A dried sludge product resulting from the treatment process of domestic wastewater. The exact processes followed for the production of the dried sludge are: Preliminary + secondary treatment of the wastewater stream, thickening, dehydration and drying of the excess sludge.

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Start and Completion Date: From 02 SEP 2021 to 17 DEC 2021
Study Initiation and Completion Date: From 01 SEP 2021 to 21 MAR 2021

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted: July 21, 1997 (OECD, 1997) corrected 26 June 2020

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

A dried sludge product resulting from the treatment process of domestic wastewater. The exact processes followed for the production of the dried sludge are: Preliminary & secondary treatment of the wastewater stream, thickening, dehydration and drying of the excess sludge.

EC Number:

943-834-9

Molecular formula:

Not applicable for UVCB substances

IUPAC Name:

A dried sludge product resulting from the treatment process of domestic wastewater. The exact processes followed for the production of the dried sludge are: Preliminary & secondary treatment of the wastewater stream, thickening, dehydration and drying of the excess sludge.

Method

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 homogenate was used as the metabolic activation system. The S9 homogenate was prepared from male Wistar rats induced with a single intra-peritoneal injection of Aroclor 1254 (0.7 mL/rat ready to use solution), 5 days prior to sacrifice. The S9 homogenate was prepared in batches and stored in a deep freezer maintained at -68 to -86 ºC.
The details of S9 homogenate used in the study are furnished as a Certificate in the report.
S9 homogenate was thawed immediately before use and mixed with the cofactor solution containing 4 mM NADP, 5 mM Glucose-6-phosphate, 8 mM MgCl2 and 33 mM KCl in PBS to achieve a final concentration of 10% S9 (v/v) in the activation mixture.
S9 Mix:
The co-factors solution was prepared by dissolving the following in 9 and
50 mL of cold PBS for the preliminary toxicity and mutation assay respectively (see Table 1). This solution was filter sterilized using a 0.2 μm disposable syringe filter.
S9 mix was prepared by mixing 1 and 5.5 mL of the S9 homogenate with
9 and 49.5 mL of the co-factors solution for the preliminary toxicity and mutation assay respectively, kept in an ice bath and used within
one hour.

Test concentrations with justification for top dose:

In the preliminary toxicity test, Salmonella typhimurium, TA 100 was exposed to test item at 7.8125, 15.625, 31.25, 62.5, 125, 250, 500 and 1000 µg/plate along with a DMSO control using direct plate incorporation method, for the selection of test doses for the mutation assay.
The test item did not precipitate on the basal agar plates up to 500 µg/plate, however it formed non-interfering precipitation at 1000 µg/plate.
It was observed that the test item is non-toxic up to 1000 µg/plate as the intensity of the bacterial background lawn as well as the mean number of revertant colonies were comparable to the DMSO control plates.
Based on these observations, it was decided to test highest test dose of 1000 µg/plate in the mutation assay.

Vehicle / solvent:

The test item was insoluble in sterile water up to 5 mg/ml and soluble in DMSO at 10 mg/mL.
The test item was found to be stable in the vehicle (DMSO) for 24 hours at room temperature at the fortification levels of 50 µg/mL and 10000 µg/mL.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Strain: WP2uvrA(pKM101) -without metabolic activation

Positive control substance:

4-nitroquinoline-N-oxide

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Strain: TA1537 -without metabolic activation

Positive control substance:

9-aminoacridine

Controls 3

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Strains: TA100, TA1535 -without metabolic activation

Positive control substance:

sodium azide

Controls 4

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Strain: TA98 -without metabolic activation

Positive control substance:

2-nitrofluorene

Controls 5

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

All strains: TA98, TA100, TA1535, TA1537, WP2urvA (pKM101) -with metabolic activation

Positive control substance:

other: 2-Aminoanthracene (CAS No. 613-13-8)

Controls 6

Negative solvent / vehicle controls:

yes

Remarks:

One hundred microliters (100 µL) of DMSO was used as the vehicle control in this study

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

Test System
Following strains of bacteria accepted under 1997 OECD guideline 471 for the assessment of point gene mutation were used:
Histidine auxotrophic strains of Salmonella typhimurium: TA98, TA100, TA1535 and TA1537.
Tryptophan auxotrophic strain of Escherichia coli: WP2uvrA (pKM101).

