2,6-dibromo-4-cyanophenyl octanoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Target substance:

Key, M-162493-01-2; Ames (similar to OECD 471, GLP); S. typhimurium TA 98, TA 100, TA 1535, TA 1537, and TA 1538; 33 – 10000 µg/plate (± S9 mix): negative

 

Source substance:

Key, source, RA-A, CAS 1689-84-5, M-251569-01-1; MLA (similar to Commission Directive 88/302/EEC, non-GLP); mouse lymphoma L5178Y cells:  15.6 – 250 µg/mL (-S9 mix), negative; 3.91 – 62.5 µg/mL (+S9 mix), positive

Key, source, RA-A, CAS 1689-84-5, M-280128-01-1; HPRT (similar to OECD 476, GLP); Chinese hamster V79 cells; 4.88 – 1250 µg/mL (± S9 mix): negative

Key, source, RA-A, CAS 1689-84-5, M-280151-01-1; HPRT (similar to OECD 476, GLP); CHO cells; 100 – 1000 µg/mL (± S9 mix): negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 Jul - 19 Jul 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1983

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 2020

Deviations:

yes

Remarks:

no strain with AT base pair at the primary reversion site used (e.g. TA 102 or E. coli WP2 uvrA), no justification for choice of vehicle and no historical control data provided

GLP compliance:

yes

Type of assay:

bacterial forward mutation assay

Target gene:

His operon (Salmonella strains)

Species / strain / cell type:

other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Fischer 344 rats treated with Aroclor 1254 i.p. at a dose of 500 mg/kg bw in corn oil; the cofactor solution was composed as follows: 0.05 M phosphate buffer (pH 7.4), 8 mM MgCI2x6H2O, 33 mM KCI, 25 mM glucose-6-phosphate (disodium salt), 4 mM NADP (disodium salt). The S9 mix was composed of 9 parts Cofactor solution and 1 part S9 fraction. The final concentration in culture was approximately 1.85% for S9 fraction and 18.5% for S9 mix.

Test concentrations with justification for top dose:

Toxicity experiment with TA 100 only:
with and without metabolic activation: 33, 100, 333, 1000, 3333, 10000 µg/plate

Mutation experiment 1 and 2:
with and without metabolic activation: 33, 100, 333, 1000, 3333, 10000 µg/plate
No toxicity to the bacteria was observed up to 10000 µg/plate, but precipitation of the test material was observed at this concentration. Therefore, based on the results of the cytotoxicity experiment, the highest dose selected for the mutation test was 10000 µg/plate.

Vehicle / solvent:

- Vehicle/solvent used: Dimethyl sulphoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene (2-AAN): TA 1535 and TA 1537 2 µg/plate and TA 1538, TA 98 and TA 100 0.5 µg/plate; + S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

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

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: approximately 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies per plate and/or by a thinning or disappearance of the bacterial background lawn

Rationale for test conditions:

according to OECD guideline 471

Evaluation criteria:

Acceptability
A test was considered acceptable if for each strain:
- the bacteria demonstrated their typical responses to crystal violet, ampicillin and UV light
- at least 2 of the vehicle control plates were within the following ranges: TA 1535, 4-30; TA 1537, 1-20; TA 98, 10-60; TA 100, 60-200 and TA 1538, 5-35
- on at least 2 of the positive control plates there were x 2 the mean vehicle control mutant numbers per plate, or in the case of TA 100, x 1.5 the mean vehicle control mutant numbers per plate. If the mean colony count on the vehicle control plates was less than 10 then a value of 10 was assumed for assessment purposes. In such cases a minimum count of 20 was required on at least 2 of the positive control plates.
- no toxicity or contamination was observed in at least 4 dose levels
- in cases where a mutagenic response was observed, that no more than one dose level was discarded before the dose which gave the highest significant mean colony number

Evaluation
A significant mutagenic response was recorded if:
- for S. typhimurium strains TA 1535, TA 1537, TA 1538 and TA 98, at least a doubling of the mean concurrent vehicle control values at some concentrations of the test substance and, for S. typhimurium strain TA 100, a 1.5-fold increase over the control value were observed. If the mean colony count on the vehicle control plates was less than 10 then a value of 10 was assumed for assessment purposes. In such cases a minimum count of 20 was required before a significant mutagenic response was identified.
- a dose related response was observed, although at high dose levels this relationship could be inverted because of, e.g. (1) toxicity to the bacteria generally, (2) specific toxicity to the mutants and (3) inhibition of foreign compound metabolizing enzymes where mutagens require metabolic activation by the liver.
- a reproducible effect in independent tests was observed.

