Isobutyl 4-chloro-3,5-diaminobenzoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro:

- Gene mutation (Bacterial reverse mutation assay / Ames test): S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 1538, ± S9, 2 grades of purification tested (similar to OECD 471): positive in TA1537, TA1538, TA98 +S9 (Frameshift mutations), negative in TA1535 and TA100 (basepair substitutions); purified compound was negative in TA1537, TA1538, TA98 +S9, non-purified compound was positive in the two highest tested concentrations in TA1537, TA1538, TA98 –S9

- Gene mutation (Bacterial reverse mutation assay / Ames test): S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 1538, ± S9 (similar to OECD 471): positive in TA1537, TA1538, TA98 ±S9 (Frameshift mutations), negative in TA1535 and TA100 ± S9 (basepair substitutions)

- Gene mutation (Bacterial reverse mutation assay / Ames test): S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 1538, ± S9 (similar to OECD 471): positive in TA1537, TA1538, TA98 +S9 (Frameshift mutations), negative in TA1537, TA1538, TA98 –S9, negative in TA1535 and TA100 ±S9 (basepair substitutions)

- Gene mutation (Bacterial reverse mutation assay / Ames test): S. typhimurium TA 1535, TA 1537, TA 98, TA 100, ± S9 (similar to OECD 471): positive in TA100 + S9 (basepair substitutions), negative in TA 100 –S9, positive in TA1537 ± S9 (Frameshift mutations, when cytotoxicity occurred), positive in TA98 + S9 (Frameshift mutations), negative in TA98 –S9, negativ in TA1535± S9(basepair substitutions)

- Gene mutation (Mammalian cell forward gene mutation assay, mouse lymphoma assay): mouse lymphoma L5178Y cells ± S9 (similar to OECD 490): negative ± S9

- Gene mutation (Mammalian cell forward gene mutation assay, HPGRT assay):CHO-K1cells ± S9 (similar to OECD 476): negative ± S9

- DNA damage and repair (UDS assay): primary rat hepatocytes, -S9 (similar to OECD 482), up to 752 µg/ml: negative

- In vitro transformation in mammalian cells: BHK 21 C13 of Syrian hamster kidney cells, ± S9, up to 1000 µg/ml: no evidence of cell transforming potential ± S9

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1978-09-29 - 1978-10-17

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Well-documented bacterial reverse mutation assay, conducted on the registered subtance itself, similar to OECD 471. TA 1538 was used as fifth tester strain instead of E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102. However, as these strains were induced to primarily detect cross-linking mutagens, and this is not the expected mode of action for a potential genotoxicity of isobutyl 4-chloro-3,5-diaminobenzoate, this deviation is not expected to have any impact on the reliability of the results. Also, minor parts of the test report were not available.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not applicable

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

his

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: see "any other information on materials and methods"

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: see "any other information on materials and methods"

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

10, 50, 100, 500, 1000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Remarks:

bacteria only / S9 fraction

Negative solvent / vehicle controls:

yes

Remarks:

DMSO 100 µL

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

9-aminoacridine

2-nitrofluorene

benzo(a)pyrene

other: N-methyl-N-nitro-N-nitrosoguanidine (MNNG), 6-Aminochrysene (6-AC), 2- Aminoanthracene (2-AA), Aflatoxin B1, 2-Aminofluorene (2-AF)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

SELECTION AGENT (mutation assays): histidine-free media

NUMBER OF REPLICATIONS: triplicates

Evaluation criteria:

All 3 control plates must have approximately equal numbers of revertant colonies. The solvent controls must have approximately the same number of colonies as spontaneous reversion controls. Positive mutagens must give at least 3x the number of colonies as the controls for spontaneous reversion. Any test with a strain which does not meet these criteria must be repeated on a separate day.
All criteria noted above must be met before results with an unknown agent can be evaluated. To be considered positive, an unknown agent should exhibit a dose response effect. That is, there should be an increasing number of mutants with increased amounts of test agent. For strains TA1535, TA1537, TA1538 and TA98, the first positive dose must produce 3x the number of colonies as the negative control. For strain TA100, at least one dose tested must produce 3.5x the number of mutants as the spontaneous reversion control.

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:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: none stated

Conclusions:

Interpretation of results: ambiguous

The study was well-performed according a method similar to OECD 471, there is no indication given that the results are not reliable. Hence, they can be used to assess the potential of isobutyl 4-chloro-3,5-diaminobenzoate to induce gene mutations in bacteria. The test agent did not induce a significant increase in the number of point mutations in Salmonella typhimurium strains in the absence and presence of the activating system for strains TA1535, TA100. Hence, it should be regarded as negative is this test. On the other side, it did induce a significant increase in the number of point mutations in Salmonella typhimurium strains in the absence and presence of the activating enzyme system of strains TA1537, TA1538, TA98. The increase was dose-dependent and ≥ 3 times over control, hence, it should be regarded as positive in this test. So, only based on this data, no conclusions can be drawn for the genotoxic potential of isobutyl 4-chloro-3,5-diaminobenzoate. The results should be considered as ambiguous, and additional data is required for a definitive assessment.

Executive summary:

In a reverse gene mutation assay in bacteria (similar to OECD 471), strains TA1535, TA100, TA1537, TA1538, TA98 of S. typhimurium were exposed to of isobutyl 4-chloro-3,5-diaminobenzoate in DMSO at concentrations of 10, 50, 100, 500, 1000 µg/plate in the presence and absence of mammalian metabolic activation in plate incorporation.

The positive controls induced the appropriate responses in the corresponding strains. There was partial evidence and a concentration related positive response of induced mutant colonies over background.

The test agent did not induce a significant increase in the number of point mutations in Salmonella typhimurium strains in the absence and presence of the activating system for strains TA1535, TA100. It did induce a significant increase in the number of point mutations in Salmonella typhimurium strains in the absence and presence of the activating enzyme system of strains TA1537, TA1538, TA98. The increase was dose-dependent and ≥ 3 times over control. The results should be considered as ambiguous.

This study is classified as acceptable.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1978-09-29 - 1978-10-17

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Well-documented bacterial reverse mutation assay, conducted on the registered subtance itself, similar to OECD 471. TA 1538 was used as fifth tester strain instead of E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102. However, as these strains were induced to primarily detect cross-linking mutagens, and this is not the expected mode of action for a potential genotoxicity of isobutyl 4-chloro-3,5-diaminobenzoate, this deviation is not expected to have any impact on the reliability of the results. Also, minor parts of the test report were not available.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not applicable

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

his

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: see "any other information on materials and methods"

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: see "any other information on materials and methods"

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

10, 50, 100, 500, 1000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Remarks:

bacteria only / S9 fraction

Negative solvent / vehicle controls:

yes

Remarks:

DMSO 100 µl

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

9-aminoacridine

2-nitrofluorene

benzo(a)pyrene

other: N-methyl-N-nitro-N-nitrosoguanidine (MNNG), 6-Aminochrysene (6-AC), 2- Aminoanthracene (2-AA), Aflatoxin B1, 2-Aminofluorene (2-AF)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

SELECTION AGENT (mutation assays): histidine-free media

NUMBER OF REPLICATIONS: triplicates

Evaluation criteria:

All 3 control plates must have approximately equal numbers of revertant colonies. The solvent controls must have approximately the same number of colonies as spontaneous reversion controls. Positive mutagens must give at least 3x the number of colonies as the controls for spontaneous reversion. Any test with a strain which does not meet these criteria must be repeated on a separate day.
All criteria noted above must be met before results with an unknown agent can be evaluated. To be considered positive, an unknown agent should exhibit a dose response effect. That is, there should be an increasing number of mutants with increased amounts of test agent. For strains TA1535, TA1537, TA1538 and TA98, the first positive dose must produce 3x the number of colonies as the negative control. For strain TA100, at least one dose tested must produce 3.5x the number of mutants as the spontaneous reversion control.