Storage:
Stock cultures of tester strains were stored in Oxoid nutrient broth No. 2 in the test facility as frozen permanents in liquid nitrogen. Laboratory stocks were maintained on respective minimal glucose agar plates as master plates of each strain, for a maximum period of 2 months and refrigerated between 2 and 8 ºC. The master plates prepared on 09 July 2021 and 25 October 2021 were used in the study.

Genotypic Characterization of Test System: see Table 2.

Test Medium and Solutions: see Table 3.

Mutation Assay:
In the initial mutation assay, which was a plate incorporation mode of exposure, the bacterial suspensions were exposed to the test item, vehicle and the positive controls in the presence and absence of an exogenous metabolic activation system. These bacterial suspensions were then mixed with overlay agar and plated immediately onto minimal medium viz., his- for Salmonella typhimurium and trp- for Escherichia coli, respectively.
In the confirmatory assay, which was a pre-incubation mode of exposure, the test constituents were mixed with the bacteria inside a tube, incubated in an incubator shaker, mixed with overlay agar and plated immediately onto minimal medium his- for Salmonella typhimurium and trp- for Escherichia coli, respectively.
After 68 hours of incubation, the revertant colonies were counted and compared with the number of spontaneous revertants in the vehicle control plates.

Test Strain Cultures:
Each of the tester strains from the master plates were inoculated into the
respective tubes containing Oxoid Nutrient broth No. 2 and the tubes were
incubated at 37 ± 1 °C for approximately 17 hours.

Test Doses:
Based on the observations of the preliminary toxicity test, the following five test doses were selected for testing in the mutation assay:
a) 10, b) 32, c) 100, d) 316, and e) 1000 µg/plate

Test Item Stock and Dilutions:
For both the initial and confirmatory mutation assay, a stock solution of
approximately 10000 μg/mL was prepared by mixing 250 mg test item in DMSO, sonicated and making up the volume to 25 mL in a volumetric flask with DMSO.
The above stock was diluted further in DMSO as follows to prepare 4 concentrations of the test item and 100 μL was added to cultures for the following final exposure amounts (see Table 4).

No. of Replicates:Three replicate plates were maintained for the initial as well as the confirmatory mutation assays.

Plating Procedure
Initial mutation assay was conducted using the direct plate incorporation method as follows:

A. Presence of Metabolic Activation
a) 2.0 mL soft agar containing histidine-biotin/tryptophan
b) 100 µL test dose/vehicle/appropriate positive control
c) 100 µL bacterial culture
d) 500 µL S9 mix

B. Absence of Metabolic Activation
a) 2.0 mL soft agar containing histidine-biotin/tryptophan
b) 100 µL test dose /vehicle/appropriate positive control
c) 100 µL bacterial culture
d) 500 µL of PBS

These test constituents were transferred into sterile test tubes, mixed and
overlaid onto VB agar plates and allowed to set. The plates were then
incubated at 37 ± 1 °C for 68 hours. Revertant colonies were counted manually and the plates were examined for bacterial background lawn.
The confirmatory mutation assay was conducted using the pre-incubation
method as follows:
A. Presence of Metabolic Activation
a) 100 µL test dose /vehicle/appropriate positive control
b) 100 µL bacterial culture
c) 500 µL S9 mix

B. Absence of Metabolic Activation
a) 100 µL test dose/vehicle/appropriate positive control
b) 100 µL bacterial culture
c) 500 µL PBS

These test constituents were transferred into sterile test tubes and kept in an
incubator shaker for 20 minutes at 37ºC and 110 rpm. After this period, 2 mL
soft agar containing histidine-biotin/tryptophan were added to each of the
tubes. The tube contents were mixed and overlaid onto VB agar plates and
allowed to set. The plates were then incubated at 37 ± 1 °C for 68 hours.
Revertant colonies were counted manually and the plates were examined for
bacterial background lawn.

Evaluation criteria:

To determine a positive result, there should be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of the test item either in the presence or absence of the metabolic activation system.
The test will be judged positive, if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value for strains TA98, TA100 and WP2uvrA (pKM101) or equal to or greater than 3 times the mean vehicle control value for strains TA1535 and TA1537.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test and Justification for the Selection of Target Top Dose:
Results of the preliminary toxicity test are presented in Table 1.
The test item did not cause precipitation up to 500 µg/plate, however it caused non-interfering precipitation at 1000 µg/plate.
The test item did not show toxicity to the tester strain at any of the tested doses up to 1000 µg/plate as the mean number of revertant colonies and bacterial background lawn were comparable with vehicle control, both in the presence and absence of metabolic activation.
Based on these observations, it was decided to test the highest test dose of 1000 µg/plate in the mutation assay.

