
Cytotoxicity Evaluation Criteria
Six analysable doses were available to evaluate the assay data. Cytotoxicity was detected by a decrease in the number of revertant colonies per plate and/or by a thinning of the bacterial background lawn (Teo et al., 2003).

Assay Evaluation Criteria
A result was considered positive if concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
Biological relevance of the result was considered first.
- Strains TA1535, TA1537
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0 times the mean negative control value.
- Strains TA98, TA100 and TA102
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0 times the mean negative control value.
Statistics:
Statistical analysis was used as an aid in the evaluation of dose response.
A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) was not evaluated as positive.
Negative results obtained in the initial toxicity-mutation test were confirmed by a second experiment, using the same method as specified above, with an alteration in concentration spacing.
- Statistical Analysis of the Results: Simple linear regression analysis was performed for TA1537, TA1535, TA98, TA100 and TA102, separately, to assess the dose dependent nature of revertant colonies.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Ames test:
- Signs of toxicity : No
- Individual plate counts : yes
- Mean number of revertant colonies per plate and standard deviation : yes

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: yes
- Negative (solvent/vehicle) historical control data: yes

Any other information on results incl. tables

Negative Control

Results of this study indicate that values of the negative control in all tester strains were within the historical ranges of respective strains.

 

Mean Count of His+ Revertant Colonies in Negative Control, Positive Controlsand Treatment Plates in the Absence of Metabolic Activation (Initial Toxicity-Mutation Test:

Concentration of

2-Amino-5-chloro-N,3-dimethylbenzamide

 (µg/plate)

His+Revertant Colonies/Plate (Absence of Metabolic Activation)

TA1537

TA1535

TA98

TA100

TA102

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

NC (DMSO)

9.00

3.61

16.33

1.53

25.00

1.73

132.00

6.08

229.67

5.03

1.5

8.33

1.53

16.00

2.00

24.00

1.00

123.00

5.57

225.33

11.72

5

7.00

3.00

16.00

2.00

25.00

4.58

130.00

15.13

226.67

7.09

15

8.00

3.46

16.00

3.61

24.00

2.00

125.33

3.21

232.67

17.01

50

5.67

2.08

14.33

3.79

24.33

3.21

119.00

7.00

222.00

7.55

150

7.33

3.21

15.67

5.03

23.67

1.15

124.67

5.13

225.67

10.50

500

8.00

1.00

14.33

4.04

25.67

2.08

134.00

4.58

223.33

7.02

1500

9.00

3.00

14.33

3.51

25.00

3.00

115.67

13.32

225.00

5.00

5000

7.67

2.08

15.00

1.73

25.00

2.00

128.00

11.79

225.00

6.00

PC

245.33

75.30

277.00

32.51

500.00

51.03

735.33

28.11

1028.33

135.55

2Aa

-

-

-

-

-

-

122.67

6.66

-

-

Key: SD = Standard Deviation, NC = Negative Control, DMSO =Dimethyl sulfoxide, PC = Positive Control {TA1537 = 9-Aminoacridine Hydrochloride Monohydrate (75 µg/plate), TA1535 = Sodium Azide (0.5 µg/plate), TA98 = 2-Nitrofluorene (7.5 µg/plate), TA100 = Sodium Azide (5 µg/plate), TA102 = Mitomycin-C (0.5 µg/plate)}, 2-Aa = 2-Aminoanthracene (5 µg/plate for TA100), - = Not Applicable.

Mean Count of His+ Revertant Colonies in Negative Control, Positive Control and Treatment Plates in the Presence of Metabolic Activation (Initial Toxicity-Mutation Test):

Concentration of

2-Amino-5-chloro-N,3-dimethylbenzamide

(µg/plate)

His+Revertant Colonies/Plate

[Presence of Metabolic Activation (5% v/v S9 mix)]

TA1537

TA1535

TA98

TA100

TA102

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

NC (DMSO)

8.33

2.31

16.33

3.51

25.67

1.53

128.00

6.24

223.67

9.50

1.5

6.33

1.53

16.00

3.00

25.00

3.61

129.00

7.00

219.33

3.79

5

7.67

1.15

15.00

1.73

24.33

2.08

125.67

6.03

226.33

4.04

15

7.00

2.65

15.67

1.53

25.00

1.00

123.67

9.61

227.67

3.21

50

9.00

1.00

17.33

1.15

24.00

4.00

125.67

9.29

222.67

5.13

150

8.00

1.00

15.33

3.06

24.33

2.52

130.67

2.31

223.33

3.06

500

8.33

3.21

14.33

2.52

25.33

1.15

125.00

7.55

225.33

11.02

1500

7.00

0.00

15.67

1.15

25.33

2.52

126.67

7.09

223.00

7.21

5000

7.00

1.00

15.00

4.36

26.00

3.00

132.33

4.04

225.00

1.73

PC

219.33

21.50

309.33

24.54

501.33

53.46

779.67

64.38

988.00

67.36

Key: SD = Standard Deviation, NC = Negative Control, DMSO = Dimethyl sulfoxide, PC = Positive control {2Aa = 2-Aminoanthracene (10 µg/plate for TA1537, TA1535, TA102 and 5 µg/plate for TA98 and TA100)}.