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:

valid

Positive controls validity:

valid

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:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: none stated

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results: ambiguous

The study was well-performed according a method similar to OECD 471, there is no indication given that the results are not reliable. Hence, they can be used to assess the potential of isobutyl 4-chloro-3,5-diaminobenzoate to induce gene mutations in bacteria. The test agent did not induce a significant increase in the number of point mutations in Salmonella typhimurium strains in the absence and presence of the activating system for strains TA1535, TA100. Hence, it should be regarded as negative is this test. On the other side, it did induce a significant increase in the number of point mutations in Salmonella typhimurium strains in the absence and presence of the activating enzyme system of strains TA1537, TA1538, TA98. The increase was dose-dependent and ≥ 3 times over control, hence, it should be regarded as positive in this test. So, only based on this data, no conclusions can be drawn for the genotoxic potential of isobutyl 4-chloro-3,5-diaminobenzoate. The results should be considered as ambiguous, and additional data is required for a definitive assessment.

Executive summary:

In a reverse gene mutation assay in bacteria (similar to OECD 471), strains TA1535, TA100, TA1537, TA1538, TA98 of S. typhimurium were exposed to of isobutyl 4-chloro-3,5-diaminobenzoate in DMSO at concentrations of 10, 50, 100, 500, 1000 µg/plate in the presence and absence of mammalian metabolic activation in plate incorporation.

The positive controls induced the appropriate responses in the corresponding strains. There was partial evidence and a concentration related positive response of induced mutant colonies over background.

The test agent did not induce a significant increase in the number of point mutations in Salmonella typhimurium strains in the absence and presence of the activating system for strains TA1535, TA100. It did induce a significant increase in the number of point mutations in Salmonella typhimurium strains in the absence and presence of the activating enzyme system of strains TA1537, TA1538, TA98. The increase was dose-dependent and ≥ 3 times over control. The results should be considered as ambiguous.

This study is classified as acceptable.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1978-10-03 - 1978-10-17

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Well-documented bacterial reverse mutation assay, conducted on the registered subtance itself, similar to OECD 471. TA 1538 was used as fifth tester strain instead of E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102. However, as these strains were induced to primarily detect cross-linking mutagens, and this is not the expected mode of action for a potential genotoxicity of isobutyl 4-chloro-3,5-diaminobenzoate, this deviation is not expected to have any impact on the reliability of the results. Also, minor parts of the test report were not available.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not applicable

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

his

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: see "any other information on materials and methods"

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: see "any other information on materials and methods"

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

10, 50, 100, 500, 1000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Remarks:

bacteria only / S9 fraction

Negative solvent / vehicle controls:

yes

Remarks:

100 µL

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

9-aminoacridine

2-nitrofluorene

benzo(a)pyrene

other: N-methyl-N-nitro-N-nitrosoguanidine (MNNG), 6-Aminochrysene (6-AC), 2- Aminoanthracene (2-AA), Aflatoxin B1, 2-Aminofluorene (2-AF)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

SELECTION AGENT (mutation assays): histidine-free media

NUMBER OF REPLICATIONS: triplicates

Evaluation criteria:

All 3 control plates must have approximately 'equal numbers of revertant colonies. The solvent controls must have approximately the same number of colonies as spontaneous reversion controls. Positive mutagens must give at least 3x the number of colonies as the controls for spontaneous reversion. Any test with a strain which does not meet these criteria must be repeated on a separate day.
All criteria noted above must be met before results with an unknown agent can be evaluated. To be considered positive, an unknown agent should exhibit a dose response effect. That is, there should be an increasing number of mutants with increased amounts of test agent. For strains TA1535, TA1537, TA1538 and TA98, the first positive dose must produce 3x the number of colonies as the negative control. For strain TA100, at least one dose tested must produce 3.5x the number of mutants as the spontaneous reversion control.

Species / strain:

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

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: none stated

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results: ambiguous

The study was well-performed according a method similar to OECD 471, there is no indication given that the results are not reliable. Hence, they can be used to assess the potential of isobutyl 4-chloro-3,5-diaminobenzoate to induce gene mutations in bacteria.
The test agent did not induce a significant increase in the number of point mutations in Salmonella typhimurium strains in the absence of the activating system for strains TA1535, TA1537, TA1538, TA100, TA98. It also did not induce a significant increase in the number of point mutations with the addition of an exogenous source of liver enzymes for metabolic activation in strains TA1535, TA100. Hence, it should be regarded as negative is this test.
On the other side, the test agent did induce a significant increase in the number of point mutations with the addition of an exogenous source of liver enzymes for metabolic activation in strains TA1537, TA1538, TA98. The increase was dose-dependent and ≥ 3 times over control, hence, it should be regarded as positive in this test. So, only based on this data, no conclusions can be drawn for the genotoxic potential of isobutyl 4-chloro-3,5-diaminobenzoate. The results should be considered as ambiguous, and additional data is required for a definitive assessment.

Executive summary:

In a reverse gene mutation assay in bacteria (similar to OECD 471), strains TA1535, TA100, TA1537, TA1538, TA98 of S. typhimurium were exposed to isobutyl 4-chloro-3,5-diaminobenzoate in DMSO at concentrations of 10, 50, 100, 500, 1000 µg/plate in the presence and absence of mammalian metabolic activation in plate incorporation.

The positive controls induced the appropriate responses in the corresponding strains. There was partial evidence and a concentration related positive response of induced mutant colonies over background.

The test agent did not induce a significant increase in the number of point mutations in Salmonella typhimurium strainsin the absence of the activating system for strains TA1535, TA1537, TA1538, TA100, TA98, and in the presence of the S9 mix in strains TA1535, TA100.

It did induce a significant increase in the number of point mutations in Salmonella typhimurium strains in the presence of the activating enzyme system of strains TA1537, TA1538, TA98. The increase was dose-dependent and ≥ 3 times over control. The results should be considered as ambiguous.

This study is classified as acceptable.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Oct+Nov 1978 (experimental phase)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Well-documented bacterial reverse mutation assay, conducted on the registered subtance itself, similar to OECD 471. E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 as fifth tester strains were lacking. However, as these strains were induced to primarily detect cross-linking mutagens, and this is not the expected mode of action for a potential genotoxicity of isobutyl 4-chloro-3,5-diaminobenzoate, this deviation is not expected to have any impact on the reliability of the results.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

recommending only four tester strains

Deviations:

not applicable

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

n/a

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

Test concentrations with justification for top dose:

Used concentrations: 0, 4, 20, 100, 500, 2500 µg/plate
Justification for top dose: toxicity

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (test item, trypaflavin) water, demineralized (Endoxan)

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (test item, trypaflavin) water, demineralized (Endoxan)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Trypaflavin

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation, method according to Ames et al.

