Formaldehyde, oligomeric reaction products with 4,4'-isopropylidenediphenol and m-phenylenebis (methylamine)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, EK195 did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9. The study was concluded to be negative without conducting a confirmatory (independent repeat) assay because the results were clearly negative; hence, no further testing was warranted.

Under the conditions of the assay described in the report, EK195 was concluded to be negative for the induction of structural and numerical chromosomal aberrations in the S9-activated 4-hour and non-activated 20-hour test systems in the in vitro mammalian chromosomal aberration test using CHO cells. In the non-activated 4-hour test system, EK195 was concluded to be negative for the induction of structural aberrations, but positive for the induction of numerical aberrations.

Under the conditions of the assay described in the report, EK195 was concluded to be negative for the induction of forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro L5178Y/TK+/- mouse lymphoma assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Target gene:

thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Non-activated 4 hours 0.38, 0.75, 1.5, 3, 6, 8 and 10
Non-activated 24 hours 0.38, 0.75, 1.5, 3, 6, 8 and 10
S9-activated 4 hours 2.19, 4.38, 8.75, 17.5, 35, 36, 37, 38 and 40

Based upon the results of the preliminary toxicity assay

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

methylmethanesulfonate

Details on test system and experimental conditions:

The preparation and addition of the test substance dose formulations was carried out under filtered lighting during the exposure period. Treatment was carried out by combining 100 μL of test substance dose formulation, vehicle or positive control dose formulation and F0P medium or S9 mix (as appropriate) with 6 x 10^6 L5178Y/TK+/- cells in a total volume of 10 mL. All pH adjustments were performed prior to adding S9 or target cells to the treatment medium. Each S9-activated 10-mL culture contained 4 mL S9 mix (final S9 concentration of 1.0%). Cultures were capped tightly and incubated with mechanical mixing at 37 ± 1°C for 4 or 24 hours.

For the preliminary toxicity assay only, after a 4-hour treatment in the presence and absence of S9, cells were washed with culture medium and cultured in suspension for two days post-treatment, with cell concentration adjustment on the first day. After a 24-hour treatment in the absence of S9, cells were washed with culture medium and immediately readjusted to 3 x 10^5 cells/mL. Cells were then cultured in suspension for an additional two days post-treatment with cell concentration adjustment on the first day.

For the definitive assay only, at the end of the exposure period, the cells were washed with culture medium and collected by centrifugation. The cells were resuspended in 20 mL F10P on Day 1 and in 10 mL F10P on Day 2, and incubated at 37 ± 1°C for two days following treatment. Cell population adjustments to 3 x 10^5 cells/mL were made as follows:
• 4 hour treatment – 1 and 2 days after treatment.
• 24 hour treatment – immediately after test substance removal, and 2 and 3 days after treatment.

Evaluation criteria:

• A result was considered positive if a concentration-related increase in mutant frequency was observed in the treated cultures and one or more treatment conditions with 10% or greater total growth exhibited induced mutant frequencies of ≥90 mutants/10^6 clonable cells (based on the average mutant frequency of duplicate cultures). If the average vehicle control mutant frequency was >90 mutants/10^6 clonable cells, a doubling of mutant frequency over the vehicle would also be required (Mitchell et al., 1997).
• A result was considered negative if the treated cultures exhibited induced mutant frequencies of less than 90 mutants/10^6 clonable cells (based on the average mutant frequency of duplicate cultures) and there was no concentration-related increase in mutant frequency.

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

Positive controls validity:

valid

Conclusions:

Under the conditions of the assay described in this report, EK195 was concluded to be negative for the induction of forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro L5178Y/TK+/- mouse lymphoma assay.

Executive summary:

The test substance, EK195, was evaluated for its ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the initial preliminary toxicity assay, the concentrations tested were 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 μg/mL. The maximum concentration evaluated approximated the limit dose for this assay. Visible precipitate was observed at concentrations ≥625 μg/mL at the beginning and end of treatment. Relative suspension growth (RSG) was 97% at concentrations of 19.5 μg/mL (4-hour treatment with S9). RSG was 0% at all higher concentrations using all treatment conditions. Based upon the steep toxicity profile observed, the concentrations chosen for the repeat of preliminary toxicity assay were 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40 μg/mL (4-hour treatment with S9) and 0.039, 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10 and 20 μg/mL (4-hour treatment without S9 and 24-hour treatment without S9).

