Nonane-1,9-diol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

When tested in vitro, 1,9 -Nonanediol was negative in an Ames test and a mammalian cell gene mutation assay with mouse lymphoma L5178Y cells, with positive results returned in the chromosome aberrations study conducted in isolated human lymphocytes.

 

1,9-Nonandiol did not induce mutation in four histidine-requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium, and one tryptophan-requiring strain (WP2uvrA ) of Escherichia coli when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/mL (the maximum recommended concentration according to current regulatory guidelines, and in several cases a toxic treatment concentration), in the absence and presence of a rat liver metabolic activation system (S9) using both plate incorporation and pre-incubation methodologies.

 

1,9-Nonanediol did not induce mutation at the tk locus of L5178Y mouse lymphoma cells when tested up to the limit of toxicity in the absence (4 hours) and presence (4 hours) of a rat liver metabolic activation system (S-9) following a single mutation experiment.

 

It is concluded that 1,9-Nonanediol did not show an increase in the incidence of chromosome aberrations in isolated human lymphocytes. These conditions included treatments up to the maximum recommended concentration (10 mM) in accordance with current in vitro genotoxicity test guidelines in the absence (4 hours [+ 20 hours recovery]) and presence (4 h [+20 h]) of a rat liver metabolic activation system (S9) following a single experiment.

 

In a separate experiment, in the absence of S9 (24 h [+24 h]), 1,9-Nonanediol increased the incidence of chromosome aberrations in isolated human lymphocytes when tested up to the limit of toxicity (relative mitotic index 47%).

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:

2003-09-03 to 2003-11-14

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 (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Chemical name: 1,9-Nonanediol
- CAS no.: 3937-56-2
- Source and lot/batch No.of test material: : BASF / 42638
- Expiration date of the lot/batch: March 2003
- Molecular weight: 160.254 g/mol
- Purity: 98.8%

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced S9 (prepared in house)

Test concentrations with justification for top dose:

1st Experiment (plate incorporation) and Experiment 2 (pre-incubation): 0, 20, 100, 500, 2500, 5000 ug/plate (the maximum recommended concentration in accordance with current regulatory guidelines for in vitro bacterial genotoxicity assays)
Due to toxicity observed at 5000 ug/plate in Experiment 2, a further pre-incubation test was undertaken (Experiment 3): 0, 250, 500, 750, 1000 and 1500 ug/plate (maximum concentration limited by toxicity)

Vehicle / solvent:

Dimethyl sulphoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: MNNG, NOPD, AAC, 4-NQO

Details on test system and experimental conditions:

Test tubes containing 2 mL of soft agar (overlay agar) kept at 45°C in a water bath had the following added: 0.1 mL test article formulation (or vehicle) ; 0.1 mL fresh bacterial culture; 0.5 mL S9 mix (or PBS in the absence of metabolic activation).
After mixing Salmonella typhimurium containing tubes were poured onto Vogel-Bonner agar plates (minimal glucose agar plates). E.coli containing tubes were poured onto minimal agar plates.
Agar plates were incubated at 37°C for 48-72 h in the dark for the bacterial colonies (his+ or typ+ revertants) counted.
Pre-incubation:
0.1 mL of test article formulation (or vehicle), 0.1 mL bacterial suspension and 0.5 mL S9 mix (or PBS in the absence of metabolic activation) were incubated at 37°C for the duration of ~20 minutes. Subsequently, 2 mL of soft agar was added and samples were poured onto the agar plates.
In both cases, agar plates were incubated at 37°C for 48-72 h in the dark for the bacterial colonies (his+ or typ+ revertants) counted.

The background lawns of the plates were examined for signs of toxicity. Other toxicity indicators that may have been noted included a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response.

Evaluation criteria:

The test chemical was considered positive in this assay if the following criteria were met:
- dose-related and reproducible increase in the number of revertant colonies (i.e. doubling of the spontaneous mutation rate in at least one tester strain either –S9 or +S9)

A test substance was generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Statistics:

Statistics not warranted

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

A. Mutation assay:

Experiment 1 (plate incorporation) treatments of all the tester strains were performed in the absence and presence of S-9, using final concentrations of 1,9-Nonanediol at 20, 100, 500, 2500, 5000 µg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity (evidenced by reduction in revertant numbers) was observed on the mutation plates treated at 5000 µg/mL with TA1537 and TA98 in the presence of S9. 1,9-Nonandiol was not mutagenic following a plate incorporation methodology

Experiment 2 (pre-incubation) treatments of all the tester strains were performed in the absence and presence of S-9 using the same dose concentrations as Experiment 1. Toxicity was evident at concentrations of 2500 ug/plate and greater. Consequently with only 4 scorable dose levels, a further pre-incubation experiment was undertaken to better define mutagenicity in the absence of overt toxicity.