DURATION
- Exposure duration: 48h

SELECTION AGENT (mutation assays): his-negative medium

NUMBER OF REPLICATIONS: 4

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A reproducible dose-dependent two-fold increase in mutation colonies compared to negative control is considered a positive result.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in dose 2500 µg/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in dose 2500 µg/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in dose 2500 µg/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in dose 2500 µg/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in dose 2500 µg/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in dose 2500 µg/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

The study was well-performed according a method similar to OECD 471 with acceptable deficiencies, there is no indication given that the results are not reliable. Hence, they can be used to assess the potential of isobutyl 4-chloro-3,5-diaminobenzoate to induce gene mutations in bacteria.
The test item induced in the Salmonella/Microsome test after incubation with dosages of 2500 µg/plate bacteriotoxic effects. Lower doses did not lead to a decrease of the titre. Assessment of the single dosage groups showed with regard to the relevant parameters dose effect and two-fold increase over background in 3 of 4 strains clear differences to the respective negative controls, which need to be regarded as strong mutagenic effects of the test item. Positive controls increased similarly clearly the number of mutant colonies two-fold over background, clearly indicating the sensitivity of the system. Hence, due to this sensitivity, clear indications for a mutagenic activity of the test item at dosages of up to 2500 µg/plate in the used tester strains in the Salmonella/Microsome test could be found.

Executive summary:

In a reverse gene mutation assay in bacteria similar to OECD 471, strains TA 1535, TA1537, and TA98, TA100 of S. typhimurium were exposed to isobutyl 4-chloro-3,5-diaminobenzoate in DMSO at concentrations of 4, 20, 100, 500, and 2500 µg/plate in the presence and absence of mammalian metabolic activation in plate incorporation. The substance was tested up to cytotoxic concentrations.

The positive controls induced the appropriate responses in the corresponding strains.  There was evidence and a concentration related positive response of induced mutant colonies over background in all strains except TA1535.

This study is classified as acceptable.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1987-06-01 - 1987-08-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

HGPRT assay only

Deviations:

not applicable

GLP compliance:

yes

Remarks:

The described study was performed in accordance with Good Laboratory Practice regulations, as set forth in the Code of Federal Regulations (21CFR 58, 40CFR 792 and 40CFR 160)

Type of assay:

other: in vitro mammalian cell forward mutation test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at room temperature at darkness

Target gene:

HGPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The hypodiploid CHO cell line was originally derived from the ovary of a female Chinese hamster (Cricetulus griseus). The particular clone used in this assay was CHO-Kl-BH4.
- Suitability of cells: The CH0-K1 cell line was originally selected by Dr. T.T. Puck (University of Colorado Medical Center, Denver, Colorado) for high clonability (approximately 85%) and rapid doubling time (11-14 hours). The BH4 subclone of CH0-K1 cells, isolated by Dr. A.W. Hsie (Oak Ridge National Laboratory, Oak Ridge, Tennessee), has been demonstrated to be sensitive to many chemical mutagens.
- Methods for maintenance in cell culture if applicable: CHO-Kl-BH4 stocks were maintained in liquid nitrogen. Laboratory cultures were maintained as monolayers at 37±2°C in a humidified atmosphere containing approximately 5% CO2.
- Normal (negative control) cell cycle time: 11-14h

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The cells used during experimental studies were maintained in Ham's Nutrient Mixture F10 supplemented with L-glutamine, penicillin G, streptomycin sulfate, and fetal bovine serum (10% by volume), hereafter referred to as culture medium. Medium used for reducing the spontaneous frequency of HGPRT- mutants prior to experimental studies consisted of culture medium supplemented with 5.0x10exp(-6) thymidine, 1.0x10exp(-5) M hypoxanthine, 2.0x10exp(-5) M glycine, and 3.2x10exp(-6) M aminopterin. The selection medium for mutants was hypoxanthine-free F10 medium containing 10 µg/ml of TG and the fetal bovine serum component reduced to 5% by volume.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: To reduce the negative control frequency (spontaneous frequency) of HGPRT- mutants to as low a level as possible, the cell cultures were exposed to conditions which selected against the HGPRT- phenotype and were subsequently returned to normal growth medium for three or more days prior to use in the assay.

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

After the selection of dimethyl sulfoxide (DMSO) as a suitable vehicle, a wide range of test article concentrations was tested for cytotoxicity, starting with a maximum applied dose of 1 mg/mL. Ten concentrations spanning a 3-log concentration range were used.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Indicated by the sponsor, tested to be suitable

Untreated negative controls:

yes

Remarks:

F10 culture medium

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

other: 5-Bromodeoxyuridine (BrdU)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): about 4x10exp6 cells in a 250-mL flask

DURATION
- Exposure duration: 4h
- Expression time (cells in growth medium): 6-7 days
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine

NUMBER OF REPLICATIONS: 1 flask split into 12 dishes

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The colonies were fixed with alcohol and stained with Giemsa.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

See "Any other information on materials and methods" due to limitations of this free-text field

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: yes

Conclusions:

The study was conducted similar to OECD guideline 476 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of Isobutyl 4-chloro-3,5-diaminobenzoate to induce gene mutations in Chinese hamster ovary (CHO) cells.
The test material was assayed for mutagenic activity at the HGPRT locus in CHO cells from 100.0 µg/mL to 375.0 µg/mL without activation and from 100.0 µg/mL to 1000.0 µg/mL with activation. Under both conditions a wide range of toxicities was induced as seen by decreases in both relative survival and relative population growth. In the activation part insoluble material was observed at 600.0 µg/mL and higher concentrations which may explain the somewhat fluctuating toxicities at these doses. In all assays small increases in mutant frequency were induced that were statistically significantly elevated over the concurrent vehicle controls, but within or near the range that is typical of vehicle control variation between trials (1-15x10exp(-6)). The lack of a dose-related response and the non-reproducible nature of the observed random increases in mutant frequency were of overriding importance in evaluating the test material as nonmutagenic in the CHO/HGPRT Forward Mutation Assay, with and without metabolic activation, according to the evaluation criteria.
The recorded data in this study declare the test item Isobutyl 4-chloro-3,5-diaminobenzoate as a non-mutagen.

Executive summary:

The objective of this in vitro assay similar to OECD 476 under GLP was to evaluate the ability of Isobutyl 4-chloro-3,5-diaminobenzoate to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in CH0-K1-BH Chinese hamster ovary cells, under conditions with and without metabolic activation.

The test article, Isobutyl 4-chloro-3,5-diaminobenzoate, was considered negative for inducing forward mutations at the HGPRT locus in Chinese hamster ovary cells both with and without metabolic activation, according to the evaluation criteria.

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

until January 1981

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

n/a

Deviations:

not applicable

GLP compliance:

not specified

Remarks:

The report has been audited by Quality Assurance personnel according to the appropriate Standard Operating Procedure

Type of assay:

other: in vitro mammalian cell forward mutation assay

Target gene:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: L5178Y mouse lymphoma cells heterozygous at the TK locus were obtained from D. Clive, Research Triangle Park, North Carolina, U.S.A.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced male Fischer rats liver S9

Test concentrations with justification for top dose:

200 µg/mL, 67 µg/mL, 20 µg/mlL 6.7 µg/mL and 2.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Remarks:

solvent controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 3 * 10exp6 cells in 5 mL

DURATION
- Exposure duration: 3h
- Expression time (cells in growth medium): 3d
- Selection time (if incubation with a selection agent): 7-10d

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 1 incubation was split into 2*3 petri dishes

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

The criterion used by Clive (Clive et al., 1979) to describe a positive result in this test was a doubling of the mutation frequency over the solvent treated control (spontaneous) value. In addition to this, a dose response shown by the induced mutation frequencies of at least 2 concentrations of test compound was considered necessary before a significant positive result could be recorded.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation occurred at 1000 µg/mL

ADDITIONAL INFORMATION ON CYTOTOXICITY:
All the mouse lymphoma cells were killed by 1000 µg/mL.