Statistics:

No statistical analysis was done.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

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 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 3333 µg/plate in the presence of S9 and tested up to precipitating concentrations

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 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 3333 µg/plate in the presence of S9 and tested up to precipitating concentrations

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:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 10000 µg/plate in the presence of S9 and tested up to precipitating concentrations

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:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 10000 µg/plate in the presence of S9 and tested up to precipitating concentrations

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: Precipitation of the test material occurred at 10000 µg/plate in all conditions.

RANGE-FINDING/SCREENING STUDIES:
In the cytotoxicity experiment, no toxicity to the bacteria was observed up to 10000 µg/plate.

STUDY RESULTS
All criteria for a valid experiment were met. At 10000 µg/plate, cytotoxicity to the bacteria occurred in the presence of S9 mix only, in all strains, except TA1535. Cytotoxicity was also observed at 3333 µg/plate in TA1537 in both experiments and TA1538 in the second experiment only.
The test substance did not induce any significant, dose-dependent increases in the revertant numbers of any strain at any of the concentrations studied. For details, please refer to the attachment 1.
The solvent control induced the expected results and the standard positive controls induced marked increases in revertant numbers, thus, confirming the suitability and sensitivity of the assay.

HISTORICAL CONTROL DATA
Detailed historical control data were not provided.

Conclusions:

The study was performed in compliance with GLP and according to OECD guideline 471. Main deviations to the current OECD guideline 471 (adopted 2020) are related to the use of bacterial strains with only GC base pair at the primary reversion site (instead of TA 102 or E. coli WP2 uvrA) and the lack of historical control data. However, the study is considered reliable and valid. Under the conditions of the assay, the test item was not mutagenic up to 10000 µg/plate in the S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 with and without metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

Summary of available data used for the endpoint assessment of the target substance

Adequacy of study:

key study

Justification for type of information:

refer to the analogue justification provided in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

Source: CAS 1689-84-5, 1991, HPRT

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

Source: CAS 1689-84-5, 1985, HPRT

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

refer to the analogue justification provided in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

Source: CAS 1689-84-5, 1982, MLA

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

Source: CAS 1689-84-5, 1982, MLA

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Key, M-227072-01-1; MNT (similar to OECD 474, GLP); Mouse bone marrow; 52 – 267 mg/kg bw: negative

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Feb - 11 Apr 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 1983

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 2016

Deviations:

yes

Remarks:

No justification for route, duration and choice of vehicle provided, historical control data given without standard deviations, highest dose did not produce toxicity in bone marrow, only 1000 instead of 4000 immature erythrocytes per animal scored

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Limited, Kent, UK
- Age at study initiation (MNT part): 7 - 8 weeks
- Weight at study initiation (MNT part): 23 - 37 g males and 18 - 31 g females
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: individually in polypropylene and stainless steel cages measuring 48 cm x 15 cm x 13 cm.
- Diet: SOS Rat and Mouse Maintenance Diet No. 1 (Special Diet Services Limited, Essex, UK), ad libitum
- Water: ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 21
- Humidity (%): 37 - 69
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: (MNT part) from: 11 Mar 1991 To: 14 Mar 1991

Route of administration:

oral: gavage

Vehicle:

- Vehicle/solvent used: corn oil (Mazzola R)
- Amount of vehicle: 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Immediately prior to dosing, the test compound was dissolved in corn oil to give the required test concentration. All dosing solutions were kept on a magnetic stirrer during the dosing procedures.

Duration of treatment / exposure:

Single treatment

Frequency of treatment:

Once

Post exposure period:

Test substance:
- low dose: 24 h
- mid dose: 24 h
- high dose: 24, 48, 72 h
Control animals: 24, 48, 72 h
Positive control: 24, 48, 72 h

Dose / conc.:

52 mg/kg bw (total dose)

Remarks:

for male mice

Dose / conc.:

105 mg/kg bw (total dose)

Remarks:

for male mice

Dose / conc.:

183 mg/kg bw (total dose)

Remarks:

for male mice

Dose / conc.:

76 mg/kg bw (total dose)

Remarks:

for female mice

Dose / conc.:

153 mg/kg bw (total dose)

Remarks:

for female mice

Dose / conc.:

267 mg/kg bw (total dose)

Remarks:

for female mice

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: gavage
- Doses / concentrations: 80 mg/kg bw
Cyclophosphamide was prepared fresh as an 8 mg/mL solution in distilled water and administered to the positive control animals in standard dose volumes of 10 mL/kg bw.