Viable counts of all the tester strains were within the required range of
1-2 x 109 CFU/mL for the initial as well as the confirmatory mutation assays. (Table2).

Mutation Assay:
Initial mutation assay was conducted using the direct plate incorporation procedure and the confirmatory mutation assay was conducted using a pre-incubation procedure.
Viable counts of all the tester strains were within the required range of
1-2 x 109 CFU/mL for the initial as well as the confirmatory mutation assays

Summarized results of the Initial Mutation Assay are presented in Tables 3 and 4.
Summarized results of the Confirmatory Mutation Assay are presented in Tables 5 and 6 .
The mean numbers of revertant colonies/plate in the DMSO control was comparable to the in-house spontaneous revertant counts for all the tester strains in both the initial and the confirmatory mutation assay.
The test item did not cause precipitation up to 316 µg/plate, however it formed non-interfering precipitation at 1000 µg/plate.
The test item was not toxic to the tester strains up to 1000 μg/plate as the mean number of revertant colonies and intensity of the bacterial background lawn was comparable to the respective DMSO control plates, both in the presence and absence of metabolic activation.
The tested doses showed no positive mutagenic increase in the mean number of revertant colonies for any of the tester strains when compared to the respective vehicle control plates, either in the presence or absence of metabolic activation.
Positive control chemicals tested simultaneously produced a more than
3-fold increase in the mean numbers of revertant colonies for all the strains when compared to the respective vehicle control plates. No toxicity was observed in the positive controls as the intensity of the bacterial background lawn of all the tester strains was comparable to that of the respective vehicle control plates.

Viable Counts

The bacterial suspension of each tester strain was diluted up to 10-6 dilution in 9 ml of PBS. One hundred microlitres from the 10-6 dilution of each tester
strain was mixed with 2 mL of soft agar and plated onto nutrient agar plates in triplicate. The plates were incubated at 37 ± 1 °C for 68 hours for the initial as well as the confirmatory mutation assays. After incubation, the number of
colonies in each plate were manually counted and expressed as the number of colony forming units per mL of the bacterial suspension.

Any other information on results incl. tables

Table 1. Results of Preliminary Toxicity Test

Treatment (mg/plate)	TA 100 revertant colonies/plate^*
	Presence of Metabolic Activation			Absence of Metabolic Activation
	Mean Revertant Colony Count	Background Lawn Intensity**	Precipitation**	Mean Revertant Colony Count	Background Lawn Intensity**	Precipitation**
DMSO	96	4+	NPO	95	4+	NPO
7.8125	94	4+	NPO	92	4+	NPO
15.625	94	4+	NPO	91	4+	NPO
31.25	91	4+	NPO	94	4+	NPO
62.5	93	4+	NPO	87	4+	NPO
125	92	4+	NPO	92	4+	NPO
250	94	4+	NPO	91	4+	NPO
500	90	4+	NPO	95	4+	NPO
1000	93	4+	NP	92	4+	NP

 

Table 2.Viable Counts of the Overnight Culture of the Tester Strains

Tester Strains	Viable Counts (x 10^9 CFU/mL*)
	Initial Mutation Assay	Confirmatory Mutation Assay
TA98	1.56	1.57
TA100	1.55	1.55
TA1535	1.55	1.55
TA1537	1.54	1.54
WP2uvrA (pKM101)	1.67	1.65

 

* Required Cell count: 1-2x10⁹ Colony Forming Units (CFU)/mL

Table 3.Summary Results of Initial Mutation Assay in the Presence of Metabolic Activation

