p-anisylamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in vitro:

Ames test:

Gene mutation toxicity wad predicted for 4-Methoxybenzylamine using data from structurally and functionally similar read across chemicals. The studies are as mentioned below:

Gene mutation toxicity study (Plate incorporation method) was performed to determine the mutagenic nature of the 70 -80% test chemical. The method was designed to meet the requirements of the OECD Guidelines for testing of the Chemicals No. 471 “Bacterial Reverse Mutation Test”, Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines. Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test material using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the first experiment (plate incorporation) was determined in a preliminary toxicity assay and was 100 to 5000µg/plate. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Based on the observations made, the test chemical was considered to be non mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 with and without metabolic activation in the Plate incorporation assay and hence is likely to be non-mutagenic.

In the same study, gene mutation toxicity study (Pre-incubation method) was also performed to determine the mutagenic nature of the 70 -80% test chemical . The method was designed to meet the requirements of the OECD Guidelines for testing of the Chemicals No. 471 “Bacterial Reverse Mutation Test”, Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines. Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test material using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the first experiment (plate incorporation) was determined in a preliminary toxicity assay and was 100 to 5000µg/plate. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. The pre-incubation modification was employed for the second experiment. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Based on the observations made, the test chemical was considered to be non mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 with and without metabolic activation in the pre-incubation assay and hence is likely to be non-mutagenic.

Gene mutation toxicity study was also performed to determine the mutagenic nature of another 70 -80% test chemical. The study was performed using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 with and without S9 metabolic activation system. The test was performed as per the preincubation assay at six different concentrations with 5.0 mg/plate being the maximum concentration. The chemical was dissolved in DMSO. Preincubation was performed for 20 mins and the exposure duration was for 48 hrs. The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated). The test chemical did not induce a doubling of revertant colonies over the control using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Based on the predicted data available for the target chemical and data from its structurally and functionally similar read across chemicals, 4-Methoxybenzylamine (CAS no 2393 -23 -9) does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Chromosome aberration test:

Gene mutation toxicity for the target chemical 4-Methoxybenzylamine was predicted on the basis of the data from read across chemicals. The studies are as mentioned below:

Chromosomal aberration study was performed to determine the mutagenic nature of 70 -80% structurally and functionally similar test chemical. The cells were exposed to the test material at three different doses with 0.5 mg/mL being the maximum concentration for48hr. Colcemid (final concn 0.2µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution for 12-15 min. A hundred well-spread metaphases were observed under the microscope. In the present studies, no metabolic activation systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. The incidence of polyploid cells for 48hr after treatment was 0.0%. The incidence of cells with structural chromosomal aberrations at 0.4mg/ml for 48hr after treatment was 47.0%. Also positive at 0.3 mg/ml at 24hr (11.0 %) and at 48hr (30.0%). D20 was 0.25 mg/ml (the dose at which structural aberrations were detected in 20 % of theMetaphases observed). TR value (the frequency of cells with exchange-type aberrations per unit dose(mg/ml) ) was 0.35. The test chemical did not induce chromosomal aberration in Chinese hamster fibroblast cell line CHL in the absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant.