Experiment 3 (pre-incubation) treatments of all the tester strains were performed in the absence and presence of S-9, using final concentrations of 1,9-Nonanediol at 250, 500, 750, 1000, 1500 µg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity (evidenced by both a reduction in revertant numbers and background lawn) was observed on the mutation plates treated at 1500 µg/mL with TA98 and WP2uvrA in the absence of S9. 1,9-Nonanediol was not mutagenic following a pre-incubation methodology

The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.

The positive controls induced an acceptable increase in revertant colony numbers,thereby demonstrating the sensitivity and specificity of the test system.

Following1,9-Nonandioltreatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were equal to or greater than 2-fold above the concurrent vehicle control. This study was therefore considered to have provided no evidence of any mutagenic activity in this assay system (refer to Table 7.6.1/01-1, -2, -3).

Table 7.6.1/01-1:
Bacterial (reverse) gene mutation plate incorporation data – Experiment 1

Conc (µg/plate)	TA98		TA100		TA1535			TA1537		WP2uvrA
	-S9	+S9	-S9	+S9	-S9	+S9		-S9	+S9	-S9	+S9
0	25	39	118	109	18	17		9	11	32	36
20	26	36	121	122	15	15		8	7	30	30
100	26	35	132	141	15	14		10	8	^29	^32
500	28	33	134	123	14	14		8	9	26	29
2500	23	36	127	128	10	10		6	6	24	29
5000	20	19	93	84	11	11		6	6	19	29
+ve	689	545	1117	986	907	128		459	113	614	253
+ve controls: -S9 (absence of metabolic activation): TA98: 4-nitro-o-phenylendiamine (NOPD) TA100, TA1535: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) TA1537: 9-aminoacridine (AAC) WP2uvrA: 4-nitroquinoline-N-oxide (4-NQO)							+S9 (presence of metabolic activation): All strains: 2-aminoanthracene

Table 7.6.1/01-2:
Bacterial (reverse) gene mutation pre-incubation data – Experiment 2

Conc (µg/plate)	TA98		TA100		TA1535			TA1537		WP2uvrA
	-S9	+S9	-S9	+S9	-S9	+S9		-S9	+S9	-S9	+S9
0	27	31	107	107	17	16		10	9	30	30
20	22	32	106	102	19	16		10	7	31	28
100	21	29	108	98	15	13		8	7	34	25
500	22	24	100	99	15	13		5	6	30	28
2500	10B	16B	43B	93B	2B	11B		1B	6B	26B	22
5000	0B	12B	0B	56B	0B	5B		0B	2B	0B	18B
+ve	802	603	805	846	719	91		379	118	634	242
B: reduced background lawn +ve controls: -S9 (absence of metabolic activation): TA98: 4-nitro-o-phenylendiamine (NOPD) TA100, TA1535: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) TA1537: 9-aminoacridine (AAC) WP2uvrA: 4-nitroquinoline-N-oxide (4-NQO)							+S9 (presence of metabolic activation): All strains: 2-aminoanthracene

Table 7.6.1/01-3:
Bacterial (reverse) gene mutation pre-incubation data – Experiment 3

Conc (µg/plate)	TA98		TA100		TA1535			TA1537		WP2uvrA
	-S9	+S9	-S9	+S9	-S9	+S9		-S9	+S9	-S9	+S9
0	26	32	108	116	19	16		8	9	30	37
250	22	27	102	110	18	15		8	8	26	37
500	30	34	112	104	16	13		6	7	20	30
750	21	31	102	102	14	14		7	7	26	29
1000	26	25	101	105	16	14		7	7	23	27
1500	14	19	101	75	14	10		5	5	12	22
+ve	620	792	717	842	717	106		415	126	628	232
+ve controls: -S9 (absence of metabolic activation): TA98: 4-nitro-o-phenylendiamine (NOPD) TA100, TA1535: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) TA1537: 9-aminoacridine (AAC) WP2uvrA: 4-nitroquinoline-N-oxide (4-NQO)							+S9 (presence of metabolic activation): All strains: 2-aminoanthracene

B. Deficiencies:

A single positive control, 2-aminoanthracene was used to evaluate the sensitivity of the assay in both the presence of S9-mix. This positive control may be activated by enzymes other than the microsomal cytochrome P450 family. Therefore it could be concluded that the S9 activity has not been demonstrated. However, S9 was prepared in house and the efficacy of the S9 mix was characterised with benzo[a]pyrene. Overall it is concluded that the S9 activity was adequately demonstrated.