Treatment [µg/plate]	No. of  revertants/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA (pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
DMSO	26	1	NA	94	2	NA	15	1	NA	16	1	NA	145	2	NA
10	24	1	0.94	92	3	0.98	15	1	0.98	14	1	0.87	143	4	0.99
32	25	2	0.95	94	2	1.00	14	2	0.96	13	1	0.85	141	4	0.97
100	24	1	0.91	89	2	0.94	13	1	0.84	14	2	0.87	140	3	0.97
316	24	2	0.91	90	3	0.96	14	2	0.91	13	1	0.85	138	2	0.96
1000	24	1	0.91	90	3	0.95	13	1	0.89	13	1	0.83	138	2	0.95
Positive control	373^c	9^c	14.36^c	775^c	7^c	8.22^c	167^c	4^c	11.13^c	177^c	5^c	11.28^c	590^d	7^d	4.08^d
^a Values are means of three replicates calculated from individual values of Appendix 3 and are rounded off to the nearest whole number              ^b Ratio of treated/Vehicle control (mean revertants per plate). The presentation was made using the mean values with decimals before rounding off to the nearest whole number. Hence, some of the values may not match if calculated using the rounded-off mean values of this summary table. ^c TA98, TA100, TA1535, TA1537: 2-Aminoanthracene (4 µg/plate) ^d WP2uvrA (pKM101): 2-Aminoanthracene (30 µg/plate)                                   SD: Standard deviation            NA: Not applicable

Table 4.Summary Results of Initial Mutation Assay in the Absence of Metabolic Activation

Treatment [µg/plate]	No. of  revertants/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA (pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
DMSO	25	2	NA	94	2	NA	15	1	NA	15	2	NA	145	2	NA
10	25	1	0.99	92	4	0.99	14	1	0.95	13	1	0.91	141	3	0.97
32	24	1	0.95	91	3	0.98	13	1	0.91	13	2	0.91	142	4	0.98
100	23	1	0.89	93	3	0.99	12	1	0.84	14	1	0.95	141	3	0.97
316	25	2	0.97	92	2	0.98	13	1	0.89	13	1	0.89	138	2	0.95
1000	22	1	0.88	89	3	0.95	13	1	0.89	13	2	0.86	139	1	0.96
Positive control	269^c	4^c	10.63^c	575^d	6^d	6.14^d	163^d	8^d	11.11^d	172^e	7^e	11.73^e	585^f	10^f	4.05^f
^a Values are means of three replicates calculated from individual values of Appendix 4 and are rounded off to the nearest whole number              ^b Ratio of treated/Vehicle control (mean revertants per plate). The presentation was made using the mean values with decimals before rounding off to the nearest whole number. Hence, some of the values may not match if calculated using the rounded-off mean values of this summary table. ^c TA98: 2-Nitrofluorene (2 µg/plate),                                                                                                             ^d TA100, TA1535: Sodium azide (1 µg/plate)                                                       ^eTA1537: 9-Aminoacridine (50 µg/plate)                                              ^f WP2uvrA (pKM101): 4-Nitroquinoline-1-oxide (4 µg/plate)                          SD: Standard deviation              NA: Not applicable

Table 5.Summary Results of Confirmatory Mutation Assay in the Presence of Metabolic Activation

Treatment [µg/plate]	No. of  revertants/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA (pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
DMSO	27	1	NA	95	2	NA	15	1	NA	15	1	NA	146	2	NA
10	25	1	0.94	94	1	0.99	13	1	0.84	13	1	0.91	141	2	0.96
32	24	1	0.89	90	2	0.95	14	1	0.96	14	2	0.98	140	2	0.96
100	24	2	0.89	94	1	0.99	13	1	0.87	13	1	0.89	141	2	0.96
316	24	2	0.89	89	2	0.93	14	1	0.91	14	1	0.95	139	2	0.95
1000	23	1	0.85	90	3	0.95	13	1	0.87	14	1	0.93	139	3	0.95
Positive control	364^c	8^c	13.64^c	766^c	15^c	8.06^c	165^c	7^c	10.98^c	168^c	5^c	11.43^c	593^d	5^d	4.06^d
^a Values are means of three replicates calculated from individual values of Appendix 5 and are rounded off to the nearest whole number              ^b Ratio of treated/Vehicle control (mean revertants per plate). The presentation was made using the mean values with decimals before rounding off to the nearest whole number. Hence, some of the values may not match if calculated using the rounded-off mean values of this summary table. ^c TA98, TA100, TA1535, TA1537: 2-Aminoanthracene (4 µg/plate) ^d WP2uvrA (pKM101): 2-Aminoanthracene (30 µg/plate)                                   SD: Standard deviation            NA: Not applicable

Table 6.Summary Results of Confirmatory Mutation Assay in the Absence of Metabolic Activation

Treatment [µg/plate]	No. of  revertants/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA (pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
DMSO	26	1	NA	95	2	NA	16	1	NA	15	1	NA	144	2	NA
10	25	2	0.96	92	4	0.97	14	1	0.89	13	1	0.87	140	3	0.97
32	24	1	0.94	90	2	0.95	14	2	0.91	14	1	0.93	141	2	0.98
100	24	2	0.95	93	2	0.98	12	1	0.79	13	1	0.87	139	3	0.96
316	24	1	0.94	90	2	0.94	14	1	0.87	13	1	0.89	139	2	0.97
1000	24	1	0.92	91	4	0.96	13	1	0.83	13	1	0.87	138	2	0.96
Positive control	269^c	6^c	10.49^c	569^d	3^d	5.97^d	165^d	7^d	10.55^d	171^e	8^e	11.38^e	592^f	6^f	4.10^f
^a Values are means of three replicates calculated from individual values of Appendix 6 and are rounded off to the nearest whole number              ^b Ratio of treated/Vehicle control (mean revertants per plate). The presentation was made using the mean values with decimals before rounding off to the nearest whole number. Hence, some of the values may not match if calculated using the rounded-off mean values of this summary table. ^c TA98: 2-Nitrofluorene (2 µg/plate),                                                                                                              ^d TA100, TA1535: Sodium azide (1 µg/plate)                                                       ^eTA1537: 9-Aminoacridine (50 µg/plate)                                              ^f WP2uvrA (pKM101): 4-Nitroquinoline-1-oxide (4 µg/plate)                          SD: Standard deviation              NA: Not applicable

 

Applicant's summary and conclusion

Conclusions:

All criteria for a valid study were met as described in the study plan. It is concluded that the test item, Dried Sludge from Domestic Wastewater, was not mutagenic in this bacterial reverse mutation test up to the dose of 1000 µg/plate under the conditions of testing employed.

Executive summary:

Dried Sludge From Domestic Wastewater was tested for its mutagenic potential in the bacterial reverse mutation assay. The study was conducted using TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium and WP2uvrA (pKM101) strain of Escherichia coli. The study consisted of a preliminary toxicity test and two mutation assays comprising three independent experiments. The bacterial tester strains were exposed to the test item in the presence and absence of a metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

The test item was found to be stable in the vehicle (DMSO) for 24 hours at room temperature at the fortification levels of 50 µg/mL and 10000 µg/mL.

 

The test item was insoluble in sterile water up to 5 mg/ml and soluble in DMSO at 10 mg/mL.

 

In the preliminary toxicity test, Salmonella typhimurium, TA 100 was exposed to test item at 7.8125, 15.625, 31.25, 62.5, 125, 250, 500 and 1000 µg/plate along with a DMSO control using direct plate incorporation method, for the selection of test doses for the mutation assay.

 

The test item did not precipitate on the basal agar plates up to 500 µg/plate, however it formed non-interfering precipitation at 1000 µg/plate.

It was observed that the test item is non-toxic up to 1000 µg/plate as the intensity of the bacterial background lawn as well as the mean number of revertant colonies were comparable to the DMSO control plates.

Based on these observations, it was decided to test highest test dose of 1000 µg/plate in the mutation assay.

In mutation assay the bacterial tester strains were exposed to Dried Sludge From Domestic Wastewater in triplicate exposure at 10, 32, 100, 316, and 1000 µg/plate using the direct plate incorporation mode of exposure in the initial mutation assay and using the pre-incubation mode of exposure in the confirmatory mutation assay. The vehicle control (DMSO) and the appropriate positive controls (2-aminoanthracene, 2-nitrofluorene, sodium azide, 9-aminoacridine, and 4-nitroquinoline-1-oxide) were tested simultaneously. The mean and standard deviation of numbers of revertant colonies were calculated for each test dose and the controls for all the tester strains.

The results from the initial as well as from the confirmatory assays, indicated that the tested doses showed no positive mutagenic increase in the mean numbers of revertant colonies for all tester strains when compared to the respective vehicle control plates, either in the presence or absence of metabolic activation.

The results of the concentration analysis of the dose formulation samples of both the initial and confirmatory mutation assays confirmed that the top dose level of 1000 µg/plate was achieved in both assays and the results support the validity of the study conclusion.

The study indicated that Dried Sludge From Domestic Wastewater was not mutagenic in this Bacterial Reverse Mutation Assay up to the highest dose of 1000 µg/plate based on cytotoxicity, under the conditions of testing employed.