Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item cadmium selenide had no mutagenic activity in Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA)

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:
18 September 2017 to 28 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine and tryptophan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat S9 fraction
Test concentrations with justification for top dose:
Examined concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate.
Examined concentrations in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate

Concentrations were selected on the basis of the Preliminary Solubility Test and Preliminary Range Finding Test (Informatory Toxicity Test).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 1% methyl cellulose
- Justification for choice of solvent/vehicle: Based on the available data, due to its chemical nature, a suspension (not a solution) was detected at concentration level of 100 mg/mL using 1% (w/v) methyl cellulose aqueous solution (1% MC). Other vehicles did not give a solution, therefore, 1% MC was selected as vehicle of the study. To ensure the suspension homogeneity (lower the sedimentation speed), the suspension was stirred continuously during the formulation and treatment.

The test item was freshly formulated at a concentration of 100 mg/mL in the vehicle (1% methyl cellulose), on the day of administration. The formulation container was magnetic stirred continuously up to the end of dose administration procedures.

Vehicle:
1 % (w/v) Methyl cellulose
Dispense code: S11136 / S11138
Manufacturer: CiToxLAB Hungary Ltd.
Expiry Date: 23 September 2017 / 22 October 2017
Storage condition: Refrigerated
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-nitro-1,2-phenylene- diamine, 2-aminoanthracene
Details on test system and experimental conditions:
The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test and a Confirmatory Mutation Test. In the Preliminary Concentration Range Finding Test as well as in the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used.

Procedure for Exposure in the Initial Mutation Test
The Initial Mutation Test followed the standard plate incorporation procedure. Bacteria were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.
Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).

The content of the tubes:
top agar 2000µL
vehicle or test item formulation (or reference controls) 50µL
overnight culture of test strain 100µL
phosphate buffer (pH 7.4) or S9 mix 500µL
This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48±1 hours.
Examined concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50, and 15.81 μg/plate in the absence and presence of metabolic activation.

Procedure for Exposure in the Confirmatory Mutation Test
The Confirmatory Mutation Test followed the standard pre-incubation procedure since no biologically relevant increase in the number of revertant colonies was observed in the Initial Mutation Test.
Bacteria were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45°C.
Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 min at 37ºC in a shaking incubator.
After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48±1 hours.
Examined concentrations in the Confirmatory Mutation Test were 5000, 1581, 500,
158.1, 50, 15.81 and 5 μg/plate in the absence and presence of metabolic activation.

EVALUATION OF EXPERIMENTAL DATA
The colony numbers on the untreated / negative (solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.

* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.









Rationale for test conditions:
As per guidelines.
Evaluation criteria:
Criteria for Validity:

The study was considered valid if:
- the number of revertant colonies of the negative (vehicle/solvent) and positive controls are in the relevant historical control range, generated at the test facility, in all tester strains of the main tests (with or without S9-mix);
- at least five analysable concentrations are presented in all strains of the main tests;

Criteria for a Positive Response:

A test item was considered mutagenic if:
- a concentration-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

Criteria for a Negative Response:

A test article was considered non-mutagenic if:
- the total number of revertants in tester strain Salmonella typhimurium TA98, TA100 or Escherichia coli WP2 uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strain Salmonella typhimurium TA1535 or TA1537 is not greater than three times the concurrent vehicle control;
- the negative response should be reproducible in at least one follow up experiment.

Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
INITIAL AND CONFIRMATORY MUTATION TESTS
In the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used. The Initial Mutation Test and Confirmatory Mutation Test were carried out using four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and the Escherichia coli WP2 uvrA strain. The Initial Mutation Test and Confirmatory Mutation Test were performed in the presence and absence of a metabolic activation system. Each test was performed with appropriate untreated, negative (solvent) and positive controls. In the main tests each sample (including the controls) was tested in triplicate.

Based on the results of the preliminary experiment, the examined test concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate and in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.

Slight precipitate was observed in the main tests in all examined bacterial strains with and without metabolic activation on the plates at 5000 μg/plate concentration.

An inhibitory, cytotoxic effect of the test item was not detected in the Initial Mutation Test and Confirmatory Mutation Tests.

In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect.

In the Initial Mutation Test (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 158.1 μg/plate concentration without metabolic activation (the observed mutation factor value was: MF: 1.46). However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the number of revertant colonies was within the historical control range.

In the Confirmatory Mutation Test (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain at 5000 μg/plate concentration without metabolic activation (the observed mutation factor value was: MF: 1.31). However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.

Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.

Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.

VALIDITY OF THE TESTS

Untreated, negative (vehicle/solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains. At least five analysable concentrations were presented in all strains with and without metabolic activation.

The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test. The study was considered to be valid.

PRELIMINARY RANGE FINDING TEST (INFORMATORY TOXICITY TEST)

In the Preliminary Range Finding Test, the plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (solvent) and positive controls. Each sample (including the controls) was tested in triplicate.

The following concentrations were examined: 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate.

In the preliminary experiment, the numbers of revertant colonies were mostly in the normal range (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system).

Slight precipitate was observed in the Preliminary Range Finding Test in all examined bacterial strains with and without metabolic activation on the plates at 5000 μg/plate concentration.

Inhibitory or toxic effects of the test item were not detected in the preliminary experiment.

Based on the results of the Range Finding Test and the solubility findings, the maximum final concentration to be tested in the main experiments was 5000 µg/plate.


Conclusions:
In conclusion, the test item cadmium selenide had no mutagenic activity in the bacterial strains under the test conditions used in this study.
Executive summary:

The test item, cadmium selenide, was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone- induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).

Based on the available data (CiToxLAB study code: 17/257-001P), the test item was dissolved  in  1%  (w/v)  methyl  cellulose  at  a  concentration  of  100 mg/mL. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 µg/plate were examined in the Range Finding Test in tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate, and in the Confirmatory Mutation Test were 5000,1581, 500, 158.1, 50, 15.81 and 5 μg/plate.

Slight precipitate was observed in the Preliminary Range Finding Test and in the main tests in all examined bacterial strains with and without metabolic activation on the plates at the 5000 μg/plate concentration.

An inhibitory, cytotoxic effect of the test item was not detected in the Preliminary Range Finding Test, in the Initial Mutation Test and in the Confirmatory Mutation Tests.

In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any treatment-related effect.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, and the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests; the examined concentration range was considered to be adequate. The study was considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The test item, cadmium selenide, was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone- induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).

Based on the available data (CiToxLAB study code: 17/257-001P), the test item was dissolved  in  1%  (w/v)  methyl  cellulose  at  a  concentration  of  100 mg/mL. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 µg/plate were examined in the Range Finding Test in tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate, and in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.

Slight precipitate was observed in the Preliminary Range Finding Test and in the main tests in all examined bacterial strains with and without metabolic activation on the plates at the 5000 μg/plate concentration.

An inhibitory, cytotoxic effect of the test item was not detected in the Preliminary Range Finding Test, in the Initial Mutation Test and in the Confirmatory Mutation Tests.

In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any treatment-related effect.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, and the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests; the examined concentration range was considered to be adequate. The study was considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Justification for classification or non-classification

The substance is not expected to meet the classification criteria according to Regulation (EC) 1272/2008 for mutagenicity based on the negative results obtained from the ames test.