Conclusions:

The study was conducted similar to OECD guideline 490 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of Isobutyl 4-chloro-3,5-diaminobenzoate to induce gene mutations in mouse lymphoma cells.
Although increases in TFT mutant numbers and frequencies were recorded sporadically at various doses of Isobutyl 4-chloro-3,5-diaminobenzoate in both the presence and absence of S-9 mix, these increases always were small. It is concluded that the test compound showed no relevant mutagenic activity in the mouse lymphoma forward mutation assay.
The recorded data in this study declare the test item Isobutyl 4-chloro-3,5-diaminobenzoate as a non-mutagen.

Executive summary:

The compound Isobutyl 4-chloro-3,5-diaminobenzoate was tested for potential mutagenicity in the mouse lymphoma L5178Y specific locus mutation test. The primary criterion used for a significant positive effect in this test was a doubling of the mutation frequency at the thymidine kinase locus over a solvent treated negative control. Mutation was scored as resistance to trifluorothymidine.

An initial toxicity test was carried out in the absence of supplementary activation conditions and with a dose range of 1 ,000 µg/mL to 0.1 µg/mL test compound. Isobutyl 4-chloro-3,5-diaminobenzoate killed all cells when tested at 1,000 µg/mL, but cultures survived all other doses used. A top dose of 200 µg/mL was selected for the first mutation test.

Three mutation tests were carried out in both the absence and presence of a post-mitochondrial supernatant fluid prepared from the livers of Aroclor 1254-induced adult, male rats and the co-factors required for mixed function oxidase reactions (S-9 mix). For both types of treatment, isolated doses of Isobutyl 4-chloro-3,5-diaminobenzoate induced increases in mutant colony numbers in 2 experiments out of 3. The compound, however, did not show any clear mutagenic activity.

Reference 7

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1986-11-11 - 1987-01-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

not applicable

GLP compliance:

yes

Remarks:

conducted in compliance with the Good Laboratory Practice regulations as set forth in the Code of Federal Regulations (21CFR 58, 40CFR 792, and 40CFR 160)

Type of assay:

other: Primary Rat Hepatocyte Unscheduled DNA Synthesis Assay

Target gene:

n/a

Species / strain / cell type:

hepatocytes: rat

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The indicator cells for this assay were hepatocytes obtained from an adult male Fischer 344 rat (150-300 g), which was purchased from Charles River Breeding Laboratories, Incorporated. The cells were obtained by perfusion of the liver in situ with a collagenase solution

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Monolayer cultures were established on plastic coverslips in culture dishes and were used the same day for initiation of the UDS assay. All cultures were maintained as monolayers at about 37°C in a humidified atmosphere containing approximately 5% C02.
The cell cultures were established in Williams' Medium E supplemented with 5% fetal bovine serum, 2mM L-glutamine, 2.4uM dexa-methasone, 90 U/ml penicillin, 90 µg/ml streptomycin sulfate, and 140 µg/ml gentamicin. After the establishment period, the dexa-methasone and serum components were removed. This latter culture medium is referred to simply as WME.

Metabolic activation:

without

Test concentrations with justification for top dose:

Dose Selection
The dose selection procedure was an integral part of the UDS assay in order to select appropriate doses for a particular, fresh primary culture of hepatocytes. A range of fifteen concentrations was applied initially to the cells, starting at approximately 1000 µg/mL (or 1000 nanoliters/mlL and diluting in approximately two-fold steps to about 0.025 µg/mL (or 0.025 nanoliters/mL). A viable cell count (trypan blue exclusion) was then obtained about twenty to twenty-four hours after initiation of the treatments. If possible at least six concentrations were chosen for analysis of nuclear labeling, starting with the highest dose that resulted in a sufficient number of survivors with intact morphologies and proceeding to successively lower doses. If the test material was freely soluble and relatively nontoxic, a maximum concentration of 5 mg/mL (or 5 microliters/mL) or another limit specified by the sponsor was tested.
Resulting doses were 752, 501, 376, 250, 100, 50.1, 25 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was dissolved, if possible, at the highest desired concentration in WME containing 1% serum, and lower concentrations were then prepared by serial dilution with WME plus 1% serum. If the test material was incompletely soluble in WME, dimethylsulfoxide (DMSO) was investigated as the solvent.

Untreated negative controls:

yes

Remarks:

solvent controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO 1%

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): approximately 0.5 x 10exp6 viable cells in 3 mL of WME plus dexamethasone and 5% serum per dish

DURATION
- Preincubation period: An attachment period of 1.5 to 2 hours at approximately 37°C in a humidified atmosphere containing about 5% CO2 was used to establish the cell cultures.
- Exposure duration: 18-19h

NUMBER OF REPLICATIONS: 5 cultures incl. 2 for cytotoxicity determinations

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The nuclei in the labeled cells were swollen by placement of the coverslips in 1% sodium citrate for eight to ten minutes, and then the cells were fixed in acetic acid:ethanol (1:3) and dried for at least twenty-four hours. The coverslips were mounted on glass slides (cells up), dipped in an emulsion of Kodak NTB2, and dried. The coated slides were stored for seven to ten days at 4°C in light-tight boxes containing packets of Drierite. The emulsions were then developed in D19, fixed, and stained with Williams' modified hematoxylin and eosin procedure.

NUMBER OF CELLS EVALUATED: The net nuclear grain count was determined for fifty randomly selected cells on each coverslip. The mean net nuclear grain count was determined from the triplicate coverslips (150 total nuclei) for each treatment condition.

DETERMINATION OF CYTOTOXICITY
- Method: Viable cell counts (trypan blue exclusion) were determined to estimate cell survival relative to the negative control.

Evaluation criteria:

The test material is considered active in the UDS assay at applied concentrations that cause:
1. An increase in the mean net nuclear grain count to at least six grains per nucleus after subtraction of the concurrent negative control value, and/or
2. An increase in the percent of nuclei having six or more net grains to at least 10% of the analyzed population after subtraction of the concurrent negative control value, and/or
3. The percent of nuclei with twenty or more grains to reach or exceed 2% of the analyzed population.

Key result

Species / strain:

hepatocytes: rat

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A good range of toxicities was induced by this assay. Treatment with 1000 µg/mL of the test material was lethal. The 752 µg/mL treatment was highly toxic (23.7% survival) and the cells had a round morphology unlike the flat morphology of the solvent controls. The survival increased to 66.0% of the solvent control value at 501 µg/mL and the cells still had a round appearance. At 376 µg/mL, the survival (62.8%) was similar to the 501 µg/mL treatment but the cells were flat indicating less toxicity. When the concentration of the test material was reduced to 100 µg/ml, the cell number and appearance were indistinguishable from the solvent controls. Seven treatments from 752 µg/mL to 25.0 µg/mL were prepared for UDS analysis in order to cover a more than adequate concentration range to look for possible DNA repair synthesis. However, the 752 µg/mL treatment was too toxic for analysis and the remaining six dose levels were analyzed. The positive control treatment of 0.10 µg/mL of 2-AAF was weakly toxic in this assay, which is in the range of the historical data.