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The doses were selected based on a dose range-finding test with 5 groups of 2 male and female CD-1 mice. Mice were orally treated once with doses ranging from 100 to 700 mg/kg bw of the test substance. These dose level were chosen based on acute oral LD50 information in rats. The mice were observed for clinical signs or mortality at frequent intervals (1 min, 0.5 h, 1 h, 2 h and 4 h) post dosing, then daily until the end of the observation period. Surviving animals were killed 14 days after dosing, by C02 asphyxiation. In the main toxicity test, 4 groups of 5 male and 5 female mice were orally treated on a single occasion with doses ranging from 125 to 500 mg/kg bw. Analogously to the range-finding test, animals were observed at the time of dosing and at 1 min, 0.5 h, 1 h, 2 h and 4 h after treatment, then daily until the end of the observation period for clinical signs or mortality. Surviving animals were killed 14 days after dosing by C02 asphyxiation. All animals were examined for gross pathology. Based on the results of this study, the doses for the micronucleus test were selected.

TREATMENT AND SAMPLING TIMES:
In the micronucleus test, 5 groups of male and female CD-1 mice were dosed once at 0 h with test or control agents, then marrow samples taken at time intervals of 24 h. In addition, bone marrow was also prepared after 48 and 72 h from animals treated with the positive and vehicle control and the high dose of the test substance. The mice were killed by cervical dislocation.

DETAILS OF SLIDE PREPARATION:
The femora were dissected out and freed of adherent tissue. A small hole was made in the neck of one femur and the marrow flushed, using a 1 mL syringe fitted with a gauge 25 needle, into a heparinized centrifuge tube containing 3 mL of a 1:1 mixture of fetal calf serum and 0.8% trisodium citrate in Sorenson's buffer, pH 6.8. This mildly hypotonic treatment served to make the micronuclei clearly visible and to distinguish them from surrounding artefacts. The contents of the tubes were briefly agitated on a vortex mixer to allow separation of the cells. The tubes were centrifuged to pellet the cells.
The main supernatant fluid was discarded. The cells were then resuspended on a vortex mixer in this residual amount of supernatant liquid. A drop of the suspension was placed at one end of the slide and a smear made by drawing the top of a Pasteur pipette horizontally along the slide. Two slides were prepared from each tube and animal. The smear was left to air dry, fixed in methanol and stained with 1% May-Grunwald in methanol and Sorenson's buffer and counterstained in 15% Giemsa (Gurr) in Sorenson's buffer. The stained smears were rinsed in 3 changes of distilled water and air dried. Finally, the smears were cleared in Histo-clear and permanent slide preparations obtained by sealing
glass coverslips onto the microscopic slides using DPX mountant.

METHOD OF ANALYSIS:
Micronuclei were analyzed with a light microscope. 1000 polychromatic erythrocytes (PCE) were counted per animal. The number of micronucleated normochromatic erythrocytes (NCE) was also recorded. The PCE/NCE ratio was determined for each animal by counting the number of immature (PCE) per mature (NCE) erythrocytes in a minimum total of 500 cells (PCE + NCE).

Evaluation criteria:

Evaluation of the results were based on Salamone et al. (1980) and Brue and Heddle (1979) and laboratory historical control data. Based on a mean frequency of micronucleated PCE of 0.128% per mouse (Salamone et al. 1980a), a positive response was suspected if the total numbers of micronuclei within any one sample group of a given number of mice exceeded the corresponding value of the historical control data (shown in attachment 1). A micronucleus within a polychromatic or normochromatic erythrocyte was defined as a distinct Giemsa-positive body of evident chromosomal origin and enclosed by the cytoplasm of the harboring cell. A negative response was recorded if the number fell below the corresponding historical control data. The cumulative historical micronucleus incidence in vehicle control dosed mice (distilled water, corn oil or 0.5% carboxymethylcellulose) in this laboratory has been determined as 0.121%, which is in line with the mean frequency of micronucleated PCE of 0.128% per mouse by Salamone et al.

Statistics:

Statistical analysis was not performed

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Two females treated with 267 mg/kg bw died, but no clinical signs or changes in the PCE/NCE ratios were observed

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
Treatment-related deaths were observed at 250, 400, 550 and 700 mg/kg bw. No abnormal clinical signs were observed prior to deaths. No unusual findings apart from bright red lungs in one male and one female mouse were observed by gross post mortem examination. Based on the results, animals in the main toxicity test were dosed with 125, 250, 375 and 500 mg/kg bw of the test substance.

