N-[3-(dimethylamino)propyl]stearamide monoacetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two valid studies are available for the test item NDPSa (Cas n.: 13282-70-7,other identifier: H-31339). 

 

Chromosome Aberration; 28 hours, OECD TG 473; negative; Shambu Roy (2016)

 

Reverse Mutation test on bacteria; OECD TG 471; negative; Myhre A., M.S. (2015)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2014-11-25 To 2015-01-21

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

Target gene:

Bacterial Reverse Mutation Test using the plate incorporation method. Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and Escherichia coli strain WP2uvrA were tested in the absence and presence of an exogenous metabolic activation system (Aroclor-induced rat liver S9).

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Liver homogenate (S9) prepared from male Sprague-Dawley rats induced with Aroclor 1254 was purchased commercially (Moltox Inc., Boone, North Carolina, U.S.A.). The S9 protein content was: 41.0, 42.1, and 40.1 mg/mL for lots 3315, 3327, and 3331, respectively.
The S9 was thawed and the 10% S9 mix prepared immediately prior to its use. The S9 mix was held on ice at all times.
The S9 mix contained proportionate volumes of the following components:

Molecular-grade water = 2.4 mL
0.825 M KCl/0.2 M MgCl2 = 0.4 mL
0.2 M phosphate buffer, pH 7.4 = 5.0 mL
0.25 M glucose-6-phosphate = 0.2 mL
0.04 M NADP = 1.0 mL
S9 = 1.0 mL
Total Volume = 10 mL

The sham mix was 100 mM phosphate buffer at pH 7.4.

Test concentrations with justification for top dose:

In the initial toxicity-mutation test (trial T-1) the dose levels tested were 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 μg/plate. Tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA were tested in both the absence and presence of S9 metabolic activation. The negative control values for tester strain TA98 were outside of the acceptable limits.
In the second toxicity-mutation test (trial T-2) the dose levels tested were 5000, 3333, 1000, 667, 333, 100, 66.7, 33.3, 10, 6.67, 3.33 μg/plate with tester strain TA98 in both the absence and presence of S9 metabolic activation; and 66.7, 33.3, 10, 6.67, 3.33 μg/plate for tester strains TA100, TA1535, and TA1537 in the absence of S9 metabolic activation. No positive mutagenic responses were observed at any dose level in any tester strain in the absence or presence of
S9 metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:sterile water (CAS 7732-18-5, molecular grade, Mediatech Inc.)

- Justification for percentage of solvent in the final culture medium: Sterile water was chosen as the dosing vehicle based on the solubility of the test substance and compatibility with the target cells. The test substance was soluble in water at 50 mg/mL, the highest stock concentration that was prepared for use on this study.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

sterile water (CAS 7732-18-5, molecular grade, Mediatech Inc.)

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene (CAS 613-13-8, Moltox Inc.)

Details on test system and experimental conditions:

The tester strains were the Salmonella typhimurium histidine auxotroph tester strains TA98, TA100, TA1535, and TA1537, and the Escherichia coli tryptophan auxotroph WP2uvrA.(1,2,3) All tester strains were obtained from Moltox Inc. (Boone, North Carolina, U.S.A.). These strains were selected due to their ability to revert to histidine and tryptophan independence when exposed to a mutagen.
In addition to a mutation in either the histidine or tryptophan operons, the tester strains contain additional mutations that enhance their sensitivity to some mutagens. A mutation of either the uvrA or uvrB gene results in a deficient DNA excision repair system. Since the uvrB deletion extends through the bio gene, the Salmonella typhimurium tester strains also require the vitamin biotin for growth.
The Salmonella typhimurium tester strains also contain the rfa wall mutation which results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide (LPS) barrier that forms the surface of the bacterial cell wall. The resulting cell wall deficiency increases permeability to certain classes of chemicals such as those containing large ring systems that would otherwise be excluded by a normal intact cell wall.
Tester strains TA98 and TA100 also contain the pKM101 plasmid, which further increases the sensitivity of these strains to some mutagens.
Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independent (prototrophy) by frameshift mutagens. Tester strain TA100 is reverted by both frame shift and base substitution mutagens. Tester strains TA1535 and WP2uvrA are reverted from auxotrophy to prototrophy by base substitution mutagens.