Conclusions:

The study was conducted on the registered substance itself similar to OECD guideline 482 under GLP. The method is to be considered scientifically reasonable with no deficiencies in documentation, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of Isobutyl 4-chloro-3,5-diaminobenzoate to induce unscheduled DNA synthesis in primary rat hepatocytes. The minimum criteria for UDS in this assay were a mean net nuclear grain count exceeding 6.55, or at least 10.7% of the nuclei containing six or more grains, or at least 2% of the nuclei containing twenty or more grains. None of the treatments with the test material samples caused nuclear labeling significantly different from the solvent control. Furthermore, no dose-related trend was evident. In contrast, the 2-AAF treatment induced large increases in nuclear labeling that greatly exceeded all three criteria used to indicate UDS. Since the assay system was demonstrated to be highly responsive to 2-AAF, the test results were considered to provide conclusive evidence for the lack of UDS induction by the test samples.

Executive summary:

In the In Vitro Primary Rat Hepatocyte Unscheduled DNA Synthesis (UDS) assay similar to OECD 482 under GLP, Isobutyl 4-chloro-3,5-diaminobenzoate did not induce significant increases in UDS. In the assay described in this report, primary rat hepatocytes were exposed for 18 hours to Isobutyl 4-chloro-3,5-diaminobenzoate at concentrations from 1000 µg/mL to 10.0 µg/mL. However treatments at 1000 µg/mL and 752 µg/mL were excessively toxic and treatments from 501 µg/mL to 25.0 µg/mL were prepared for UDS analysis. The test material was insoluble above 250 µg/mL and a good range of toxicities was induced (66.0% to 107.0% survival). None of the criteria used to indicate UDS were approached by the treatments, and no dose-related response was observed. Therefore, the test material was evaluated as inactive in the Primary Rat Hepatocyte UDS Assay.

Reference 8

Endpoint:

in vitro transformation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

until November 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test: The assay is based on the observations that, following exposure to a carcinogen, malignantly transformed cells unlike their non-malignant counter-parts, will undergo sufficient divisions in a soft agar medium to produce macroscopically visible colonies (Shin, et al 1975 and di Mayorca et al 1973).
- Short description of test conditions: Samples of BHK 21 C13 cells were exposed to the test chemical. After a 7 day incubation period, the colonies growing in the tissue culture dishes were fixed and cell viability determined. After 21 days incubation, the cells growing in soft agar were examined with a New Brunswick Biotran II Automatic Colony Counter and the number of transformants regarded as colonies with a diameter of > 0.22 mm, were counted.
- Parameters analysed / observed: From the results of the transformation and survival assays the transformation frequency (number of transformed colonies/10exp5 surviving cells) was calculated for each dose.

GLP compliance:

not specified

Remarks:

The report has been audited by Quality Assurance personnel according to the appropriate Standard Operating Procedure

Type of assay:

in vitro mammalian cell transformation assay

Target gene:

not applicable

Species / strain / cell type:

mammalian cell line, other: BHK 21 C13 cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: These were a subline from a sample of BHK 21 C13 of Syrian hamster kidney cells provided by the Imperial Cancer Research Fund.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: For cell cultivation, the medium was Dulbecco's Modification of Eagle's Minimum Essential Medium (DMEM), with a concentration of sodium bicarbonate of 2 g/l to permit equilibration with a gas phase of 5% C02 in air, it was supplemented with newborn calf serum (10%, v/v), selected by pre-screening a number of serum samples for cloning efficiency, and contained gentamicin (50 µg/ml) .
For incubation with the compound under test, the cells, in order to prevent clumping during exposure, were suspended in Eagle's Minimum Essential Medium modified for suspension cultures (MEMS), buffered with 20 mM HEPES, the S-9 mixture was added to ascertain if the carcinogenic potential of the compound was affected by the metabolic activity of liver enzymes.
Culture media and sera were obtained from Flow Laboratories, Irvine, Scotland and other chemicals from Sigma London Chemical Company Limited.
For subcultivation, the medium was removed and the cells treated for one minute in a solution of 0.25% trypsin in phosphate buffered balanced salt solution containing EDTA (0.002% w/v). After removal of the enzyme solution, the flasks were left to incubate at 37°C until the cells began to detach from the plastic. 5 ml of fresh culture medium was then added and the cells brought into suspension by repeated aspiration through a sterile 10 ml pipette. Aliquots of the cell suspension were then added to medium in fresh culture flasks, the usual ratio for division of monolayers being 1:30.
- Properly maintained: yes
- Periodically 'cleansed' against high spontaneous background: Cells were grown as monolayers in Nunc flasks and, in order to minimise selection for spontaneously transformed variants which accumulate when stocks reach confluence (Kao et al 1975), the cells were never grown to confluency.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

1000, 500, 250, 125, 62.5 µg/mL
From the results of the initial toxicity test a top dose of 1.000 µg/mL was selected for the transformation assays.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

10µL DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

other: N-methyl-N'-nitro-N-nitroso-guanidine (MNNG)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium or on agar
- Cell density at seeding (if applicable): 10exp6 cells/mL

DURATION
- Exposure duration: 4h
- Expression time (cells in growth medium): 7 days (cell viability) or 21 days (transformation)

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: colony count

Evaluation criteria:

In the cell transformation test described by Styles (1977), a transformation frequency, at LC50 of the compound under test, of 5 times that for the spontaneous transformation frequency is considered to indicate potential carcinogenicity. Using this method in tests on 120 compounds 91% of carcinogens and 97% of non-carcinogens were correctly distinguished, Purchase et al (1976). This is one criterion applied in the present study to indicate potential carcinogenicity of the test agent. In addition, if the test compound is relatively non-toxic or is insufficiently soluble to achieve a concentration resulting in 50% toxicity, a 2-fold increase in the absolute number of transformed colonies per dish at 2 doses is considered to indicate a positive response.
The clone of BHK 21 CI3 cells used in these studies may in particular experiments give very low spontaneous transformation levels i.e. 1 or 2. If this occurs the values obtained with the treated cells are related instead to the average spontaneous transformation rate (5 transformed colonies) found from a series of tests.

Key result

Species / strain:

mammalian cell line, other: BHK 21 C13 cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Conclusions:

The study was conducted on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of Isobutyl 4-chloro-3,5-diaminobenzoate to induce cell transformations in mammalian cells. Three experiments were performed in the presence of S-9. No consistent increase in the absolute number of transformed colonies was observed, and calculation of the transformation frequencies at the LC50 indicated no significant increase over negative control values. In the absence of S-9 mix, a 4-fold increase in transformation frequencies at the LC50 was noted in one experiment, but no increase was noted in the first experiment. By the criteria of Styles (1977) a 5-fold increase in transformation frequencies at the LC50 is required to indicate potential carcinogenicity. Thus, by the criteria used in this assay, Isobutyl 4-chloro-3,5-diaminobenzoate showed no evidence of cell transforming potential.

Executive summary:

Isobutyl 4-chloro-3,5-diaminobenzoate was tested for potential carcinogenicity in a cell culture transformation assay.

Cells treated with Isobutyl 4-chloro-3,5-diaminobenzoate in the presence and absence of S-9 mix showed some increase in the transformation frequency over the control values. However the frequencies at the LC50 levels did not approach the value of 5 times that of negative controls considered to indicate potential carcinogenicity.