RESULTS OF THE MAIN CYTOTOXICITY TEST
No deaths occurred at 125 mg/kg bw, whereas deaths were observed in the higher dose groups as follows:
250 mg/kg bw: 1/5 males and 0/5 females
375 mg/kg bw: 5/5 males and 3/5 females
500 mg/kg bw: 5/5 males and 4/5 females
These deaths were preceded by dose-related clinical signs comprising reduced activity, piloerection and subdued behavior (please also refer to the attachment 2). However, no changes were observed at the gross post mortem examination. Based on these results, the LD50 values were calculated for males (262 mg/kg bw) and for females (382 mg/kg bw). Therefore, the highest dose used in the micronucleus test was 183 mg/kg bw for males and 267 mg/kg bw for females (equal to 0.7 x LD50 dosages). For details, please refer to the attachment 2.

RESULTS OF THE MICRONUCLEUS TEST
- Clinical signs: No adverse reactions were observed following treatment.
- Mortality: 2 female mice died following exposure to 267 mg/kg bw of the test substance.
- Body weight: There was no evidence of body weight loss in the treated mice compared to the control animals.
- Induction of micronuclei: No difference were observed between control and treatment groups. The highest frequency of micronucleated polychromatic erythrocytes observed in the test groups was 0.16%, which was identical to the highest vehicle control frequency.
- Ratio of PCE/NCE: No difference were observed between control and treatment groups at any time point.
- Positive control: Substantial induction of bone marrow micronuclei were observed in animals treated with 80 mg/kg bw cyclophosphamide, notably in samples taken 24 and 48 h after dosing. The positive control also caused a decline in PCE/NCE ratios compared to the vehicle control.
- Vehicle control: The numbers of micronucleated bone marrow polychromatic erythrocytes in the control mice were within the in-house historical control range for negative control exposed mice at all experimental points.
For details, please refer to the attachment 3.

Conclusions:

The study was performed according to OECD guideline 474 and compliant with GLP. Under the conditions of this study, the test substance was found to be devoid of micronucleus inducing potential when tested to lethal doses in male and female CD-1 mice using a single oral exposure and multiple sampling time protocol.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

No data available.

Additional information

Several studies are available with respect to genotoxicty of the target substance (CAS 1689-99-2). In addion, a read-across approach to the analogue substance (CAS 1689-84-5) was performed. The read-across is based on (bio) transformation to common compound(s), which thus have the same biological target(s) and therefore cause the same effects. A detailed justification for read-across is provided in the technical dossier (see IUCLID section 13.2).

To characterize the mutagenic/genotoxic potential of the target substance, several in vitro and in vivo studies have been conducted on the target as well as on the source substance, following testing protocols equivalent or similar to currently acknowledged test guidelines.

 

Genetic toxicity in vitro

Bacterial mutagenicity of the target substance

The target substance (CAS 1689-99-2) was investigated in an Ames test, considered as key study, with respect to bacterial mutagenicity (M-162493-01-1). The study was performed in accordance with GLP and similar to OECD 471 (2020) with a standard battery of tester strains including 5 Salmonella typhimurium strains (TA 98, TA 100, TA 1535, TA 1537, TA 1538). There were two noteworthy deviations from the current version of OECD 471, namely the fact that no strain with AT base pair at the primary reversion site (e.g. TA 102 or E. coli WP2uvrA) has been included in the study protocol and that no historical control data are available for the testing laboratory. However, the study is considered valid and acceptable for the assessment. The target substance solved in dimethyl sulfoxide (DMSO) and tested in the plate incorporation assay at concentrations ranging from 33 to 10000 µg/plate in the presence and in the absence of S9 in two independent mutagenicity tests. Analytical determination of the test solutions (low and high dose) gave 96.9 to 107.2% of the intended nominal concentrations. Cytotoxicity to the bacteria occurred in all strains, except TA 1535 at a concentration of 10000 µg/plate in the presence of S9 only. Cytotoxicity was also observed at 3333 µg/plate in TA1537 in both experiments and TA 1538 in the second experiment only. Precipitation of the test material occurred at 10000 µg/plate under all test conditions. The target substance did not induce any significant, dose-dependent increases in the revertant numbers of any strain at any of the concentrations studied. The solvent control induced the expected results and the standard positive controls induced marked increases in revertant numbers, thus, confirming the suitability and sensitivity of the assay. Under the conditions of the assay, the test item was not mutagenic up to 10000 µg/plate in the S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 with and without metabolic activation.