.

Mean Count of His+ Revertant Colonies in Negative Control, Positive Controls and Treatment Plates in the Absence of Metabolic Activation (Confirmatory Mutation Test):

Concentration of

2-Amino-5-chloro-N,3-dimethylbenzamide(µg/plate)

His+Revertant Colonies/Plate

[Absence of Metabolic Activation]

TA1537

TA1535

TA98

TA100

TA102

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

NC (DMSO)

9.67

2.08

16.00

4.58

21.33

3.21

136.00

11.53

224.67

16.26

156.25

8.00

3.61

16.67

4.93

19.33

3.21

133.67

3.79

235.33

11.68

312.5

7.33

2.31

15.67

1.53

21.67

4.73

132.00

17.09

228.00

10.15

625

8.33

3.79

15.33

2.08

21.00

4.00

127.67

3.06

231.67

6.43

1250

8.00

3.00

16.33

3.21

22.00

4.00

129.67

7.51

220.33

19.04

2500

9.33

2.89

14.67

1.53

22.33

3.79

134.33

10.07

231.00

6.24

5000

7.67

3.21

15.33

3.51

22.33

4.51

128.33

17.50

231.33

13.58

PC

280.00

28.62

300.33

33.56

523.67

36.12

809.00

21.17

918.67

92.26

2Aa

-

-

-

-

-

-

133.33

6.43

-

-

Key: SD = Standard Deviation, NC = Negative Control, DMSO = Dimethyl sulfoxide, PC = Positive Control {TA1537 = 9-Aminoacridine Hydrochloride Monohydrate (75 µg/plate), TA1535 = Sodium Azide (0.5 µg/plate), TA98 = 2-Nitrofluorene (7.5 µg/plate), TA100 = Sodium Azide (5 µg/plate), TA102 = Mitomycin-C (0.5 µg/plate)}, 2-Aa = 2-Aminoanthracene (5 µg/plate for TA100), - = Not Applicable.

Mean Count of His+ Revertant Colonies in Negative Control, Positive Control and Treatment Plates in the Presence of Metabolic Activation (Confirmatory Mutation Test):

Concentration of

2-Amino-5-chloro-N,3-dimethylbenzamide(µg/plate)

His+Revertant Colonies/Plate

[Presence of Metabolic Activation (10% v/v S9 mix)]

TA1537

TA1535

TA98

TA100

TA102

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

NC (DMSO)

9.33

3.06

18.67

3.51

27.33

6.11

134.00

5.57

234.33

20.03

156.25

8.33

2.89

17.33

3.79

25.00

5.29

136.33

9.71

235.00

10.82

312.5

8.33

2.08

17.33

2.08

25.33

4.16

124.00

10.58

224.33

14.01

625

8.00

3.61

16.33

3.51

25.33

1.15

132.67

15.57

230.67

2.31

1250

9.00

2.65

18.33

3.06

26.33

5.86

132.67

9.87

230.67

24.34

2500

8.00

2.65

16.67

2.08

25.67

4.16

134.00

5.00

234.33

9.07

5000

10.33

2.08

18.00

4.58

23.67

2.52

136.00

10.44

234.00

7.94

PC

263.33

52.37

310.00

31.51

589.00

69.76

769.33

47.50

992.00

85.25

Key: SD = Standard Deviation, NC = Negative Control, DMSO = Dimethyl sulfoxide, PC = Positive Control {2Aa = 2-Aminoanthracene (10 µg/plate for TA1537, TA1535, TA102 and 5 µg/plate for TA98 and TA100)}.

Positive Controls

2-Aminoanthracene was used as the positive control, in the presence of the metabolic activation system for all tester strains during the initial toxicity and the confirmatory mutation test. The large historical control data of this laboratory proved the efficiency and suitability of 2-aminoanthracene as a positive control, in the presence of the metabolic activation system. The batch of S9 used in this study was characterised with benzo(a)pyrene by the supplier which required the metabolic activation system with microsomal enzymes.

 

Positive controls exhibited a clear increase in the number of the revertant colonies when compared with that of the concurrent negative controls. This demonstrated the efficiency of the test system and suitability of procedures employed in the assay.