In the repeat of preliminary toxicity assay, no visible precipitate was observed at the beginning or end of treatment. Relative suspension growth (RSG) was 19, 41 and 24% at concentrations of 36 μg/mL (4-hour treatment with S9), 5 μg/mL (4-hour treatment without S9) and 5 μg/mL (24-hour treatment without S9), respectively. RSG was or approximated 0% at all higher concentrations using all treatment conditions. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 2.19, 4.38, 8.75, 17.5, 35, 36, 37, 38 and 40 μg/mL (4-hour treatment with S9), 0.38, 0.75, 1.5, 3, 6, 8 and 10 μg/mL (4-hour treatment without S9) and 0.38, 0.75, 1.5, 3, 6, 8 and 10 μg/mL (24-hour treatment without S9).

In the definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment. Cultures treated at concentrations of 2.19, 4.38, 8.75 and 17.5 μg/mL (4-hour treatment with S9), 0.38, 0.75, 1.5 and 3 μg/mL (4-hour treatment without S9) and 0.38, 0.75, 1.5 and 3 μg/mL (24-hour treatment without S9) exhibited 42 to 93%, 66 to 103% and 60 to 108% RSG, respectively, and were cloned. Relative total growth of the cloned cultures ranged from 40 to 86% (4-hour treatment with S9), 62 to 126% (4-hour treatment without S9) and 48 to 95% (24-hour treatment without S9). No increases in induced mutant frequency ≥90 mutants/106 clonable cells were observed under any treatment condition.

These results indicate EK195 was negative for the ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and Muriel (1976).

Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976).

Salmonella tester strains were derived from Dr. Bruce Ames’ cultures; E. coli tester strains

Evaluation criteria:

All Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene. Cultures of tester strains TA98 and TA100 must demonstrate the presence of the pKM101 plasmid R-factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene.
All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls as follows (inclusive): TA98, 10 - 50; TA100, 80 - 240; TA1535, 5 - 45; TA1537, 3 - 21; WP2 uvrA, 10 - 60.

To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3x109 cells/mL.
The mean of each positive control must exhibit at least a 3.0-fold increase in the number of revertants over the mean value of the respective vehicle control.
A minimum of three non-toxic dose levels is required to evaluate assay data. A dose level is considered toxic if one or both of the following criteria are met: (1) A >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) At least a moderate reduction in the background lawn (background code 3, 4 or 5).

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, EK195 did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9. The study was concluded to be negative without conducting a confirmatory (independent repeat) assay because the results were clearly negative; hence, no further testing was warranted.

Executive summary:

The test substance, EK195, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 μg per plate. Precipitate was observed at 5000 μg per plate. Toxicity was observed beginning at 33.3, 66.7, 100, 333 or 667 μg per plate. Based upon these results, the maximum dose tested in the mutagenicity assay was 1000 μg per plate for tester strains TA98, TA1537 and WP2 uvrA (in the presence and absence of S9 activation) and TA1535 (in the presence of S9 activation) and 333 μg per plate for tester strains TA100 (in the presence and absence of S9 activation) and TA1535 (in the absence of S9 activation).

In the mutagenicity assay, the dose levels tested were 3.33, 10.0, 33.3, 100, 333 and 1000 μg per plate for tester strains TA98, TA1537 and WP2 uvrA (in the presence and absence of S9 activation) and TA1535 (in the presence of S9 activation) and 0.333, 1.00, 3.33, 10.0, 33.3, 100 and 333 μg per plate for tester strains TA100 (in the presence and absence of S9 activation) and TA1535 (in the absence of S9 activation). No precipitate was observed. Toxicity was observed beginning at 33.3, 100, 333 and 1000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