By the criteria used in this test Isobutyl 4-chloro-3,5-diaminobenzoate showed no evidence of cell transforming potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Genetic toxicity in vivo:

- in vivo: Chromosome aberration (micronucleus assay), NMRI mice m/f, 20 g/kg, 24, 48, or 72 h after gavage,femur bone marrow erythroblasts (similar to OECD 474): 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:

weight of evidence

Study period:

October 1986 - March 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not applicable

GLP compliance:

yes

Remarks:

OECD-Grundsätze der Guten Laborpraxis (Bundesanzeiger Nr. 42a vom 4. Februar 1983)

Type of assay:

other: mammalian soma cell micronucleus assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerated
- Stability under test conditions: confirmed to be stable

Species:

mouse

Strain:

NMRI

Remarks:

Bor:NMRI (SPF Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Breeder Winkelmann, Borchen
- Age at study initiation: ca. 9-12 weeks
- Weight at study initiation: 31-44 g
- Assigned to test groups randomly: yes
- Fasting period before study: not stated
- Housing: in groups of max. 3 resp. 5 mice, divided according to sex and treatment groups, in macrolone cages type I resp. II, on wooden soft bedding (Fa. Bogner GmbH, 5650 Solingen-Ohligs)
- Diet (e.g. ad libitum): "fixed -formula" ― Standarddiat Altromin 1324; Hersteller Altromin GmbH, Lage) ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22°C (set as 22±2°C)
- Humidity (%): 45-54% (set as min. 50%)
- Air changes (per hr): at least 10/h
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: polyethylene glycol (Lutrol)
- Amount of vehicle (if gavage or dermal): 40 mL/kg initially, which were reduced to 24 mL/kg as the first treated animals died due to the inherent toxicity of Lutrol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item was suspended in Lutrol and stirred with a magnetic stirrer until application.

Duration of treatment / exposure:

single gavage, animals were decapitated either 24, 48, or 72 h after gavage

Frequency of treatment:

single gavage

Post exposure period:

animals were decapitated either 24, 48, or 72 h after gavage

Dose / conc.:

20 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5/sex/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: oral: gavage
- Doses / concentrations: 20 mg/kg

Tissues and cell types examined:

femur bone marrow erythroblasts

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Preliminary toxicity test with doses of 5, 10, 20, and 25 g/kg. All doses led to symptoms as apathy, diminished motility, ruffled fur, face-down position, cramps, difficulties breathing, orange coloured urine, diarrhea and lacking defecation. In the 25 g/kg dose group, 3 of 5 animals died.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Single treatment, tissue was collected 24, 48, or 72 h after gavage (Controls only after 24h).
Sampling and preparation was done according to Schmid (SCHMID, W. Der Mikrokerntest. Deutsche Forschungsgemeinschaft. Kommission fur Mutagenitätsfragen. Mitteilung II 53—61 1975; Schmid W. The micronucleus—test for cytogenetic analysis. In: Hollander, A. (ed.), Chemical Mutagens, Principles and Methods for their Detection, Vol. 4 31-53 Plenum Press New York (1976)

DETAILS OF SLIDE PREPARATION:
Of each treated animal (no pretreatment with a spindle poison), at least one femur was preparated, in brief, using a syringe with calf serum and purgeing the bone. The collected cells were centrifuged and the sediment was spread onto the slide. Slides were stained with a staining machine Ames Hema-Tek Slide stainer by the company Miles.

METHOD OF ANALYSIS: As a rule, 1000 polychromatic erythrocytes per animals were analysed for micronuclei in a meandering course. In adition it is reasonable to determine the ratio of polychromatic and normochromatic cells. In case there were way more than 3000 normochromatic cells with 1000 polychromatic ones, without similar effects in other animals of the same treatment group, a pathologic, non-substance-related event is detected and the animal is excluded from assessment.
In addition to the number of normochromatic cells the number of normochromatic cells with micronuclei was determined.

Evaluation criteria:

The treatment group with the highest mean value, provided that this value was above the value of the negative control group, as well as the positive control group, were evaluated with the distribution-free Wilcoxon rank test, with regard to the amount of polychromatic cells with micronuclei and of normochromatic cells. A difference is statistically significantly different if the error probability is <5% that the value of the treatment group is higher than the one of control. Evaluation of the rate of normochromatic cells with micronuclei was performed if already an elevated rate of polychromatic cells with micronuclei was seen. In this case, the group with the highest mean value was compared to control with the one-sided Chi²-Test.
In addition, the 1s ranges for all mean values were calculated.

Statistics:

See evaluation criteria

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Treated mice showed signs of toxicity incl. apathy, diminished motility, ruffled fur, diarrhea and orange stained urine, 2/30 animals died.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In the preliminary toxicity test with doses of 5, 10, 20, and 25 g/kg, all doses led to symptoms as apathy, diminished motility, ruffled fur, face-down position, cramps, difficult breathing, orange coloured urine, diarrhea and lacking defecation. In the 25 g/kg dose group, 3 of 5 animals died.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Not significantly over control
- Ratio of PCE/NCE (for Micronucleus assay): Treatment with the test item caused an inhibition of erythropoesis, Ratios of PCE/NCE are:
1000 : 1783 (1s = 771) (negative control)
1000 : 3189 (1s = 1741) (treatment group, 24h)
1000 : 2224 (1s = 1077) (treatment group, 48h)
1000 : 1755 (1s = 688) (treatment group, 72h)
- Appropriateness of dose levels and route: Treated mice showed signs of toxicity incl. apathy, diminished motility, ruffled fur, diarrhea and orange stained urine, 2/30 animals died.

Conclusions:

The study was conducted similar to OECD guideline 474 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of Isobutyl 4-chloro-3,5-diaminobenzoate to induce micronuclei in vivo in NMRI mice.
There were no biologically or statistically significant differences between the treatment groups (20 g/kg test item) and the negative control with regard to polychromatic cells with micronuclei, relevant biological differences were not observed.
Hence, Isobutyl 4-chloro-3,5-diaminobenzoate should be regarded as non-clastogenic in vivo.

Executive summary:

Using the micronucleus test similar to OECD TG 474, Isobutyl 4-chloro-3,5-diaminobenzoate was investigated in male and female mice for a possible clastogenic effect on the chromosomes of bone marrow erythroblasts. The known clastogen and cytostatic agent cyclophosphamide served as a positive control.

The animals received a single administration. 24, 48 and 72 hours after the administration the femoral marrow of the Isobutyl 4-chloro-3,5-diaminobenzoate groups was prepared. Negative and positive control were sacrificed after 24 hours only. The dose of Isobutyl 4-chloro-3,5-diaminobenzoate was 20000 mg/kg body weight, and of the positive control cyclophosphamide 20 mg/kg. The test substance and the positive control cyclophosphamide were administered orally.

The animals treated with Isobutyl 4-chloro-3,5-diaminobenzoate showed lasting symptoms of toxicity after the administration. 2 of 30 treated animals died until the end of the test.

Erythrocyte formation, as measured by the ratio polychromatic to normochromatic erythrocytes, was time-dependent affected.

No indications of a clastogenic effect of Isobutyl 4-chloro-3,5-diaminobenzoate were found after a single treatment with 20000 mg/kg per os.