Sister chromatid exchanges test was performed to determine the mutagenic nature of 70 -80% structurally and functionally similar test compound. The test chemical was studied at a dose level of 160-1600µg/mL using Chinese hamster ovary cells (CHO-W-B1) both in the presence and absence of S9 metabolic activation system. 5-Bromodeoxyuridine (BrdUrd; 10 pM) was added 2 hr after addition of the test chemical (without S9) or immediately after the S9 mix plus chemical had been removed. The chemical treatment periods were appoximately 25 hr without S9 and 2 hr with S9. The total incubation time with BrdUrd was 25-26 hr, with colcemid (0.1µg/ml) present during the final 2-3 hr. Immediately before the cells were harvested, the cell monolayers were examined, and the degree of confluence and availability of mitotic cells were noted. Cells were collected by mitotic shake-off at doses up to the maximum considered likely to yield sufficient metaphase cells for analysis; supernatant medium was returned to appropriate flasks so that subsequent harvests could bemade from the same cultures if necessary. Because all mitotic cells were removed in the initial harvest, cells collected during subsequent harvests had come into mitosis during the period between harvests and thus had been exposed to colcemid for an average of 4 hr. After 1-3 min treatment with hypotonic solution (75 mM KCl), cells were fixed in 3: 1 methano1: glacial acetic acid (V/V). For a preliminary assessment of cell cycle delay, test slides were prepared from cells treated at the highest dose levels to see if later harvests were necessary. These test slides were stained with “dilute” Hoeschst 33258 (0.5µg/ml in Sorensen’s buffer, pH 6.8) and examined by fluorescence microscopy to assess cell cycle kinetics. In control cultures, almost all cells completed two cycles in BrdUrd (M2 cells) in 25-26 hr, whereas, in treated cultures, cell cycle delay was common. In cases of severe delay, additional harvests were made from the same cultures at a later time to obtain sufficient second metaphase (M2) cells for SCE analysis. After staining for 10 min in “concentrated” Hoechst 33258 (5µg/ml in pH 6.8 buffer) and exposure to “black light” at 55 to 60°C for about 5 min, slides were stained in Giemsa. All slides were coded, and 50 cells per dose were scored from the three highest doses at which sufficient M2 cells were available. When cell cycle delay was noted, cell kinetics were recorded by classifying each of 100 metaphases as M1, M1+, or M2, i.e., having completed one (M1), two (M2), or between one and two (M1 +) cell cycles in BrdUrd. Delay was noted at the top dose with S9 (5 mg/ml), and the culture was harvested 2 hr later than the controls. There were increases in aberrations, but these were statistically significant only without S9. The test compound did not induce an increase in the number of Sister chromatid exchanges in the Chinese hamster ovary cells (CHO-W-B1) in the presence of S9 metabolic activation system but it induced sister chromatid exchanges in the absence of S9 metabolic activation system.

Gene mutation toxicity study for 4-Methoxybenzylamine (CAS no 2393 -23 -9) as predicted using data from read across chemicals with mammalian cell lines in the presence and absence of S9 metabolic activation system is negative and hence the chemical is predicted to not classify as a gene mutant in vitro.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

experimental data of read across substances

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar read across chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

WoE derived based on the predicted data for the target chemical and experimental data from structurally and functionally similar read across chemicals

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other:

Remarks:

1. Predicted data/Target

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium, other: TA100, 1535, 102, 98, 1537

Remarks:

RA 2/3

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium, other: TA92, TA1535, TA100, TA1537, TA94 and TA98

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

not applicable

Metabolic activation system:

No data

Test concentrations with justification for top dose:

1. No data
2/3. 0, 100, 333, 1000, 2500 and 5000 µg/plate
4. 6 different concentrations were used; 5.0 mg/plate was the maximum concentration

Vehicle / solvent:

1. No data
2/3/4.
- Vehicle(s)/solvent(s) used: Dimethyl suphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

1. Target predicted data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

RA 2/3

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

RA 4

Details on test system and experimental conditions:

1. No data
2.
METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): No data

DURATION
- Preincubation period: No data
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): Triplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: No data

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data

3.
METHOD OF APPLICATION: preincubation
- Cell density at seeding (if applicable): No data

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Triplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: No data

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data

4. METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

Rationale for test conditions:

1. No data
2/3. No data.
4. No data

Evaluation criteria:

1. Prediction was done considering a dose dependent increase in the number of revertants/plate

2/3. The test material may be considered positive in this test system if the following criteria are met :
The test material should have induced a reproducible, dose related and statistically(Dunnett’s method of linear regression) significant increase in the revertant count in at least one strain of bacteria.

4. The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated).

Statistics:

1. No data
2/3. Dunnett’s method of linear regression
4. No data

Species / strain:

S. typhimurium, other:

Remarks:

Target predicted data

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium, other: TA10, 98, 1535, 1537, 102

Remarks:

RA 2/3

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA92, TA1535, TA100, TA1537, TA94 and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

1. No data
2/3. No data
4. ADDITIONAL INFORMATION ON CYTOTOXICITY: The maximum dose for negative results represents the highest non-cytotoxic dose used in the experiment

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

Gene mutation toxicity study for 4-Methoxybenzylamine as predicted using data from read across chemicals for Salmonella typhimurium bacterial strains in the presence and absence of S9 metabolic activation system is negative and hence the chemical is predicted to not classify as a gene mutant in vitro.