Conclusions:

It was concluded that 1,9-Nonanediol did not induce mutation in four histidine-requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium, and one tryptophan-requiring strain (WP2uvrA) of Escherichia coli when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/mL (the maximum recommended concentration according to current regulatory guidelines, and in several cases a toxic treatment concentration), in the absence and presence of a rat liver metabolic activation system (S9) using both plate incorporation and pre-incubation methodologies.

Executive summary:

In a reverse gene mutation assay in bacteria, S. typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2uvrA were exposed to1,9-Nonanediol formulated in dimethyl sulphoxide.Both plate incorporation and pre-incubation methodologies were used.

Experiment 1 (plate incorporation) treatments of all the tester strains were performed in the absence and presence of S-9, using final concentrations of 1,9-Nonandiol at 20, 100, 500, 2500, 5000 µg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity (evidenced by reduction in revertant numbers) was observed on the mutation plates treated at 5000 µg/mL with TA1537 and TA98 in the presence of S9. 1,9-Nonandiol was not mutagenic following a plate incorporation methodology

Experiment 2 (pre-incubation) treatments of all the tester strains were performed in the absence and presence of S-9 using the same dose concentrations as Experiment 1. Toxicity was evident at concentrations of 2500 ug/plate and greater. Consequently with only 4 scorable dose levels, a further pre-incubation experiment was undertaken to better define mutagenicity in the absence of overt toxicity.

Experiment 3 (pre-incubation) treatments of all the tester strains were performed in the absence and presence of S-9, using final concentrations of 1,9-Nonandiol at 250, 500, 750, 1000, 1500 µg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity (evidenced by both a reduction in revertant numbers and background lawn) was observed on the mutation plates treated at 1500 µg/mL with TA98 and WP2uvrA in the absence of S9. 1,9-Nonandiol was not mutagenic following a pre-incubation methodology

The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.

The positive controls induced an acceptable increase in revertant colony numbers, thereby demonstrating the sensitivity and specificity of the test system.

Following 1,9-Nonanediol treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were equal to or greater than 2-fold above the concurrent vehicle control. This study was therefore considered to have provided no evidence of any mutagenic activity in this assay system.

It was concluded that 1,9-Nonanediol did not induce mutation in four histidine-requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium, and one tryptophan-requiring strain (WP2uvrA) of Escherichia coli when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/mL (the maximum recommended concentration according to current regulatory guidelines, and in several cases a toxic treatment concentration), in the absence and presence of a rat liver metabolic activation system (S9) using both plate incorporation and pre-incubation methodologies.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-11-29 to 2016-12-20

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)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian forward gene mutation

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Chemical name: 1,9-Nonanediol
- CAS no.: 3937-56-2
- Source and lot/batch No.of test material: Kuraray / 72732
- Expiration date of the lot/batch: 19 April 2018
- Molecular weight: 160.254 g/mol
- Purity: 99.23%

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital / beta-naphthoflavone

Test concentrations with justification for top dose:

Range-finder:
4 h -S9: 0, 4, 8, 16, 40, 80, 160, 401, 801, 1202, 1603 (equivalent to 10 mM, the maximum recommended concentration in accordance with current regulatory guidelines for in vitro mammalian genotoxicity assays in the absence of toxicity) ug/mL

Main mutation assay:
4 h -S9: 0, 80, 160, 401, 801, 962, 1122, 1282, 1442 ug/mL (maximum concentration limited by toxicity)
4 h +S9: 0, 16, 40, 80, 160, 401, 801, 1202, 1603 ug/mL (equivalent to 10 mM, the maximum recommended concentration which also induced an aceeptable level of toxicity)

Vehicle / solvent:

cell culture medium (RPMI)

Untreated negative controls:

yes

Remarks:

RPMI cell culture media

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

methylmethanesulfonate

Details on test system and experimental conditions:

Preliminary cytotoxicity assay:
Cells were exposed to test article formulations, solvent controls for 4 h in the absence of S9 only. Duplicate cultures were used for the vehicle control and single cultures were used the test article formulation cultures. At the end of treatment cultures were washed, cell culture counted, adjusted (where necessary to 3 x 105 cells/mL) transferred to cell culture flasks and incubated for 2 days, with cell adjustment made every 24 hours. After 2-days growth period the relative suspension growth (RSG) of the treated cell cultures was calculated to estimate toxicity.
Osmolality and pH were measured on post-treatment media. As no marked changes are observed, further measurements were not deemed necessary for the mutation experiments.

Mutation assay:
The mutation assay was conducted as detailed above for the preliminary cytotoxicity assay, with the following exceptions:
- Positive controls included.
- S9 was included
- At the end of the 2-days of growth, cells were sub-cultured to assess cytotoxicity and to initiate the phenotypic expression.
- Mutation frequency was determined by plating ~2000 cells/well in cell culture medium containing 4 µg TFT/mL. Plates were incubated for 10-14 days. After this period the number of well without growth was counted to provide CE in TFT. Wells with growth in indicated evidence of TFT-resistance mutants. Colony sizing was performed on negative and positive controls.
- Cloning efficiency was determined by plating ~1.6 cells/well into two 96 well plates. Plates were incubated for 10-14 days. After this period the number of wells without growth of cells was counted.
Solubility of the test article in culture medium was assessed, by eye, at the beginning and end of treatment.

Rationale for test conditions:

refer to justifcation of dose levels

Evaluation criteria:

Acceptance criteria:
The assay was considered acceptable if all the following criteria (as defined by Moore et al ) were met:
- The mean mutant frequencies in the vehicle control cultures fell within the normal range (50 to 170 mutants/106 viable cells)
- At least one positive control showed either an absolute increase in mean total MF of at least 300 x 10-6 (at least 40% of this should be in the small colony MF), or an increase in small colony mutant frequency of at least 150 x 10-6 above the concurrent vehicle control
- The RTG for the positive controls was greater than 10%
-The mean cloning efficiency of the vehicle controls from the Mutation Experiments were between the range 65% to 120%
The mean SG of the vehicle controls from the Mutation experiments were between the range of 8 to 32 following 3 hour treatments.

Evaluation criteria:
The test article was considered mutagenic in this assay (as defined by Moore et al1) if :
- The MF of any test concentration exceeded the sum of the vehicle control mutant frequency plus GEF
- The linear trend test was statistically significant
- Any observed response is reproducible under the same treatment conditions.
The test article was considered positive in this assay if both of the above criteria were met.

Statistics:

not warranted

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Preliminary cytotoxicity assay:

The maximum concentration tested in the preliminary test was 1603 µg/mL (equivalent to 10 mM) in the absence of S9. No precipitate was observed.

In the absence of S9, no overttoxicity was observed such that the highest concentration analysed (1603 µg/mL) gave 86% RSG.

Osmolality and pH were measured on post-treatment media. As no marked changes are observed, further measurements were not deemed necessary for the mutation experiments.

Main mutation assay:

-S9:

The maximum concentration tested was 1442 µg/mL. Toxicity, expressed in terms of relative total growth (RTG) gave 14% RTG.

In the absence of S9, no increases in mutant frequency (MF) which exceeded the sum of the Global Evaluation Factor (GEF) of 126 + the vehicle control MF, were observed in any treated cultures (refer to Table 7.6.1/02-1).

Table 7.6.1/02-1:
Mouse lymphoma toxicity and mutant frequency data: 4 h –S9

Conc.		-S9		% of small colony mutants
(µg/mL)	(mM)	%RTG	MF
0	0	100	54.7	12.8
80	0.5	124	56.4	-
160	1.0	108	56.6	-
401	2.5	87	72.8	-
801	5.0	89	51.4	-
962	6.0	68	67.5	-
1122	7.0	66	12.3	10.2
1282	8.0	39	9.0	19.4
1442	9.0	14	12.3	18.4
EMS 300		76	502.4^$	-
MMS 10		55	520.0^$	58.4
+ve controls: EMS – ethyl methanesulphonate; MMS - methyl methanesulphonate $ Sum of the vehicle control mutant frequency (MF) + GEF (126) exceeded [180.7]

 

+S9:

The maximum concentration tested was 1603 µg/mL. Toxicity, expressed in terms of RTG gave 11% RTG.