Cyclophosphamide, the positive control, had a clear clastogenic effect, as can be seen from the biologically relevant increase in polychromatic erythrocytes with micronuclei. An inhibition of erythropoiesis was not found here.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

1. Problem formulation

There are several studies available dealing with a possible genotoxicity of isobutyl 4-chloro-3,5-diaminobenzoate. Commonly genotoxicity testing begins with testing for gene mutation in bacteria and, if required, continues with testing for gene mutations and chromosome aberrations in mammalian cells in vitro. In case any further need for information is identified, testing continues in vivo and other supporting information on genotoxicity is considered. This approach is e.g. (tonnage-driven) foreseen under REACH. There were four Ames tests conducted with partially positive results. In order to “de-risk” these findings, two in vitro gene mutation assays in mammalian cells (HPGRT, MLA), generally being of higher significance for human risk assessment, were conducted, revealing negative results. Furthermore, an UDS assay in primary rat hepatocytes, an in vitro transformation study in mammalian cells, and even in vivo studies were conducted, i.e. a micronucleus assay in mice and an orienting dermal carcinogenesis study in mice, all revealing negative results. Hence, the present evaluation aims to demonstrate that isobutyl 4-chloro-3,5-diaminobenzoate does not need to be regarded as mutagen.

 

 

2. Hypothesised Mode of action Statement

Based on the available partially positive or ambiguous genotoxicity, as will be summarized below, no conclusion on the mode of action can be drawn. Given data indicate that these results were due to genotoxic impurities of the test items. As these data were gained with various products which were manufactured by different companies decades ago, no certificate of analysis could be retrieved of the batches in question. However, the present manufacturing process does not result in contaminated products and only the registered substance is formed, so the partially positive or ambiguous results are considered not to be relevant any more for the current product. Further, all other genotoxicity data (in vitro and in vivo) including carcinogenesis data indicate that these results are not relevant in vivo. Based on the physico-chemical properties of the registered substance (solid, logPow = -0.97, water solubility = 444.5 mg/L at 20°C and pH 5.4, molecular weight = 242.7 g/mol), a lacking absorption can be excluded as a reason, the substance is considered to be bioavailable at least to a certain extent.

 

 

3. Summary of data for use in Mode of Action Analysis

 

System	Results	Comments
Ames test, S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 1538, ± S9	2 grades of purification tested: positive in TA1537, TA1538, TA98 +S9 (Frameshift mutations), negative in TA1535 and TA100 (basepair substitutions); purified compound was negative in TA1537, TA1538, TA98 +S9, non-purified compound was positive in the two highest tested concentrations in TA1537, TA1538, TA98 –S9	Both the technical product and the purified substance were negative in TA1535 and TA100, which detect transitions and transversions at the G:C basepair, which can hence be excluded. TA1537, TA1538, TA98 detect frameshifts at or near GGGGG (TA1537), or GCGCGCGC (ΔGpC, TA1538, TA98), but here only in the non-purified version, making an attribution of the effect to an irrelevant impurity very likely.
Ames test, S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 1538, ± S9	positive in TA1537, TA1538, TA98 ±S9 (Frameshift mutations), negative in TA1535 and TA100 ± S9 (basepair substitutions)	See above, transitions and transversions at the G:C basepair can be excluded, the test item only may induce frameshifts at or near GGGGG or GCGCGCGC, irrespective of metabolic activation
Ames test, S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 1538, ± S9	positive in TA1537, TA1538, TA98 +S9 (Frameshift mutations), negative in TA1537, TA1538, TA98 –S9, negative in TA1535 and TA100 ±S9 (basepair substitutions)	See above, transitions and transversions at the G:C basepair can be excluded, the test item only may induce frameshifts at or near GGGGG or GCGCGCGC, in this case however only with metabolic activation
Ames test, S. typhimurium TA 1535, TA 1537, TA 98, TA 100, ± S9	positive in TA100 + S9 (basepair substitutions), negative in TA 100 –S9, positive in TA1537 ± S9 (Frameshift mutations, when cytotoxicity occurred), positive in TA98 + S9 (Frameshift mutations), negative in TA98 –S9, negativ in TA1535 ± S9 (basepair substitutions)	In this case, also basepair substitutions at the G:C site with metabolic activation in TA100, however already in cytotoxic ranges.
MLA, mouse lymphoma L5178Y cells ± S9	negative ± S9	Although increases in TFT mutant numbers and frequencies were recorded sporadically at various doses of Isobutyl 4-chloro-3,5-diaminobenzoate in both the presence and absence of S9 mix, these increases always were small. It is concluded that the test compound showed no relevant mutagenic activity in the mouse lymphoma forward mutation assay.
HGPRT assay, CHO-K1 cells ± S9	negative ± S9	Doses ranged from 100.0 µg/mL to 375.0 µg/mL –S9 and from 100.0 µg/mL to 1000.0 µg/mL +S9. Under both conditions a wide range of toxicities was induced as seen by decreases in both relative survival and relative population growth. In all assays small increases in mutant frequency were induced that were statistically significantly elevated over the concurrent vehicle controls, but within or near the range that is typical of vehicle control variation between trials. The lack of a dose-related response and the non-reproducible nature of the observed random increases in mutant frequency were of overriding importance in evaluating the test material as nonmutagenic in the CHO/HGPRT Assay ±S9.
UDS assay, primary rat hepatocytes, -S9	up to 752 µg/ml: negative	None of the treatments with the test material samples caused nuclear labeling significantly different from the solvent control. Furthermore, no dose-related trend was evident in a concentration range of 501 µg/ml (66% survival) to 25.0 µg/mL.
In vitro transformation in mammalian cells: BHK 21 C13 of Syrian hamster kidney cells, ± S9	no evidence of cell transforming potential ± S9	No consistent increase in the absolute number of transformed colonies was observed in 3 experiments, and calculation of the transformation frequencies at the LC50 indicated no significant increase over negative control values. In the absence of S-9 mix, a 4-fold increase in transformation frequencies at the LC50 was noted in one experiment, but no increase was noted in the first experiment. A 5-fold increase in transformation frequencies at the LC50 is required to indicate potential carcinogenicity. Thus, Isobutyl 4-chloro-3,5-diaminobenzoate showed no evidence of cell transforming potential.
Micronucleus assay In vivo, NMRI mice, femur bone marrow erythroblasts	negative	Treated mice showed signs of toxicity incl. apathy, diminished motility, ruffled fur, diarrhea and orange stained urine, 2/30 animals died. There were no biologically or statistically significant differences between the treatment groups (20 g/kg test item) and the negative control with regard to polychromatic cells with micronuclei. The incidence of such cells with micronuclei were 0.6/1000 (1s = 0.5) (negative control), and 1.1/1000, (1s = 1.2), 0.4/1000 (1s = 0.7) resp. 1.6/1000 (1s = 1.2) in the test item groups. The values of the 24h and 72h groups are above the negative control, may however not be considered as relevant as there was no time-dependence observed. Furthermore, they lie in the range of the historical control data of the negative control. Relevant biological differences were not observed. No bone marrow depression was observed, neither, as the ratio of PCE/NCE was not biologically relevantly altered (1000 : 2268 (1s = 1253) compared to control, 1000 : 1783). The substance is considered to be bioavailable at least to a certain extent.
Orienting dermal carcinogenesis study	No carcinogen. Despite a total dose of 25 g/kg there were neither an elevated number of malignant tumours with a specific localisation nor tumours at the injections site which is more susceptible towards carcinogenic noxa. Weight gains, behaviour, appearance and survival times do also not indicate any impairment by the substance.	The present study was designed as orienting study, so only a group size of 18/sex instead of 50 was chosen. Also, according to e.g. OECD TG 451, the highest dose should induce toxic symptoms, which were not noted, and higher doses in theory may have led to an increased tumour incidence. However, the total dose corresponds to an approximate NOAEL of 67 mg/kg bw/d, which is already a rather high dose for life-time studies. It is considered sufficient, provided this MoA was applicable, to induce both genotoxic damage or generally modify biological pathway, and so being able to detect both genotoxic and non-genotoxic carcinogens, and to induce tumour rates over background even with the smaller group size. Further, systemic bioavailability is given. The substance was applied subcutaneously, and by being moderately soluble, a distribution through the whole body can be assumed, encapsulations or deposits at the injection site were not noted. Further, due to the initially rather high local concentration, possible tumours would have to be noted here, as this tissue is both susceptible for directly genotoxic carcinogens and possesses a vast amount of biological pathways which may be induced to support tumour growth. Further, skin also possesses a certain metabolic capacity which should have sufficed to activate the substance if required. As those effects were not seen, the substance is most likely not a carcinogen, no conclusion on a MoA can be drawn. The substance induced benign tumours over control, but still within the normal biological variations of this strain.