 

Bacterial mutagenicity of the source substance

In addition, a study on bacterial mutagenicity is available for the source substance and, in line with the test results established with the target substance, gave no indication for a mutagenic potential in bacteria. The source substance was tested in an Ames test conducted according to EPA OPP 84-2 and similar to OECD Guideline 471 (2020). The GLP-compliant study was performed with a standard battery of tester strains including 5 Salmonella typhimurium strains (TA 98, TA 100, TA 1535, TA 1537, TA 1538; M-227063-01-1). There were two noteworthy deviations from the current version of OECD 471, namely the fact that no strain with AT base pair at the primary reversion site (e.g. TA 102 or E. coli WP2uvrA) has been included in the study protocol and that no historical control data are available for the testing laboratory. However, the study is considered valid and acceptable for the assessment. The source substance (CAS 1689-84-5) was completely soluble in dimethyl sulfoxide (DMSO) and no precipitation was observed at any concentration tested. In the dose range-finding experiment, the concentration range of the test item was 6.67 – 5000 µg/plate with and without metabolic activation. Based on the range-finding study, the concentration which gave a detectable reduction of revertants per plate and/or a thinning or disappearance of the bacterial background lawn, was selected as the highest concentration of the test article in the subsequent mutagenicity assay. Therefore, the source substance was tested in the plate incorporation assay at concentrations ranging from 10 to 3330 µg/plate in the presence of S9 and 3.33 to 1000 µg/plate in the absence of S9 in two independent mutagenicity tests. Analysis of the test solutions gave 95.2 to 104% of the intended nominal concentrations, thus demonstrating a sufficient content and stability. The analogue substance did not induce relevant increases in revertant colony numbers for any tester strain neither in the absence nor presence of metabolic activation. Cytotoxicity to bacteria was observed at 1000 µg/plate, evident by the thinning or disappearance of the bacterial background lawn and/or the reduced number of revertants on the plate. The positive control produced marked increases in revertant numbers and the solvent controls induced the expected results, both confirming the suitability and sensitivity of the assay. Under the conditions of the assay, the source substance is considered to be non-mutagenic in S. typhimuirum strains TA 98, TA 100, TA 1535, TA 1537 and in TA 1538 with and without metabolic activation.

DNA repair assay in mammalian cells with the target substance

The target substance (CAS 1689-99-2) was furthermore investigated with regards to genotoxicty in an unscheduled DNA synthesis (UDS) assay in vitro, which is considered as a supporting study (M-227070-01-1). The study was performed according to GLP and EPA OPP 84-2. Primary cultures of adult rat hepatocytes were prepared from adult, male Fischer 344 rats. Cultures were exposed to the test agent for 18-20 h using concentrations of the target substance from 0.98 to 125 µg/mL in two independent experiments. The test subtacne was dissolved in DMSO and solutions were reported to be homogeneous and stable for at least 24 h at ambient temperature in the dark. Analysis conducted on the highest and lowest dosing solution demonstrated that the percentage difference from nominal concentrations was less than 10%. In both assays, toxicity was evident in cultures dosed with 31.25, 62.5 and 125 µg/mL. In the 5 highest assessable (non-toxic) concentrations of the test item, no evidence of unscheduled DNA synthesis was detected. The vehicle and untreated controls revealed expected results and the positive control produced net grain values in the normal UDS positive range. Therefore, the vehicle, negative and positive controls from both assays were considered valid. Under the experimental conditions reported, the test substance was found to be inactive in the primary rat hepatocyte UDS assay.

As the UDS test in vitro conducted with the target substance is considered to be only an "indicator or satellite" test according to the endpoint specific guidance R7a (ECHA, 2017), the data requirement according to Annex VIII of REACH regulation requiring an in vitro gene mutation study in mammalian cells was covered by read across to the analogue substance (source, CAS 1689-84-5). For the source substance three in vitro gene mutation assays in mammalian cells are available, i.e. one mouse lymphoma assay (MLA) and two HGPRT assays.

Gene mutations in mammalian cells with the source substance

The mutagenicity of the source substance (CAS 1689-84-5) was evaluated in a mouse lymphoma forward mutation assay, which complies to a considerable extent to the Commission Directive 88/302/EEC (M-251569-01-1). When the study was performed, GLP was not mandatory at that time (1982). However, a quality assurance statement was provided. The mouse lymphoma L5178Y cells were exposed to the test item for 4 h in the presence and absence of a metabolic activation system. In the absence of S9, cultures were incubated with 15.6 to 500 µg/mL of the analogue substance. In the presence of S9, cultures were treated with concentrations between 3.91 and 62.5 µg/mL. The test item, dissolved in DMSO, appeared miscible in the assay medium up to 125 µg/mL. At concentrations of > 250 µg/mL, a pale yellow color and white precipitate were noted. The negative and solvent control average cloning efficiencies ranged from 81.9% (without S9) to 79.8% (with S9), thus demonstrating good cloning conditions. In the presence and absence of S9 mix, the test substance caused a dose-dependent decrease in the percent relative growths, thus, showing moderate and high toxicity. Further, no clear increases in mutant frequencies were noted in the absence of S9 at any of the source substance concentrations studied. In the presence of S9 mix, 31.3 and 62.5 µg/mL of the test item induced mutant frequencies above the minimum criterion for significance. A 2-fold increase in the mutant frequency was observed at the weakly toxic concentration of 31.3 µg/mL (39.6% relative growth) and a 4.1-fold increase was noted at the moderately toxic concentration of 62.5 µg/mL (7.9% relative growth). The mutant frequencies of the negative and solvent controls were within the normal range. Positive control agents, ethylmethane sulphonate (-S9) and dimethylnitrosamine (+S9), yielded the expected mutant frequencies that were greatly in excess of the background. In conclusion, under the conditions of this study, the source substance was considered to be active in the mouse lymphoma forward mutation assay at weakly to moderately toxic concentrations in the presence of a metabolic activation system. However, it has to be noted that the test material used in the assay was not specified and an independent repeat experiment was not performed. Therefore, the study is considered reliable with restrictions.