In the presence of S9, no increases in mutant frequency (MF) which exceeded the sum of the Global Evaluation Factor (GEF) of 126 + the vehicle control MF, were observed in any treated cultures (refer to Table 7.6.1/02-2).

 

The positive controls induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants under both treatment conditions, thereby demonstrating the sensitivity and specificity of the test system.

Table 7.6.1/02-2:
Mouse lymphoma toxicity and mutant frequency data: 4 h +S9

Conc.		+S9		% of small colony mutants
(µg/mL)	(mM)	%RTG	MF
0	0	100	96.6	18.7
16	0.10	98	99.6	-
40	0.25	85	83.1	-
80	0.50	95	78.7	-
160	1.0	115	91.2	-
401	2.5	114	80.5	-
801	5.0	102	87.0	13.6
1202	7.5	55	75.0	13.7
1603	10	11	109.4	36.2
B[a] P 2.5		80	404.6^$	47.2
+ve controls: B[a]P – benzo[a]pyrene $ Sum of the vehicle control mutant frequency (MF) + GEF (126) exceeded [222.6]

Conclusions:

It is concluded that 1,9-Nonanediol did not induce mutation at the tk locus of L5178Y mouse lymphoma cells when tested up to the limit of toxicity in the absence (4 hours) and presence (4 hours) of a rat liver metabolic activation system (S9) following a single mutation experiment.

Executive summary:

In a mammalian cell gene mutation assay, mouse lymphoma L5178Y cells were exposed to 1,9-Nonanediol, formulated in RPMI cell culture media. Forward mutation at the thymidine kinase (tk^+/-) gene locus was measured. Dose concentrations selected for the main mutation assay were selected following a preliminary cytotoxicity test.

Osmolality and pH were measured on post-treatment media. As no marked changes are observed, further measurements were not deemed necessary for the mutation experiments.

In main mutation assay in the absence of S9 the maximum concentration tested was 1442 µg/mL. Toxicity expressed in term of relative total growth (RTG) was reduced to 12% RTG, at the maximum concentration. In the absence of S9, no increases in mutant frequency (MF) which exceeded the sum of Global Evaluation Factor (GEF, 126) + the vehicle control MF, were observed.

In main mutation assay in the presence of S9 the maximum concentration tested was 1603 µg/mL (equivalent to 10 mM, the maximum recommended concentration in accordance with current regulatory guidelines). Toxicity expressed in term of RTG was reduced to 14% RTG, at the maximum concentration. In the presence of S9, no increases in MF which exceeded the sum of GEF + the vehicle control MF, were observed.

The positive controls induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants,thereby demonstrating the sensitivity and specificity of the test system.

It is concluded that 1,9-Nonanediol did not induce mutation at the tk locus of L5178Y mouse lymphoma cells when tested up to the limit of toxicity in the absence (4 hours) and presence (4 hours) of a rat liver metabolic activation system (S9) following a single mutation experiment.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-01-12 to 2017-03-22

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)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian chromosome aberration assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Chemical name: 1,9-Nonanediol
- CAS no.: 3937-56-2
- Source and lot/batch No.of test material: Kuraray / 72732
- Expiration date of the lot/batch: 19 April 2018
- Molecular weight: 160.254 g/mol
- Purity: 99.23%

Species / strain / cell type:

lymphocytes:

Remarks:

human peripheral blood lymphocytes

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

colcemid

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital / beta-naphthoflavone

Test concentrations with justification for top dose:

Preliminary cytotoxicity test:
4h -/+S9:
0, 4.0, 8.0, 16, 40, 80, 160, 401, 801, 1202, 1603 ug/mL
Experiment 1:
4 h +/-S9: 0, 321, 641, 801, 962, 1282, 1603 ug/mL (equivalent to 10 mM, the maximum recommended concentration in accordance with current regulatory guidelines for in vitro mammalian genotoxicity assays in the absence of toxicity)

Experiment 2:
24 h -S9:
0, 40, 160, 321, 641, 801, 1122, 1282, 1603 ug/mL

Vehicle / solvent:

RPMI cell culture medium

Untreated negative controls:

yes

Remarks:

RPMI cell culture media

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Cultured CHO were exposed to the test article in the presence and absence of S9 mix (rat Aroclor 1254). Without S9 mix cells were exposed continuously for 20 hours without recovery and with S9 mix exposure was limited to 2 hours, with cells harvested 22 hours later. On the basis of the results of the preliminary toxicity test the following concentrations (in duplicate) were selected in order that an appropriate range of toxicity was observed:
-/+S9: 0, 0.06, 0.13, 0.25, 0.5, 1, 2, 4 µg/mL
Positive controls were included. From 200 metaphases/dose level with 20 centromeres, chromosome number, all chromosomes normal or some aberrant, and specific types and numbers of aberrations were recorded.