These results indicate EK195 was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Treatment Condition Treatment Time Recovery Time Doses (μg/mL) B1
Non-activated 4 hr 16 hr 5, 10, 15, 20, 25, 30, 35, 40, 45
20 hr 0 hr 5, 10, 15, 20, 25, 30, 35, 40, 45
S9-activated 4 hr 16 hr 10, 25, 50, 60, 70, 80, 90, 100, 125

Treatment Condition Treatment Time Recovery Time Doses (μg/mL) B1 repeat
Non-activated 4 hr 16 hr 1, 2.5, 5, 10, 20, 22.5, 25, 27.5, 30, 32.5, 35
S9-activated 4 hr 16 hr 10, 20, 40, 50, 60, 80, 100

Treatment Condition Treatment Time Recovery Time Doses (μg/mL) B2
Non-activated 4 hr 16 hr 1, 2.5, 5, 10, 20, 22.5, 25, 27.5, 30, 32.5, 35
S9-activated 4 hr 16 hr 10, 20, 40, 50, 60, 80, 100

Treatment Condition Treatment Time Recovery Time Doses (μg/mL) B3
Non-activated 4 hr 16 hr 3, 6, 10, 14, 20, 22.5, 25, 27.5, 30, 32.5, 35
20 hr 0 hr 3, 6, 8, 10, 12, 14, 16, 18, 20, 25
S9-activated 4 hr 16 hr 10, 20, 30, 40, 45, 47.5, 50, 55, 60

Treatment Condition Treatment Time Recovery Time Doses (μg/mL) B4
Non-activated 4 hr 16 hr 3, 6, 10, 14, 20, 22.5, 25, 27.5, 30, 32.5, 35
20 hr 0 hr 3, 6, 8, 10, 12, 14, 16, 18, 20, 25
S9-activated 4 hr 16 hr 10, 20, 30, 40, 45, 47.5, 50, 55, 60

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

methylmethanesulfonate

Details on test system and experimental conditions:

Chinese hamster ovary (CHO-K1) cells (repository number CCL 61) were obtained from American Type Culture Collection, Manassas, VA. In order to assure the karyotypic stability of the cell line, working cell stocks were not used beyond passage 15. The frozen lot of cells was tested using the Hoechst staining procedure and found to be free of mycoplasma contamination. This cell line has an average cell cycle time of 10-14 hours with a modal chromosome number of 20. The use of CHO cells has been demonstrated to be an effective method of detection of chemical clastogens (Preston et al., 1981).

Evaluation criteria:

The test substance was considered to have induced a positive response if
• at least one of the test concentrations exhibits a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
• the increase is concentration-related (p ≤ 0.05), and
• results are outside the 95% control limit of the historical negative control data.
The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Under the conditions of the assay described in this report, EK195 was concluded to be negative for the induction of structural and numerical chromosomal aberrations in the S9-activated 4-hour and non-activated 20-hour test systems in the in vitro mammalian chromosomal aberration test using CHO cells. In the non-activated 4-hour test system, EK195 was concluded to be negative for the induction of structural aberrations, but positive for the induction of numerical aberrations.

Executive summary:

The test substance, EK195, was tested to evaluate the potential to induce structural chromosomal aberrations using Chinese hamster ovary (CHO) cells in both the absence and presence of an of an exogenous metabolic activation system. CHO cells were treated for 4 hours in the absence and presence of S9, and for 20 hours in the absence of S9. Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the preliminary toxicity assay (A1), the doses tested ranged from 0.5 to 5000 μg/mL, which was the limit dose for this assay. Cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was observed at doses ≥ 50 μg/mL in the non-activated 4- and 20-hour exposure groups; and at doses ≥ 150 μg/mL in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 500 μg/mL in the non-activated 4-hour exposure group and at doses ≥ 150 μg/mL in the S9-activated 4-hour and the non-activated 20-hour exposure groups. Based upon these results, the doses chosen for the chromosomal aberration assay ranged from 5 to 45 μg/mL for the non-activated 4- and 20-hour exposure groups; and from 10 to 125 μg/mL for the S9-activated 4-hour exposure group.