In the second key study, the potential of the test substance to induce mutagenic effects was investigated in the HGPRT assay in Chinese hamster V79 cells (M-280128-01-1). This study was performed compliant to GLP and the EEC Directive 79/831, Annex V, Part B, following a test protocol similar to OECD 476 (1984). V79 cultures were exposed to the source substance (CAS 1689-84-5) for 3 h in the absence and presence of a metabolic activation system. The concentrations used in the main test were based on a preliminary cytotoxicity experiment. Therefore, in the first experiment cultures were treated with 39.0 – 625 µg/mL of the test item in the absence of S9 and with 4.88 – 1250 µg/mL in the presence of S9. In the second experiment cells were treated with 19.5 – 625 µg/mL of the analogue substance, both in the absence and presence of S9. The test item was completely soluble in DMSO, but a slight and dose-related reduction in the pH was observed ranging between 6.7 and 7.2 in the absence and presence of S9. In the preliminary cytotoxicity experiment, no cytotoxic effects were observed at concentrations up to and including 1250 µg/mL in the absence of S9. In the presence of metabolic activation, a dose-dependent decrease of the survival was observed reaching approximately 7% at 1250 µg/mL. In the main mutation assay, the average cloning efficiencies of the solvent controls were high. In the absence of S9, the analogue substance produced dose-related toxicity. The % relative survival after treatment at 625 µg/ml was 12% and 3% in the first and second trial, respectively. No significant (i.e. >5-fold) increases in mutant frequencies were noted at any of the test substance concentrations studied. In the presence of S9, the analogue substance also produced dose-related toxicity. At 1250 µg/mL the culture treated in the absence of S9 was dead and could not be plated. The % relative survival after treatment at 625 µg/mL was 17% and 20% in the first and second trial, respectively. In the first main experiment, a 5-fold increase in mutant frequency over the solvent control was observed at 4.88 µg/mL in the presence of S9. Since this increase was only found at the lowest dose-level of the test substance and was not reproduced in the second experiment, this was considered to be not related to the treatment but rather to the extremely low spontaneous mutant frequency observed for the vehicle control. No further significant increases in mutant frequencies were noted at any of the test substance concentrations investigated. The mutant frequencies of the solvent controls were found to be within the normal range. Positive control agents, ethylmethanesulphonate (-S9) and dimethylnitrosamine (+S9), yielded mutant frequencies that were greatly in excess of the background. Under the conditions of this study, the analogue substance was considered to be negative for inducing forward mutations at the HGPRT locus in Chinese hamster V79 cells in the presence and absence of a metabolic activation system.