Rationale for test conditions:

The tester strain cultures must exhibit a characteristic mean number of spontaneous revertants per plate when plated along with the negative (vehicle) control under selective conditions.

Evaluation criteria:

A minimum of 3 non-toxic scorable dose levels are required to validate the study. A dose level is considered toxic if it causes:
• A >50% reduction in the mean number of revertants per plate relative to the mean negative control value and exhibits a dose-dependent drop in the revertant count, or
• A reduction in the background lawn.
In the event that less than 3 non-toxic dose levels are achieved, the affected portion of the test
will be repeated with an appropriate change in dose levels.

Data will be judged positive if the increase in mean revertants at the highest numerical dose response is  3.0-fold the mean concurrent negative control value (vehicle control). This increase in the mean number of revertants per plate must be accompanied by a dose response associated with increasing concentrations of the test substance unless observed at the top dose level only.
Data sets will be judged positive if the increase in mean revertants at the highest numerical dose response is  2.0-fold the mean concurrent negative control value (vehicle control). This increase in the mean number of revertants per plate must be accompanied by a dose response associated with increasing concentrations of the test substance unless observed at the top dose level only.
A data set may be judged equivocal if there is a biologically relevant increased response that only partially meets criteria for a positive response. A response will be evaluated as negative if it is neither positive nor equivocal.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

with metabolic activation 667 μg/plate without metabolic activation 1000 μg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

The positive control that was used with tester stain TA98 in the presence of S9 activation was past the supplier’s expiration date; however, the positive control performed as expected with values consistent with the historical control data.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

The maximum dose prepared was 3333 mg/mL and a 100 μL plating aliquot was used.

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No positive mutagenic responses were observed at any dose level or with any tester strain in either the absence or presence of S9 metabolic activation.

Trial T-2

 

Tester Strain	Metabolic Activation	Precipitation starting at (μg/plate)	Toxicity starting at (μg/plate)   >50 reduction in revertant colonies	Toxicity starting at (μg/plate)   Background lawn reduction
TA98	- +	1000 667	66.7 667	66.7 33.3
TA100	-	None*	667	3.3.3
TA1535	-	None*	None*	66.7
TA1537	-	None*	None*	33.3

* None observed at the dose levels tested.

 

 

Dose levels used in T-2 Test.

 

Tester Strain	Metabolic Activation	Dose levels (μg/plate)
TA98	- +	100, 66.7, 33.3, 10, 6.67, 3.33 333, 100, 66.7, 33.3, 10
TA100	- +	100, 66.7, 33.3, 10, 6.67, 3.33 333, 100, 66.7, 33.3, 10
TA1535	- +	100, 66.7, 33.3, 10, 6.67, 3.33 333, 100, 66.7, 33.3, 10
TA1537	- +	100, 66.7, 33.3, 10, 6.67, 3.33 333, 100, 66.7, 33.3, 10
WP2uvrA	- +	1000, 667, 333, 100, 66.7 3333, 1000, 667, 333, 100

 

 

Mutagenicity Test

 

Tester Strain	Metabolic Activation	Precipitation starting at (μg/plate)	Toxicity starting at (μg/plate)   >50 reduction in revertant colonies	Toxicity starting at (μg/plate)   Background lawn reduction
TA98	- +	None* None*	100 333	100 333
TA100	- +	None* None*	66.7 333	66.7 None*
TA1535	- +	None* None*	100 333	66.7 333
TA1537	- +	None* None*	None* None*	66.7 333
WP2uvrA	- +	667 667	None* 3333	667 1000

* None observed at the dose levels tested.

Conclusions:

All criteria for a valid study were met. Under the conditions of this study, H-31339 showed no evidence of mutagenicity in the Bacterial Reverse Mutation Test either in the absence or presence of Aroclor-induced rat liver S9. It was concluded that the test substance was negative in this in vitro test.

Executive summary:

The test substance, H-31339, was evaluated for mutagenicity in the Bacterial Reverse Mutation Test using the plate incorporation method. Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and Escherichia coli strain WP2uvrA were tested in the absence and presence of an exogenous metabolic activation system (Aroclor-induced rat liver S9).

The test was performed in 2 phases. The first phase was the toxicity-mutation test, which established the dose range for the mutagenicity test, and provided a preliminary mutagenicity evaluation. The second phase was the mutagenicity test, which evaluated and confirmed the mutagenic potential of the test substance.

Sterile water was chosen as the dosing vehicle based on the solubility of the test substance and compatibility with the target cells. The test substance was soluble in water at 50 mg/mL, the highest stock concentration that was prepared for use on this study.

In the toxicity-mutation test, the maximum dose evaluated was 5000 μg/plate for tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA in the absence and presence of S9 metabolic activation. This dose was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot. The toxicity-mutation test was conducted in two trials. The plate incorporation method was employed.

In the initial toxicity-mutation test (trial T-1) the dose levels tested were 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 μg/plate. Tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA were tested in both the absence and presence of S9 metabolic activation. The negative control values for tester strain TA98 were outside of the acceptable limits; therefore, the data collected for this strain in trial T-1 were considered invalid. The invalid data is maintained with the study records but not included in this final report. No positive mutagenic responses were observed at any dose level in any tester strain in the absence or presence of S9 metabolic activation.

In the second toxicity-mutation test (trial T-2) the dose levels tested were 5000, 3333, 1000, 667, 333, 100, 66.7, 33.3, 10, 6.67, 3.33 μg/plate with tester strain TA98 in both the absence and presence of S9 metabolic activation; and 66.7, 33.3, 10, 6.67, 3.33 μg/plate for tester strains TA100, TA1535, and TA1537 in the absence of S9 metabolic activation. No positive mutagenic responses were observed at any dose level in any tester strain in the absence or presence of S9 metabolic activation.

Based on the toxicity-mutation test, the maximum dose evaluated in the mutagenicity test varied depending on the tester strain and metabolic activation. The maximum dose prepared was 3333 mg/mL and a 100 μL plating aliquot was used.

In the mutagenicity test the plate incorporation method was employed. No positive mutagenic responses were observed at any dose level or with any tester strain in either the absence or presence of S9 metabolic activation.

All criteria for a valid study were met. Under the conditions of this study, H-31339 showed no evidence of mutagenicity in the Bacterial Reverse Mutation Test either in the absence or presence of Aroclor-induced rat liver S9. It was concluded that the test substance was negative in this in vitro test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

in Human Peripheral Blood Lymphocytes (HPBL).

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2016-01-18 To 2016-05-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

updated and adopted 26 September 2014

Deviations:

yes

Remarks:

In the S9-activated 4-hour vehicle control group (Slide B), only 130 cells were initially scored, instead of 150 cells, as specified in the study protocol. Upon discovery, 20 additional cells were scored and documented on the same score sheet.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

chromosome aberration test

Species / strain / cell type:

mammalian cell line, other:

Details on mammalian cell type (if applicable):

Human Peripheral Blood Lymphocytes (HPBL)

Metabolic activation:

with and without

Metabolic activation system:

The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with AroclorTM 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice. The S9 (Lot No. 3563, Exp. Date: 15 Dec 2017) was purchased commercially from MolTox (Boone, NC). Upon arrival at BioReliance, the S9 was stored at -60°C or colder until used. Each bulk preparation of S9 was assayed for its ability to metabolize benzo(a)pyrene and 2-aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.

Test concentrations with justification for top dose:

At least 300 metaphases must be analyzed from at least three appropriate test substance concentrations. The number of metaphases scored may be reduced when high numbers of cells with chromosomal aberrations (≥10% metaphases) are observed as with a positive test substance or the positive control substance.

20-hr treatment with metabolic activation: 5 μg/mL.
20-hr treatment without metabolic activation: 2.5 μg/mL.
4-hr treatment with 16 hr recovery with metabolic activation: 10 μg/mL.
4-hr treatment without metabolic activation: 5 μg/mL.

Vehicle / solvent:

Water.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Treatment 20 hr without S9; 4 hr with 16 hr recovery period with and without S9. The induced value was outside the historical range of solvent control of this assay (0.0 to 1.0%). Vehicle control: Water (MMC, CP).

Details on test system and experimental conditions:

The test substance must be tested using a 4-hr treatment with and without S9, as well as a 20-hr treatment without S9. However, all three treatment conditions need not be evaluated in the case of a positive test substance response under any treatment condition.

Rationale for test conditions:

At least 300 metaphases must be analyzed from at least three appropriate test substance concentrations. The number of metaphases scored may be reduced when high numbers of cells with chromosomal aberrations (≥10% metaphases) are observed as with a positive test substance or the positive control substance.

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.

Statistics:

Statistical analysis was performed using the Fisher's exact test (p ≤ 0.05) for a pairwise comparison of the frequency of aberrant cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.

Key result

Species / strain:

mammalian cell line, other: human peripheral blood lymphocytes (HPBL)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

55 ± 5% reduction in mitotic index relative to the vehicle control observed at doses of ≥ 25 μg/mLin the non-activated 4-hour exposure group, at 30 μg/mL in the S9-activated 4-hour exposure group, and at doses ≥ 18 μg/mL in the non-activated 20-hour group

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

 

Preliminary Toxicity Assay

Cytotoxicity results of the preliminary toxicity assay are presented. Visible precipitate and hemolysis were observed in treatment medium at the following doses:

 

Treatment Condition	Treatment Time	Visible Precipitate At the beginning of treatment period	Visible Precipitate At the conclusion of treatment period	Hemolysis at the conclusion of treatment period
Non-activated	4 hr	≥ 600 μg/mL	≥ 60 μg/mL	≥ 60 μg/mL
	20 hr	≥ 600 μg/mL	≥ 200 μg/mL	≥ 200 μg/mL
S9-activated	4 hr	≥ 600 μg/mL	≥ 60 μg/mL	≥ 200 μg/mL

 

The osmolarity in treatment medium was measured as follows:

 

Dose Tested	Dose (μg/mL)	Osmolarity (mmol/kg)
Vehicle	0	288
Highest soluble	200	300
Lowest precipitating	600	299
Highest	2000	274

 

The osmolality of the test substance doses in treatment medium is acceptable because it did not exceed the osmolality of the vehicle by more than 120%. The pH of the highest dose of test substance in treatment medium was 7.5.

 

Cytotoxicity (≥ 50% reduction in mitotic index relative to the vehicle control) was observed at doses ≥ 20 μg/mL in the non-activated 4 and 20-hour exposure groups, and in the S9-activated 4-hour exposure group. Based upon the results of the preliminary toxicity assay, the doses selected for the chromosome aberration assay were as follows:

Treatment Condition	Treatment Time	Recovery Time	Doses (μg/mL)
Non-activated	4 hr	16 hr	1,2.5,5,10,12,14,16,18,20,25,30
	20 hr	0 hr	1,2.5,5,10,12,14,16,18,20,25,30
S9-activated	4 hr	16 hr	1,2.5,5,10,12,14,16,18,20,25,30

 

 

 

Preliminary Toxicity Test using H-31339 in the absence of exogenous metabolic activation 4-hours treatment, 20-hour harvest.

 

Treatment (-S9) μg/mL	Mitotic Index (%)	Percent Change (%)
Water	15.2
H-31339
0.2	14.2	-7
0.6	13.8	-9
2	13.0	-14
6	12.6	-17
20	7.4	-51
60 p	0.4	-97
200 p	0.0	-100
600 p	0.0	-100
2000 p	0.0	-100

 Treatment: Human peripheral blood lymphocyte cells were treated in the absence of an exogenous source of metabolic activation for 4 hours at 37 ± 1°C. Metaphase cells were collected following a 16-hour recovery period.

Mitotic Index = (Cells in mitosis/500 cells scored) x 100.

Percent change = (Treatment mitotic index - control mitotic index)/control mitotic index, expressed as a percentage.

p: Visible precipitate was observed in the treatment medium at the conclusion of the treatment period.

 

 

Preliminary Toxicity Test using H-31339 in the presence of exogenous metabolic activation 4-hours treatment, 20-hour harvest.

 

Treatment (-S9) μg/mL	Mitotic Index (%)	Percent Change (%)
Water	15.4
H-31339
0.2	13.0	-16
0.6	12.4	-19
2	12.0	-22
6	12.2	-21
20	7.0	-55
60 p	0.4	-97
200 p	0.0	-100
600 p	0.0	-100
2000 p	0.0	-100

Treatment: Human peripheral blood lymphocyte cells were treated in the absence of an exogenous source of metabolic activation for 4 hours at 37 ± 1°C. Metaphase cells were collected following a 16-hour recovery period.

Mitotic Index = (Cells in mitosis/500 cells scored) x 100.

Percent change = (Treatment mitotic index - control mitotic index)/control mitotic index, expressed as a percentage.

p: Visible precipitate was observed in the treatment medium at the conclusion of the treatment period.

Conclusions:

Under the conditions of the assay described in this report, H-31339 was considered as negative for the induction of structural and numerical chromosome aberrations in the presence and absence of the exogenous metabolic activation system in the in vitro mammalian chromosome aberration test using human peripheral blood lymphocytes.

Executive summary:

The test substance, H-31339, was tested to evaluate the potential to induce structural chromosomal aberrations using human peripheral blood lymphocytes (HPBL) in both the absence and presence of an of an exogenous metabolic activation system. Water was used as the vehicle.

In the preliminary toxicity assay, the doses tested ranged from 0.2 to 2000 μg/mL, which was the limit dose for this assay. Cytotoxicity (≥ 50% reduction in mitotic index relative to the vehicle control) was observed at doses ≥ 20 μg/mL in the non-activated 4 and 20-hour exposure groups, and in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 60 μg/mL in the non-activated and S9-activated 4-hour exposure groups, and at doses ≥ 200 μg/mL in the non-activated 20-hour exposure group. Based upon these results, the doses chosen for the chromosome aberration assay ranged from 1 to 30 μg/mL for all three exposure groups.

In the initial chromosome aberration assay, cytotoxicity (55 ± 5% reduction in mitotic index relative to the vehicle control), was observed at doses ≥ 25 μg/mL in the non-activated 4-hour exposure group, at 30 μg/mL in the S9-activated 4-hour exposure group, and at doses ≥ 18 μg/mL in the non-activated 20-hour exposure group. However, due to unacceptable cell growth, the chromosome aberration assay was repeated at doses ranging from 1 to 30 μg/mL for all three exposure groups.

In the repeat assay, cytotoxicity (55 ± 5% reduction in mitotic index relative to the vehicle control), was observed at doses ≥ 10 μg/mL in the non-activated 4-hour exposure group, at doses ≥ 14 μg/mL in the S9-activated 4-hour exposure group, and at doses ≥ 5 μg/mL in the non-activated 20-hour exposure group. The doses selected for evaluation of chromosome aberrations were 2.5, 5, and 10 μg/mL for the non-activated 4-hour exposure group, 5, 10, and 14 μg/mL for the S9-activated 4-hour exposure group, and 1, 2.5, and 5 μg/mL for the non-activated 20-hour exposure group.

No significant or dose-dependent increases in structural aberrations were observed in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). No significant or dose-dependent increases in numerical (polyploid or endoreduplicated cells) aberrations were observed in the S9-activated 4-hour and the non-activated 20-hour exposure groups (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

In the non-activated 4-hour exposure group, a statistically significant and dose-dependent increase in numerical aberrations (1.7%) was observed at 10 μg/mL (p ≤ 0.05; Fisher’s Exact and Cochran-Armitage tests). The induced value was outside the historical range of solvent control of this assay (0.0 to 1.0%). The numerical aberration at lower two dose levels of test substance and in solvent was 0.0% and most likely caused the dose responsiveness. The cytotoxicity was 51% at 10 μg/mL at which the 1.7% induction in numerical aberrations was observed. The next lower dose level (5 μg/mL) with 36% cytotoxicity had 0.0% aberrations. This indicates that the positive response observed at 10 μg/mL could be an isolated event, and therefore had no biological relevance. The 0.0 % aberration in solvent is further contributing to the statistical significance.

Based on these observations H-31339 was considered negative for the induction of structural and numerical chromosome aberrations in the presence and absence of the exogenous metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Not applicable as there are no adverse-effects observed.

Additional information

Chromosome Aberration Test:

 

The test substance, H-31339, was tested to evaluate the potential to induce structural chromosomal aberrations using human peripheral blood lymphocytes (HPBL) in both the absence and presence of an of an exogenous metabolic activation system. Water was used as the vehicle.

In the preliminary toxicity assay, the doses tested ranged from 0.2 to 2000 μg/mL, which was the limit dose for this assay. Cytotoxicity (≥ 50% reduction in mitotic index relative to the vehicle control) was observed at doses ≥ 20 μg/mL in the non-activated 4 and 20-hour exposure groups, and in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 60 μg/mL in the non-activated and S9-activated 4-hour exposure groups, and at doses ≥ 200 μg/mL in the non-activated 20-hour exposure group. Based upon these results, the doses chosen for the chromosome aberration assay ranged from 1 to 30 μg/mL for all three exposure groups.

In the initial chromosome aberration assay, cytotoxicity (55 ± 5% reduction in mitotic index relative to the vehicle control), was observed at doses ≥ 25 μg/mL in the non-activated 4-hour exposure group, at 30 μg/mL in the S9-activated 4-hour exposure group, and at doses ≥ 18 μg/mL in the non-activated 20-hour exposure group. However, due to unacceptable cell growth, the chromosome aberration assay was repeated at doses ranging from 1 to 30 μg/mL for all three exposure groups.

In the repeat assay, cytotoxicity (55 ± 5% reduction in mitotic index relative to the vehicle control), was observed at doses ≥ 10 μg/mL in the non-activated 4-hour exposure group, at doses ≥ 14 μg/mL in the S9-activated 4-hour exposure group, and at doses ≥ 5 μg/mL in the non-activated 20-hour exposure group. The doses selected for evaluation of chromosome aberrations were 2.5, 5, and 10 μg/mL for the non-activated 4-hour exposure group, 5, 10, and 14 μg/mL for the S9-activated 4-hour exposure group, and 1, 2.5, and 5 μg/mL for the non-activated 20-hour exposure group.

No significant or dose-dependent increases in structural aberrations were observed in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). No significant or dose-dependent increases in numerical (polyploid or endoreduplicated cells) aberrations were observed in the S9-activated 4-hour and the non-activated 20-hour exposure groups (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

In the non-activated 4-hour exposure group, a statistically significant and dose-dependent increase in numerical aberrations (1.7%) was observed at 10 μg/mL (p ≤ 0.05; Fisher’s Exact and Cochran-Armitage tests). The induced value was outside the historical range of solvent control of this assay (0.0 to 1.0%). The numerical aberration at lower two dose levels of test substance and in solvent was 0.0% and most likely caused the dose responsiveness. The cytotoxicity was 51% at 10 μg/mL at which the 1.7% induction in numerical aberrations was observed. The next lower dose level (5 μg/mL) with 36% cytotoxicity had 0.0% aberrations. This indicates that the positive response observed at 10 μg/mL could be an isolated event, and therefore had no biological relevance. The 0.0 % aberration in solvent is further contributing to the statistical significance.