In the initial chromosomal aberration assay (B1), cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control), was observed at doses ≥ 25 μg/mL in the non-activated 4-hour exposure group; at doses ≥ 50 μg/mL in the S9-activated 4-hour exposure group; and at doses ≥ 15 μg/mL in the non-activated 20-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 80 μg/mL in the S9-activated 4-hour exposure group. The doses selected for evaluation of chromosomal aberrations were 5, 10, and 25 μg/mL for the non-activated 4-hour exposure group; 10, 25, and 50 μg/mL for the S9-activated 4-hour exposure group; and 5, 10, and 15 μg/mL for the non-activated 20-hour exposure group.

In the non-activated 20-hour exposure group, no statistically significant or dose-dependent increases in structural or numerical (polyploid or endoreduplicated cells) aberrations were observed at any dose (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). The non-activated and S9-activated 4-hour exposure groups were rescored by a third slide scorer. However, due to inter-scorer variability in percent aberrant cells, the chromosomal aberration assay was repeated at doses ranging from 1 to 35 μg/mL for the non-activated 4-hour exposure group and from 10 to 100 μg/mL for the S9-activated 4-hour exposure group. Data from the initial and repeat scoring for these two treatment groups are maintained in the study file, but not reported.

In the repeat assay (B2), cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control), was observed at doses ≥ 27.5 μg/mL in the non-activated 4-hour exposure group and at doses ≥ 50 μg/mL in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 80 μg/mL in the S9-activated 4-hour exposure group. The doses selected for evaluation of chromosomal aberrations were 10, 20, and 27.5 μg/mL for the non-activated 4-hour exposure group and 10, 20, and 50 μg/mL for the S9-activated 4-hour exposure group.

In the repeat assay, no significant or dose-dependent increases in structural aberrations were observed in the non-activated and S9-activated 4-hour exposure groups (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

In the non-activated 4-hour exposure group, a statistically significant increase in numerical aberrations (8.0%) was observed at 20 μg/mL. Although the increase was outside the range of the historical 95% control limit (0.00% to 3.72%), the Cochran-Armitage test was negative for a dose-response (p > 0.05). Also, in the S9-activated 4-hour exposure group, the induction of chromosomal aberrations in the vehicle control (7.0%) was outside the range of the historical 95% control limit (0.00% to 6.35%). Therefore, the chromosomal aberration assay was repeated for the third time at doses ranging from 1 to 35 μg/mL for the non-activated 4-hour exposure group and from 10 to 100 μg/mL for the S9-activated 4-hour exposure group.

In the second repeat assay (B3), cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control), was observed at doses ≥ 30 μg/mL in the non-activated 4-hour exposure group and at doses ≥ 50 μg/mL in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 80 μg/mL in the S9-activated 4-hour exposure group. The doses selected for evaluation of chromosomal aberrations were 20, 25, and 30 μg/mL for the non-activated 4-hour exposure group and 10, 40, and 50 μg/mL for the S9-activated 4-hour exposure group.

During scoring, high numerical aberrations were observed in the non-activated 4-hour exposure group due to scorer variability between two independent slide scorers. Therefore, a third scorer was added to re-score all selected dose levels for numerical aberrations, per BioReliance SOP. The results obtained from the third slide scorer were reported. In the non-activated 4-hour exposure group, no statistically significant or dose-dependent increases in structural aberrations were observed at any dose (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). However, a statistically significant increase in numerical aberrations (7.0%) was observed at 30 μg/mL. Although the increase was outside the range of the historical 95% control limit (0.00% to 3.72%), the Cochran-Armitage test was negative for a dose-response (p > 0.05).

In the S9-activated 4-hour exposure group, no statistically significant or dose-dependent increases in structural or numerical aberrations were observed at any dose (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

Due to scorer variability in the percentage of aberrant cells in the three previous trials, the chromosomal aberration assay was repeated again at doses ranging from 3 to 35 μg/mL for the non-activated 4-hour exposure group; from 10 to 60 μg/mL for the S9-activated 4-hour exposure group; and from 3 to 25 μg/mL for the non-activated 20-hour

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)