In a further HGPRT study the mutagenic effects of the source substance on CHO cells were investigated (M-280151-01-1). This study was performed in compliance with GLP and EPA OPP 84-2 and followed a testing protocol similar to OECD 476 (1984). During the study, CHO cells were exposed to the source substance (CAS 1689-84-5) for 4 h in the presence and absence of a metabolic activation system. The concentrations used in the study were based on a cytotoxicity assay using 1.95 – 1000 µg/mL of the test item. In the main test, cells were treated with 100 – 1000 µg/mL of the test substance in the presence and absence of S9 mix. The test article was dissolved in DMSO and did not alter the pH of the treatment medium outside the range of pH 7.0 to pH 7.8 at any applied concentration. The test item remained in solution in culture medium up to a maximum applied concentration of about 600 µg/mL. The precipitate disappeared with time in at least one instance at a concentration of 1000 µg/mL. Samples of the dilutions used in the mutagenicity assay were analyzed by high performance liquid chromatography to assess the accuracy of the formulations. Analysis of the test solutions gave 96.3 to 101% of the intended nominal concentrations. The average cloning efficiencies of the solvent controls were high. With and without S9, a dose-related decrease in both relative survival and relative population growth was observed. Without metabolic activation, the concentrations of 100 to 1000 µg/mL showed a relative population growth compared to the concurrent vehicle controls of 88.1 to 4.5%. In contrast, with metabolic activation 900 µg/mL and 1000 µg/mL were lethal and not available for evaluation. 800 µg/mL caused a decrease in relative population growth of 2.7%, whereas 600 µg/mL and 500 µg/mL were moderately toxic with a relative population growth of 56.4% and 65.8% relative population growth, respectively. Under non-activation conditions, the test substance did not induce mutant frequencies that were statistically elevated over the mutant frequencies of the concurrent vehicle control cultures. In the presence of S9, four of the seven analyzed treatments had statistically elevated mutant frequencies, however, no dose-response was observed and the mutant frequencies were within the acceptable range as they were within normal assay variation observed at the testing laboratory. Therefore, the increases were not considered indicative of a positive response. The mutant frequencies of the solvent controls were found to be within the normal range. Positive control agents, 5-bromo-2'-deoxyuridine (-S9) and 3-methylcholanthrene (+S9), yielded mutant frequencies that were greatly in excess of the background. In conclusion, under the conditions of this study, the source substance was considered to be negative for inducing forward mutations at the HGPRT locus in CHO cells in the presence and absence of a metabolic activation system.

 

Conclusion on genetic toxicity in vitro:

The Ames and UDS assay with the target substance as well as the Ames and HGPRT assay with the source substance provided negative results both in bacteria (Salmonella typhimurium) and in mammalian cells (rat hepatocytes, V79 and CHO). Controversial results were obtained by the mouse lymphoma forward mutation assay conducted with the source substance. The positive findings were only observed at cytotoxic concentrations. As the mouse lymphoma assay was conducted in 1982, the study did not follow the current OECD testing guidelines. Further, the test substance was not characterized (no information on purity). Therefore, the study is considered only reliable with restrictions. As negative results were obtained in the more recent HPRT studies, assessment of mutagenicity in vitro of the analogue substance was found inconclusive. An in vitro cytogenicity study is not required as adequate in vivo studies (MNT and UDS) are available (please refer to “Genetic toxicity in vivo” M-227072-01-1 and M-202441-01-1).

 

Genetic toxicity in vivo:

Regarding the assessment of genetic toxicity of the target and source substance in vivo, a cytogenetic assay in CD-1 mouse is available (MNT, OECD 474) fulfilling the criteria of a key study. In addition, an unscheduled DNA synthesis assay in Fischer 344 rats (OECD 486) was regarded as supporting study.

 

In vivo somatic cell genotoxicity study with the target substance

In the mammalian erythrocyte micronucleaus test, the target substance (CAS 1689-99-2) was assessed for its potential to induce micronuclei in bone marrow cells of CD-1 mice by a GLP-compliant test according to OECD Guideline 474 (1983, M-227072-01-1). Groups of 5 mice per sex were once treated by gavage with vehicle alone (corn oil) or 52, 105 or 183 mg/kg bw for males and 76, 153 or 267 mg/kg bw for females. At the end of the study, animals were sacrificed 24 h following treatment and the bone marrow was prepared by removal of the femora. Additional animals treated with the vehicle or the positive control or the highest test subtsance dose were sacrificed 48 and 72 h following dosing. Dose preparations from the main micronucleus test with the test substance were retained for analysis, but an analytical report was not provided. In the main cytotoxicity test, deaths occurred at >125 mg/kg bw as 5/5 males and 4/5 females were found dead at 500 mg/kg bw. These deaths were preceded by dose-related clinical signs including reduced activity, piloerection and subdued behavior. However, no changes were observed at the gross post mortem examination. Based on these results, the LD50 values were calculated for males (262 mg/kg bw) and for females (382 mg/kg bw). Therefore, the highest dose used in the micronucleus test was 183 mg/kg bw for males and 267 mg/kg bw for females (equal to 0.7 x LD50 dosages). In the micronucleus test, two females of the high dose group died. The incidence of micronucleated polychromatic erythrocytes (MN-PCE) in the bone marrow of the test substance exposed mice was similar to negative control frequencies at all sampling times. The numbers of MN-PCE in mice dosed with the vehicle were within historical control ranges. The exposure of mice to the positive control agent resulted in substantial inductions of MN-PCE, notably in samples taken 24 and 48 h after dosing. Under the conditions of this study, the target substance was found to be devoid of micronucleus inducing potential when tested to lethal doses in male and female CD-1 mice using a single oral exposure and multiple sampling time protocol.