 

 

4. Listing of key events identified for a specific Mode of Action

All available information is denoted above. There are rather consistent increases in frameshift mutations at or near GGGGG or GCGCGCGC, which could neither be clearly verified in mammalian cells nor attributed to the test item itself, as the effect is most likely induced by an impurity, which could be delineated from experiments with the purified substance, which corresponds to the recent produced product. Even in the very unlikely case that these effects are due to the substance itself rather than due to an impurity, any genotoxic effects could not be verified in vivo (mouse micronucleus assay / carcinogenicity study). This clearly shows that both a possible Mode of Action cannot be identified, as there is most likely none, and that there is no relevance of the positive / ambiguous results for humans.

 

 

5. Weight of Evidence Analysis of available data/information for Mode of Action Analysis in experimental species

5a. Dose Response Relationships and Temporal Association

In the available Ames test, a dose response was identified in the strains / test items revealing positive results. In higher tier tests, only sporadic and so not dose related increases were noted, in the vast majority only negative results were observed. No increase of tumours over control was observed during the whole life-time carcinogenicity study, so no temporal association can be made. In the mouse micronucleus test, the numbers of cells with micronuclei of the 24h and 72h groups are above the negative control, may however also not be considered as relevant as there was no time-dependence observed. Furthermore, they lie in the range of the historical control data of the negative control. So no human relevant Dose Response Relationships and Temporal Association was observed.

5b. Consistency & Specificity – Biological Plausibility

Besides the fact that the results of the Ames tests revealed rather consistently in the non-purified samples an increase in frameshift mutations at or near GGGGG (TA1537), or GCGCGCGC (ΔGpC, TA1538, TA98), there is no further consistency or specificity for a specific mode of action observed. Already the purified, recently relevant purified sample gave negative results here. Further, clearly positive results could also not be obtained in vitro in mammalian cells, neither for mutagenicity nor other relevant endpoints, such as cell transformation or UDS. In vivo, not any evidence for relevant positive effects, neither micronucleus induction nor tumour formation, was seen. So there is actually a consistency in the absence of genotoxic effects in higher studies. There are no effects related to the initial damage of a cell, neither mutations, chromosome damage or DNA repair, nor related to the promotion of the damage; no increase in tumour formation was seen in mice. So actually given data consistently show that there is no human relevance with regard to genotoxicity.

 

 

6. Other potential Modes of Action

None identified due to the absence of effects in studies more valuable than a bacteria reverse mutation test.

 

 

7. Uncertainties/Inconsistencies and Identification of Data Gaps

None identified due to the absence of effects in studies more valuable than a bacteria reverse mutation test.

 

 

8. Conclusions in relation to problem formulation

The present evaluation aims to demonstrate that isobutyl 4-chloro-3,5-diaminobenzoate does not need to be regarded as mutagen, and that the four Ames tests conducted with partially positive results are not relevant for humans. As shown above, higher tier studies consistently revealed that there are no effects related to the initial damage of a cell, neither mutations, chromosome damage or DNA repair, nor related to the promotion of the damage; no increase in tumour formation was seen in mice. Consequently, it was shown that isobutyl 4-chloro-3,5-diaminobenzoate does not need to be regarded as mutagenic in humans.

Additional information

There is additional information on carcinogenicity available, supporting the conclusion that Isobutyl 4-chloro-3,5-diaminobenzoate does not need to be considered as mutagen:

In an orienting carcinogenesis study, 100 d old Wistar-W.64 rats were injected doses of isobutyl 4-chloro-3,5-diaminobenzoate of 500 - 1000 mg/kg as suspension in physiological saline in intervals of 1 - 5 weeks subcutaneously in the middle of the back. After 371 days the test item was applied a last time, a total dose of 25 g/kg was reached, and the animals were observed until death. Examined parameters were body weight gains, behaviour, appearance, survival time, localisation and amount of tumours. All dead animals were carefully dissected and tumours / suspected tumours histologically examined.

The average survival time of the male rats treated with isobutyl 4-chloro-3,5-diaminobenzoate was 824 ± 194 days of the study. Of the 18 male rats, 6 died with each a malignant tumour and 2 rats with each 3 benign tumours. The average survival time of the female rats treated with isobutyl 4-chloro-3,5-diaminobenzoate was 737 ± 236 days of the study. Of these 18 female rats, 4 died with in total 5 malignant tumours. Further, in 8 animals in total 14 benign tumours were observed. So of the 36 isobutyl 4-chloro-3,5-diaminobenzoate-treated rats 10 died with malignant tumours (28%). Total amount of malignant tumours were 11 (referring to the total animal number 31%), the benign 17 (47%).

The average survival time of the male control rats treated with physiological saline was 834 ± 229 days of the study, the one of the females 888 ± 177 days. Of the 25 male control rats 4 died with each one malignant tumour. Of the 25 female control rats 9 died with malignant tumours, 3 of them with a double tumour. Further, in 9 animals 14 benign tumours were observed. Of the 50 control rats hence 13 (26%) died with malignant tumours. The total number of benign tumours were 15 (30%).

Despite a total dose of 25 g/kg there were neither an elevated number of malignant tumours with a specific localisation nor tumours at the injections site which is more susceptible towards carcinogenic noxa. Weight gains, behaviour, appearance and survival times do also not indicate any impairment by the substance.

So, isobutyl 4-chloro-3,5-diaminobenzoate does not need to be regarded as carcinogen.

Justification for classification or non-classification

There were four Ames tests conducted with partially positive results, which are most likely due to a genotoxic impurity which is not relevant any more for the registered substance. Already the purified, recently relevant purified sample gave negative results here. Further, clearly positive results could also not be obtained in vitro in mammalian cells, neither for mutagenicity nor other relevant endpoints, such as cell transformation or UDS. In vivo, not any evidence for relevant positive effects, neither micronucleus induction nor tumour formation, was seen. So there is a consistency in the absence of genotoxic effects in higher studies. There are no effects related to the initial damage of a cell, neither mutations, chromosome damage or DNA repair, nor related to the promotion of the damage; no increase in tumour formation was seen in mice. So actually given data consistently show that there is no human relevance with regard to genotoxicity, the substance does not need to be considered as a mutagen.

According to Regulation 1272/2008, substances must be classified as germ cell mutagen Cat. 2, if they meet the following criteria:

Substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans The classification in Category 2 is based on:

— positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from:

— somatic cell mutagenicity tests in vivo, in mammals; or

— other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays.

Note: Substances which are positive in in vitro mammalian mutagenicity assays, and which also show chemical structure activity relationship to known germ cell mutagens, shall be considered for classification as Category 2 mutagens.

As outlined above, none of those criteria are met, and so isobutyl 4-chloro-3,5-diaminobenzoate does not need to be regarded as germ cell mutagen.