 

An increase in the revertants was not observed in the tester strain TA100 (initial toxicity-mutation test and confirmatory mutation test), treated with 2-aminoanthracene in the absence of the metabolic activation but a clear increase was observed in the presence of the metabolic activation. This demonstrated the efficiency of the S9, fraction used in this assay.

 

Initial Toxicity-Mutation Test

Cytotoxicity was characterised by the inhibition of the bacterial background lawn and/or reduction in the number of the revertant colonies.

A normal growth was observed, up to the tested concentration of 5000 µg/plate in all tester strains, in the absence and presence (5% v/v S9 mix) of the metabolic activation. Results revealed that there was no positive mutagenic effect in any tester strain, up to 5000 µg/plate of 2-amino-5-chloro-N,3-dimethylbenzamide, both in the absence and presence of the metabolic activation (5% v/v S9 mix), when compared with that of the negative control. Statistical analysis did not reveal any significant effect.

 

Based on the results of the initial toxicity mutation test , six concentrations of 156.25, 312.5, 625, 1250, 2500, and 5000 µg/plate for TA1537, TA1535, TA98, TA100 and TA102 were selected for the confirmatory mutation test, in the absence and presence of the metabolic activation system (10% v/v S9 mix).

 

Confirmatory Mutation Test

A normal bacterial background lawn, without significant reduction in the number of the revertant colonies was observed up to 5000 µg/plate in TA1537, TA1535, TA98, TA100, and TA102, both in the absence and presence of the metabolic activation system (10% v/v S9 mix).

No increase in the number of the revertant colonies was observed, either in the absence or presence of the metabolic activation system (10% v/v S9 mix) in any tester strain.

 Results revealed that there was no positive mutagenic effect in TA1537, TA1535, TA98, TA100, and TA102 at 156.25, 312.5, 625, 1250, 2500 and 5000 µg/plate of 2-amino-5-chloro-N,3-dimethylbenzamide, either in the absence or presence of the metabolic activation system (10% v/v S9 mix), when compared with that of the concurrent negative control. Statistical analysis did not reveal any significant effect.

Applicant's summary and conclusion

Conclusions:
From the results of this study, it is concluded that 2-amino-5-chloro-N,3-dimethylbenzamide is non-mutagenic to the histidine requiring strains of Salmonella typhimurium (TA1537, TA1535, TA98, TA100, and TA102) when tested under specified experimental conditions. According to EC regulation 1272/2008 the substance is not classified as mutagenic.
Executive summary:

This study was performed to evaluate the mutagenic potential of 2-amino-5-chloro-N,3-dimethylbenzamide by the bacterial reverse mutation test, using five histidine deficient mutant tester strains of Salmonella typhimurium (i.e., TA1537, TA1535, TA98, TA100, and TA102).

2-Amino-5-chloro-N,3-dimethylbenzamide was tested in two independent experiments, in the absence and presence of the metabolic activation by an Aroclor 1254-induced rat liver S-9 fraction . Bacterial cultures were exposed to the 2-amino-5-chloro-n,3-dimethylbenzamide at eight concentration levels (three plates/concentration) from 1.5 to 5000 µg/plate in the initial toxicity-mutation test. A normal bacterial background lawn was observed up to 5000 µg/plate in tester strains (TA1537, TA1535, TA98, TA100, and TA102), in the absence and presence of the metabolic activation (5% v/v S9 mix). No increase in the number of revertant colonies (no mutagenic effect) was observed, either in the absence or presence of the metabolic activation system (5% v/v S9 mix) in tester strains (TA1537, TA1535, TA98, TA100, and TA102). To confirm the negative results obtained in the initial toxicity mutation test, a confirmatory mutation test was conducted with an increased S9 concentration, i.e., 10% v/v S9 mix and modified concentration spacing. Based on the results of the initial toxicity mutation test, bacterial cultures were exposed to 2-amino-5-chloro-N,3-dimethylbenzamide at six concentration levels (three plates/concentration) from 156.25 to 5000 µg/plate for TA1537, TA1535, TA98, TA100, and TA102, in the absence and presence (10% v/v S9 mix) of the metabolic activation. After 48 hours of incubation at 37 ± 1°C, revertant colonies were enumerated.

2-Amino-5-chloro-N,3-dimethylbenzamide did not induce any significant increase in the number of the revertant colonies, in trials, with or without S9 mix, in any tester strain. All values for the negative control were within historical control ranges of the laboratory. Positive controls showed an increase in the number of the revertant colonies, demonstrating sensitivity and efficiency of the test system.

All criteria for a valid study were met, as described in the study plan. From the results of this study, under the specified experimental conditions, 2-amino-5-chloro-N,3-dimethylbenzamide is concluded to be non-mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium.