Pyridinium dichromate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item is mutagenic in the Ames test with the strains TA97a, TA98, TA100 and TA102 with and without an external metabolising system (reference 7.6.1 -1).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 July to 16 September 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his- (S. typhimurium)

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 102

Species / strain / cell type:

S. typhimurium, other: TA97a

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : A lyophilised post-mitochondrial supernatant of homogenised livers of male Sprague Dawley rats, induced with Aroclor 1254 and obtained from Fa. Moltox (11-01L, Lot No. 2465), was used as metabolic system (S9-Mix).
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL of S9-mix
- quality controls of S9: The metabolic activity of the microsomes was verified by the positive control substances of each study.

Test concentrations with justification for top dose:

0.8, 2.5, 7.4, 22, 67 and 200 µg per plate without external metabolisation, and
0.8, 2.5, 7.4, 22, 67 and 200 µg per plate with S9-mix from Aroclor 1254 induced microsomes of rat liver as an external metabolising system
The concentrations for the first experiment were set according to a preliminary toxicity test. The test substance was toxic at 5000 and 1667 µg/plate At 556 and 185 µg/plate only microcolonies were visible instead of a bacterial background lawn Therefore 200 µg/plate was chosen as highest concentration which could be in the toxic range, and a total of 6 concentrations was tested. The number and intervals of the concentrations are in accordance with the guidelines.

Vehicle / solvent:

deionised water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-o-phenylenediamine

Remarks:

10 µg, TA97a, without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

2 µg, TA98, without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100 (2 µg), TA1535 (1 µg), without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: t-Butyl-hydroperoxide

Remarks:

50 µg, TA102, without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

10 µg, TA97a, with S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

TA98 (1 µg), TA100 (2 µg), TA 1535 (2 µg), with S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 1,8-Dihydroxy-anthraquinone

Remarks:

50 µg, TA102, with S9 mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
Triplicate repetitions were run for each dose group, for the control groups six-fold repetitions were run. Due to the clear positive result of this test only one experiment was performed.

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: After the top agar had solidified, the plates were incubated at 37 °C until the colonies were visible (2 days).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

Determination of the toxicity
Additionally to the counting of colonies the bacterial background of the plates was inspected visually. The following signs of toxicity, if present, were recorded:
• A reduced bacterial background lawn (mottled instead of homogeneous).
• Microcolonies of bacteria instead of a homogeneous background lawn.
• No background lawn.
• Clearly reduced numbers of revertant colonies.

Calculations, criteria for a positive result:
Means and standard deviations were calculated for the number of mutants in every concentration group.
The criteria for a positive result are:
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
• For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: The 2½ fold of the amount of the spontaneous revertants.
• For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: The 1 2/3 fold of the amount of the spontaneous revertants.
These threshold values were derived from the variations in the control samples of our historic data of the Ames test.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 200 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

at 67 µg/plate

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 200 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

at 67 and 22 µg/plate

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 200 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

at 67 and 22 µg/plate

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 200 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA97a

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

at 67 and 22 µg/plate

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 200 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: No precipitation of the test substance was seen in any of the concentration groups.

Ames test:
- Signs of toxicity
In the preliminary test the test substance was toxic to the bacteria at 185 µg/plate and above. In the main test the test substance was again toxic to the bacteria at 200 and 67 µg/plate, resulting in the occurrence of microcolonies instead of a bacterial background or a reduced bacterial background lawn. At 22 µg/plate and beneath the bacterial background was normal.

- Genotoxicity results:
In the plates of strain TA97a, TA98, TA100 and TA102 a significant increase of the revertant colonies was observed in the higher concentrations.
In the strain TA1535 no significant increase of the revertant counts was obtained.

Table 1: Mean number of revertants per plate for strain TA97a

Without metabolisation				Metabolisation with S9-mix
conc.	revertants / plate			conc.	revertants / plate
µg/plate	mean	SD	N	µg/plate	mean	SD	N
200	0.0	0.0	3	200	39.7	1.5	3
67	119.5	57.3	2	67	344.3	26.7	3
22	177.3	26.4	3	22	182.0	22.5	3
7.4	117.3	17.9	3	7.4	136.3	0.6	3
2.5	101.0	9.9	2	2.5	123.7	14.6	3
0.8	104.7	8.5	3	0.8	124.7	27.0	3
solvent	102.5	2.1	2	solvent	162.7	7.9	6
positive	327.0	26.9	2	positive	417.7	21.1	3

 

solvent:      deionised water

positive:     without metabolisation: 4-Nitro-o-phenylene-diamine, 10 µg / plate

                  with metabolisation: 7,12-Dimethylbenz[a]anthracene, 10 µg / plate

reverse:

exceeding the threshold for mutagenicity:>167 % of the solvent controls

 

Table 2: Mean number of revertants per plate for strain TA1535

Without metabolisation				Metabolisation with S9-mix
conc.	revertants / plate			conc.	revertants / plate
µg/plate	mean	SD	N	µg/plate	mean	SD	N
200	0.0	0.0	0	200	0.0	0.0	0
67	4.0	1.0	3	67	16.0	1.0	3
22	10.7	3.8	3	22	9.3	4.0	3
7.4	11.0	1.0	3	7.4	8.3	1.5	3
2.5	8.0	2.0	3	2.5	10.0	1.7	3
0.8	7.7	3.1	3	0.8	10.0	2.6	3
solvent	7.2	3.1	6	solvent	10.0	4.1	6
positive	163.3	25.8	3	positive	217.0	9.2	3

 

solvent:      deionised water

positive:     without metabolisation: Sodium azide, 1 µg / plate

                  with metabolisation: 2-Amino-anthracene, 2 µg / plate

reverse:

exceeding the threshold for mutagenicity:>250 % of the solvent controls

Table 3: Mean number of revertants per plate for strain TA98

Without metabolisation				Metabolisation with S9-mix
conc.	revertants / plate			conc.	revertants / plate
µg/plate	mean	SD	N	µg/plate	mean	SD	N
200	0.0	0.0	3	200	0.0	0.0	3
67	6.0	3.5	3	67	48.7	2.5	3
22	29.3	5.1	3	22	22.7	1.5	3
7.4	16.3	1.5	3	7.4	12.7	2.5	3
2.5	12.3	3.2	3	2.5	15.0	1.7	3
0.8	9.0	1.7	3	0.8	11.0	1.0	3
solvent	12.7	1.4	6	solvent	15.2	2.2	6
positive	199.7	3.8	3	positive	403.0	22.3	3

 

solvent:      deionised water

positive:     without metabolisation: 2-Nitrofluorene, 2 µg / plate

                  with metabolisation: 2-Amino-anthracene, 1 µg / plate

reverse:

exceeding the threshold for mutagenicity:>250 % of the solvent controls

Table 4: Mean number of revertants per plate for strain TA100

Without metabolisation				Metabolisation with S9-mix
conc.	revertants / plate			conc.	revertants / plate
µg/plate	mean	SD	N	µg/plate	mean	SD	N
200	0.0	0.0	3	200	0.0	0.0	3
67	175.3	29.7	3	67	403.0	23.8	3
22	150.3	35.3	3	22	122.0	8.0	3
7.4	75.7	3.5	3	7.4	78.7	7.4	3
2.5	82.7	10.6	3	2.5	82.7	17.6	3
0.8	71.7	3.8	3	0.8	93.3	14.0	3
solvent	74.3	14.7	6	solvent	90.2	12.4	6
positive	303.7	9.1	3	positive	1165.3	137.7	3

 

solvent:      deionised water

positive:     without metabolisation: Sodium azide, 2 µg / plate

                  with metabolisation: 2-Amino-anthracene, 2 µg / plate

reverse:

exceeding the threshold for mutagenicity:>167 % of the solvent controls

 

Table 5: Mean number of revertants per plate for strain TA102

Without metabolisation				Metabolisation with S9-mix
conc.	revertants / plate			conc.	revertants / plate
µg/plate	mean	SD	N	µg/plate	mean	SD	N
200	0.0	0.0	3	200	174.7	94.6	3
67	413.7	79.5	3	67	554.3	43.4	3
22	296.0	39.4	3	22	272.0	13.5	3
7.4	177.7	17.7	3	7.4	177.7	30.2	3
2.5	165.0	9.5	3	2.5	179.0	17.4	3
0.8	144.7	5.1	3	0.8	205.3	26.6	3
solvent	146.8	13.0	6	solvent	150.5	20.3	6
positive	397.0	14.7	3	positive	366.0	57.3	3

 

solvent:      deionised water

positive:     without metabolisation: t-Butyl-hydroperoxide, 50 µg / plate

                  with metabolisation: 1,8-Dihydroxy-anthraquinone, 50 µg / plate

reverse:

exceeding the threshold for mutagenicity:>167 % of the solvent controls

Conclusions:

According to these results, the substance is mutagenic in the Ames test with the strains of Salmonella typhimurium TA97a, TA98, TA100 and TA102 with and without an external metabolising system.

Executive summary:

A study according OECD TG 471 was performed to determine a possible mutagenic action of the test substance with the Salmonella typhimurium reverse mutation test (Ames test). This test is sensitive to frameshift mutations as well as to base pair mutations. The performance of the test with and without an external metabolising system enables the detection of the mutagenic action of the test substance itself as well as of its metabolites.

The test substance was dissolved in deionised water. The following concentrations were tested:

0.8, 2.5, 7.4, 22, 67 and 200 µg per plate without external metabolisation, and

0.8, 2.5, 7.4, 22, 67 and 200 µg per plate with S9-mix from Aroclor 1254 induced microsomes of rat liver as an external metabolising system.

The test was performed according to the plate incorporation method. As test system the bacterial strains Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 were used. Negative and positive controls were included. Due to the clear positive result of this test only one experiment was performed.

Toxicity of the test substance to the bacteria was observed at 200 and 67 µg/plate, resulting in the occurrence of micro-colonies instead of a bacterial background or a reduced bacterial background lawn. At 22 µg/plate and beneath the bacterial background was normal.

No precipitation of the test substance was seen in any of the concentration groups.

In the plates of the strains TA97a, TA98, TA100 and TA102 an increase of the mutation frequency to more than the threshold values (250 % of the controls for strains TA98 and TA1535, 167 % of the controls for strains TA97a, TA100 and TA102) was obtained. In the strain TA1535 no significant increase of the revertant counts was obtained.

According to the results obtained in this study, the test item is mutagenic in the Ames test with the strains TA97a, TA98, TA100 and TA102 with and without an external metabolising system.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames Test

A study according OECD TG 471 was performed to determine a possible mutagenic action of the test substance with the Salmonella typhimurium reverse mutation test (Ames test). This test is sensitive to frameshift mutations as well as to base pair mutations. The performance of the test with and without an external metabolising system enables the detection of the mutagenic action of the test substance itself as well as of its metabolites.

The test substance was dissolved in deionised water. The following concentrations were tested:

0.8, 2.5, 7.4, 22, 67 and 200 µg per plate without external metabolisation, and

0.8, 2.5, 7.4, 22, 67 and 200 µg per plate with S9-mix from Aroclor 1254 induced microsomes of rat liver as an external metabolising system.

The test was performed according to the plate incorporation method. As test system the bacterial strains Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 were used. Negative and positive controls were included. Due to the clear positive result of this test only one experiment was performed.

Toxicity of the test substance to the bacteria was observed at 200 and 67 µg/plate, resulting in the occurrence of micro-colonies instead of a bacterial background or a reduced bacterial background lawn. At 22 µg/plate and beneath the bacterial background was normal.

No precipitation of the test substance was seen in any of the concentration groups.

In the plates of the strains TA97a, TA98, TA100 and TA102 an increase of the mutation frequency to more than the threshold values (250 % of the controls for strains TA98 and TA1535, 167 % of the controls for strains TA97a, TA100 and TA102) was obtained. In the strain TA1535 no significant increase of the revertant counts was obtained.

According to the results obtained in this study, the test item is mutagenic in the Ames test with the strains TA97a, TA98, TA100 and TA102 with and without an external metabolising system.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

According to Annex VI of Regulation (EC) No 1272/2008 (CLP Regulation) Chromium (VI) compounds (Index Number 024-017-00-8) are harmonised classified for carcinogenicity Cat 1B (H350). Therefore, a separate classification for mutagenicity was not conducted.