 

In vivo somatic cell genotoxicity study with the source substance

In a supporting study, the potential of the source substance (CAS 1689-84-5) to induce DNA damage followed by DNA repair was investigated in Fisher 344 rats (M-202441-01-1). This SCE assay was performed according to GLP and OECD guideline 486. Rats were administered by gavage at dose levels of 0, 12.5, 25 and 50 mg/kg bw. 0.5% methylcellulose aqueous solution served as vehicle control. After an exposure period of 2 or 16 h, rats were anesthetized followed by in situ perfusion of the liver. The hepatocytes were isolated by collagenase perfusion. Rat hepatocytes were cultured, radio-labelled, fixed and developed for autoradiography. For the DNA repair test, only the slides of the 25 and 50 mg/kg bw dose levels were chosen to be examined. Dose-dependent reduction of cell viability was observed in hepatocytes treated with the test substance. Severe cytotoxicity was observed in hepatocytes of animals treated with 50 mg/kg bw when compared to the controls. The cell viability was reduced to 32 - 33% at the 2 h sampling time and to 39 - 47% at the 16 h sampling time related to the controls. Statistically significant increases compared to control groups were observed in tritiated thymidine incorporation in groups treated with 50 mg/kg bw and at the 2 h sampling time. At this dose and sampling time, the percentage of cells under repair was in some occasions higher than 20%. In contrast, 50 mg/kg bw at the 16 h sampling time did not induce any statistically significant increases in tritiated thymidine incorporation compared with the control group. The same applies to the lower dose of 25 mg/kg bw at both sampling times (2 and 16 h). The positive controls (methylmethane sulfonate and 2-acetylaminofluorene) caused a statistically significant increase in tritiated thymidine incorporation in comparison. The percentage of cells under repair ranged between 66 and 84%. Under the experimental conditions applied, it can be concluded that the source substance induced UDS in rat liver cells only at the 2 h sampling time and at the highest dose tested (50 mg/kg bw) in presence of severe cytotoxic effects. No increase in UDS was induced at the later sampling time at the dose of 50 mg/kg bw. No dose-correlation was found as no UDS induction was observed at the level of 25 mg/kg bw.

 

Conclusion on genetic toxicity in vivo:

The target substances was clearly negative following oral administration in a mouse bone marrow micronucleus test. Positive findings were obtained for the source substance in the UDS assay with rats in the presence of severe cytotoxic effects. No dose-correlation was found since no UDS induction was observed at lower dose levels tested. Furthermore, the observed effect was only observed at the 2 h sampling time, but not at the later sampling time of 16 h. Overall, as the MNT conducted with the target substance was negative and the positive response in the UDS in vivo with the source substance was only seen at the early sampling time and at severely cytotoxic doses, the conclusion that the target substance does not possess any genotoxic potential in vivo is justified. This is supported by the negative outcome of the UDS in vitro performed with the target substance in primary hepatocytes. This assessment is in line with the conclusion drawn by the EFSA.

 

Conclusion on genetic toxicity of the test substance

Several in vitro assays (Ames, UDS, MLA and HGPRT) and in vivo studies (MNT, UDS) are available for the target (CAS 1689-99-2) and the source substance (CAS 1689-84-5). The target substance was tested in an Ames assay and in an UDS in vitro study as well as in an MNT in vivo in mice. For the source substance an Ames assay, a mouse lymphoma forward mutation test and two HGPRT studies were included in the assessment of genetic toxicity. Genetic toxicity of the source substance was investigated in an UDS study in vivo. Since some of the studies were conducted according to older versions of the OECD guidelines, they were considered to be reliable with restrictions. However, they were deemed acceptable following review and evaluation. Whereas both ames assays, the UDS in vitro and both HGPRT assays were unequivocally negative, the MLA with the source substance provided a positive response at weakly to moderately cytotoxic concentrations in the presence of a metabolic activation system. Therefore, two in vivo studies were included in the hazard assessment comprising a micronucleus test with the target substance and an unscheduled DNA synthesis test conducted with the source substance. Whereas the MNT is considered an in vivo test for the assessment of clastogenic and aneugenic potential, the UDS is an indicator test measuring DNA repair of primary damage in liver cells. The UDS can therefore also be used as an acceptable in vivo test for detecting the potential of gene mutations, under the premiss that the liver is a target organ for the test substance (Guidance on Information Requirements and Chemical Safety Assessment, R.7a, ECHA, 2017). This is the case for the source substance.

The overall assessment of the genetic toxicity in vivo data leads to the conclusion that the source and the target substance do not possess any genotoxic potential in vivo.

References not included in IUC:

Detailed information on references not included in IUC are available in the CSR and in chapter 13.

Justification for classification or non-classification

The available data on genetic toxicity of the target and source substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.