Executive summary:

Gene mutation toxicity wad predicted for 4-Methoxybenzylamine using data from structurally and functionally similar read across chemicals. The studies are as mentioned below:

Gene mutation toxicity study (Plate incorporation method) was performed to determine the mutagenic nature of the 70 -80% test chemical. The method was designed to meet the requirements of the OECD Guidelines for testing of the Chemicals No. 471 “Bacterial Reverse Mutation Test”, Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines. Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test material using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the first experiment (plate incorporation) was determined in a preliminary toxicity assay and was 100 to 5000µg/plate. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Based on the observations made, the test chemical was considered to be non mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 with and without metabolic activation in the Plate incorporation assay and hence is likely to be non-mutagenic.

In the same study, gene mutation toxicity study (Pre-incubation method) was also performed to determine the mutagenic nature of the 70 -80% test chemical . The method was designed to meet the requirements of the OECD Guidelines for testing of the Chemicals No. 471 “Bacterial Reverse Mutation Test”, Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines. Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test material using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the first experiment (plate incorporation) was determined in a preliminary toxicity assay and was 100 to 5000µg/plate. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. The pre-incubation modification was employed for the second experiment. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Based on the observations made, the test chemical was considered to be non mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 with and without metabolic activation in the pre-incubation assay and hence is likely to be non-mutagenic.

Gene mutation toxicity study was also performed to determine the mutagenic nature of another 70 -80% test chemical. The study was performed using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 with and without S9 metabolic activation system. The test was performed as per the preincubation assay at six different concentrations with 5.0 mg/plate being the maximum concentration. The chemical was dissolved in DMSO. Preincubation was performed for 20 mins and the exposure duration was for 48 hrs. The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated). The test chemical did not induce a doubling of revertant colonies over the control using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Based on the predicted data available for the target chemical and data from its structurally and functionally similar read across chemicals, 4-Methoxybenzylamine (CAS no 2393 -23 -9) does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.-

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicity in vitro:

Ames test:

Gene mutation toxicity wad predicted for 4-Methoxybenzylamine using data from structurally and functionally similar read across chemicals. The studies are as mentioned below:

Gene mutation toxicity study (Plate incorporation method) was performed to determine the mutagenic nature of the 70 -80% test chemical. The method was designed to meet the requirements of the OECD Guidelines for testing of the Chemicals No. 471 “Bacterial Reverse Mutation Test”, Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines. Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test material using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the first experiment (plate incorporation) was determined in a preliminary toxicity assay and was 100 to 5000µg/plate. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Based on the observations made, the test chemical was considered to be non mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 with and without metabolic activation in the Plate incorporation assay and hence is likely to be non-mutagenic.

In the same study, gene mutation toxicity study (Pre-incubation method) was also performed to determine the mutagenic nature of the 70 -80% test chemical . The method was designed to meet the requirements of the OECD Guidelines for testing of the Chemicals No. 471 “Bacterial Reverse Mutation Test”, Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines. Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test material using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the first experiment (plate incorporation) was determined in a preliminary toxicity assay and was 100 to 5000µg/plate. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. The pre-incubation modification was employed for the second experiment. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Based on the observations made, the test chemical was considered to be non mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 with and without metabolic activation in the pre-incubation assay and hence is likely to be non-mutagenic.

Gene mutation toxicity study was also performed to determine the mutagenic nature of another 70 -80% test chemical. The study was performed using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 with and without S9 metabolic activation system. The test was performed as per the preincubation assay at six different concentrations with 5.0 mg/plate being the maximum concentration. The chemical was dissolved in DMSO. Preincubation was performed for 20 mins and the exposure duration was for 48 hrs. The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated). The test chemical did not induce a doubling of revertant colonies over the control using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Based on the predicted data available for the target chemical and data from its structurally and functionally similar read across chemicals, 4-Methoxybenzylamine (CAS no 2393 -23 -9) does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Chromosome aberration test:

Gene mutation toxicity for the target chemical 4-Methoxybenzylamine was predicted on the basis of the data from read across chemicals. The studies are as mentioned below:

Chromosomal aberration study was performed to determine the mutagenic nature of 70 -80% structurally and functionally similar test chemical. The cells were exposed to the test material at three different doses with 0.5 mg/mL being the maximum concentration for48hr. Colcemid (final concn 0.2µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution for 12-15 min. A hundred well-spread metaphases were observed under the microscope. In the present studies, no metabolic activation systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. The incidence of polyploid cells for 48hr after treatment was 0.0%. The incidence of cells with structural chromosomal aberrations at 0.4mg/ml for 48hr after treatment was 47.0%. Also positive at 0.3 mg/ml at 24hr (11.0 %) and at 48hr (30.0%). D20 was 0.25 mg/ml (the dose at which structural aberrations were detected in 20 % of theMetaphases observed). TR value (the frequency of cells with exchange-type aberrations per unit dose(mg/ml) ) was 0.35. The test chemical did not induce chromosomal aberration in Chinese hamster fibroblast cell line CHL in the absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant.

Sister chromatid exchanges test was performed to determine the mutagenic nature of 70 -80% structurally and functionally similar test compound. The test chemical was studied at a dose level of 160-1600µg/mL using Chinese hamster ovary cells (CHO-W-B1) both in the presence and absence of S9 metabolic activation system. 5-Bromodeoxyuridine (BrdUrd; 10 pM) was added 2 hr after addition of the test chemical (without S9) or immediately after the S9 mix plus chemical had been removed. The chemical treatment periods were appoximately 25 hr without S9 and 2 hr with S9. The total incubation time with BrdUrd was 25-26 hr, with colcemid (0.1µg/ml) present during the final 2-3 hr. Immediately before the cells were harvested, the cell monolayers were examined, and the degree of confluence and availability of mitotic cells were noted. Cells were collected by mitotic shake-off at doses up to the maximum considered likely to yield sufficient metaphase cells for analysis; supernatant medium was returned to appropriate flasks so that subsequent harvests could bemade from the same cultures if necessary. Because all mitotic cells were removed in the initial harvest, cells collected during subsequent harvests had come into mitosis during the period between harvests and thus had been exposed to colcemid for an average of 4 hr. After 1-3 min treatment with hypotonic solution (75 mM KCl), cells were fixed in 3: 1 methano1: glacial acetic acid (V/V). For a preliminary assessment of cell cycle delay, test slides were prepared from cells treated at the highest dose levels to see if later harvests were necessary. These test slides were stained with “dilute” Hoeschst 33258 (0.5µg/ml in Sorensen’s buffer, pH 6.8) and examined by fluorescence microscopy to assess cell cycle kinetics. In control cultures, almost all cells completed two cycles in BrdUrd (M2 cells) in 25-26 hr, whereas, in treated cultures, cell cycle delay was common. In cases of severe delay, additional harvests were made from the same cultures at a later time to obtain sufficient second metaphase (M2) cells for SCE analysis. After staining for 10 min in “concentrated” Hoechst 33258 (5µg/ml in pH 6.8 buffer) and exposure to “black light” at 55 to 60°C for about 5 min, slides were stained in Giemsa. All slides were coded, and 50 cells per dose were scored from the three highest doses at which sufficient M2 cells were available. When cell cycle delay was noted, cell kinetics were recorded by classifying each of 100 metaphases as M1, M1+, or M2, i.e., having completed one (M1), two (M2), or between one and two (M1 +) cell cycles in BrdUrd. Delay was noted at the top dose with S9 (5 mg/ml), and the culture was harvested 2 hr later than the controls. There were increases in aberrations, but these were statistically significant only without S9. The test compound did not induce an increase in the number of Sister chromatid exchanges in the Chinese hamster ovary cells (CHO-W-B1) in the presence of S9 metabolic activation system but it induced sister chromatid exchanges in the absence of S9 metabolic activation system.

Gene mutation toxicity study for 4-Methoxybenzylamine (CAS no 2393 -23 -9) as predicted using data from read across chemicals with mammalian cell lines in the presence and absence of S9 metabolic activation system is negative and hence the chemical is predicted to not classify as a gene mutant in vitro.

Justification for classification or non-classification

Based on the predicted data available for the target chemical and data from its structurally and functionally similar read across chemicals, 4-Methoxybenzylamine (CAS no 2393 -23 -9) does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.