Based on these observations H-31339 was considered negative for the induction of structural and numerical chromosome aberrations in the presence and absence of the exogenous metabolic activation system.

 

Bacterial Reverse Mutation Test:

 

The test substance, H-31339, was evaluated for mutagenicity in the Bacterial Reverse Mutation Test using the plate incorporation method. Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and Escherichia coli strain WP2uvrA were tested in the absence and presence of an exogenous metabolic activation system (Aroclor-induced rat liver S9).

The test was performed in 2 phases. The first phase was the toxicity-mutation test, which established the dose range for the mutagenicity test, and provided a preliminary mutagenicity evaluation. The second phase was the mutagenicity test, which evaluated and confirmed the mutagenic potential of the test substance.

Sterile water was chosen as the dosing vehicle based on the solubility of the test substance and compatibility with the target cells. The test substance was soluble in water at 50 mg/mL, the highest stock concentration that was prepared for use on this study.

In the toxicity-mutation test, the maximum dose evaluated was 5000 μg/plate for tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA in the absence and presence of S9 metabolic activation. This dose was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot. The toxicity-mutation test was conducted in two trials. The plate incorporation method was employed.

In the initial toxicity-mutation test (trial T-1) the dose levels tested were 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 μg/plate. Tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA were tested in both the absence and presence of S9 metabolic activation. The negative control values for tester strain TA98 were outside of the acceptable limits; therefore, the data collected for this strain in trial T-1 were considered invalid. The invalid data is maintained with the study records but not included in this final report. No positive mutagenic responses were observed at any dose level in any tester strain in the absence or presence of S9 metabolic activation.

In the second toxicity-mutation test (trial T-2) the dose levels tested were 5000, 3333, 1000, 667, 333, 100, 66.7, 33.3, 10, 6.67, 3.33 μg/plate with tester strain TA98 in both the absence and presence of S9 metabolic activation; and 66.7, 33.3, 10, 6.67, 3.33 μg/plate for tester strains TA100, TA1535, and TA1537 in the absence of S9 metabolic activation. No positive mutagenic responses were observed at any dose level in any tester strain in the absence or presence of S9 metabolic activation.

Based on the toxicity-mutation test, the maximum dose evaluated in the mutagenicity test varied depending on the tester strain and metabolic activation. The maximum dose prepared was 3333 mg/mL and a 100 μL plating aliquot was used.

In the mutagenicity test the plate incorporation method was employed. No positive mutagenic responses were observed at any dose level or with any tester strain in either the absence or presence of S9 metabolic activation.

All criteria for a valid study were met. Under the conditions of this study, H-31339 showed no evidence of mutagenicity in the Bacterial Reverse Mutation Test either in the absence or presence of Aroclor-induced rat liver S9. It was concluded that the test substance was negative in this in vitro test.

All criteria for a valid study were met. Under the conditions of this study, H-31339 showed no evidence of mutagenicity in the Bacterial Reverse Mutation Test either in the absence or presence of Aroclor-induced rat liver S9. It was concluded that the test substance was negative in this in vitro test.

 

Justification for selection of Genetic toxicity endpoint -
two key 1 studies used

 

 

Harmonised classification:

The substance has no harmonised classification according to the Regulation (EC) No. 1272/2008 (CLP). 

 

Self-classification:

Based on the available information on test item, no self-classification is proposed according to the CLP or GHS.

 

Justification for classification or non-classification

As required by Annexes VII and VIII of REACH, two valid studies are presented (Roy S., 2016; Myhre A., M.S.,2015).

All studies indicated negative results for mutagenicity.

In accordance with Regulation (EC) No 1272/2008 (CLP), these results are conclusive but not sufficient for classification.