Rationale for test conditions:

refer to "Any other information on materials and methods incl. tables"

Evaluation criteria:

A test article was considered to be clearly positive if, in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase was dose-related when evaluated with an appropriate trend test,
c) any of the results were outside the distribution of the historical negative control data
When all of these criteria were met, the test chemical was then considered able to induce chromosomal aberrations in cultured mammalian cells in this test system.

Statistics:

Statistical significance at the 5% level (p < 0.05) was evaluated by the Fischer´s exact test

Key result

Species / strain:

lymphocytes: human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

4 h treatments

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human peripheral blood lymphocytes

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

24 h treatment

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Preliminary cytotoxicity assay

Test item concentrations between 4.0 and 1603 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity following a 4 h treatment. In the pre-test for toxicity, no precipitation of the test article was observed. No relevant influence on osmolality or pH value was observed. No over toxicity (relative MI <55%) was observed in either treatment

Cytogenetic assay:

Experiment 1:

-S9:

No clear cytotoxicity was observed up to the highest concentration (equivalent to 10 mM), with relative MIs of 105%, 89% and 91% at concentrations of 801, 1282 and 1603 µg/mL, respectively.

No biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells (excluding gaps) after treatment with the test article at concentrations of 801, 1282 and 1603 µg/mL were 4.3%, 2.7% and 2.0%, respectively. The frequency of aberrant cells (excluding gaps) was consistent with the concurrent control value (3.0%) and generally within the range of the laboratory historical solvent control data (95th percentile). Of note, whilst the frequency of aberrant cells at the lowest concentration exceeded both the concurrent control value and the historical control range (0 – 3.47%); this increase was not considered biologically relevant as it was neither dose dependent, statistically significant nor accompanied by an appropriate trend test.

Table 7.6.1/03-1:
Experiment 1: 4 h [+20 h]) –S9 cytotoxicity and chromosomal aberration data

Conc		Relative MI (%)	Cytotoxicity (%)	Mean (%) aberrant cells^1	Historical control Mean (%) aberrant cells^1,2
(mM)	(µg/mL)
0	0	100	-	3.0	0 – 3.47%
5	801	105	-	4.3
8	1282	89	11	2.7
10	1603	91	9	2.0
EMS (600 µg/mL)		87	13	7.6*	1.07 – 28.59
*p=0.05 1   Excluding gaps 2   lower control limit - upper control limit (95%, mean +/-2 S.D. collected 2011 - 2016

+S9:

No clear cytotoxicity was observed up to the highest concentration (equivalent to 10 mM), with relative MIs of 74%, 90% and 85% at concentrations of 801, 1282 and 1603 µg/mL, respectively.

No biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells (excluding gaps) after treatment with the test article at concentrations of 801, 1282 and 1603 µg/mL were 3.3%, 3.7% and 2.0%, respectively. The frequency of aberrant cells (excluding gaps) was consistent with the concurrent control value (3.0%) and generally within the range of the laboratory historical solvent control data. However the historical control range stated was a range covering the minimum and maximum values. It is not appropriate to use the simple observed range of the accumulated historical data for an assessment. Rather, the distribution of the data together with appropriate descriptive statistics should be considered (i.e.confidence intervals, 95-99% percentiles, as per Hayashi et al (2011)). Taking this approach, the 95^th percentiles are 0 – 3.42%. Taking this approach the frequency of aberrant cells at the intermediate concentration exceeded both the concurrent control value and the historical control range (0 – 3.42%); this increase was not considered biologically relevant as it was neither dose dependent, statistically significant nor accompanied by an appropriate trend test.

Table 7.6.1/03-2:
Experiment 1: 4 h [+20 h]) +S9 cytotoxicity and chromosomal aberration data

Conc		Relative MI (%)	Cytotoxicity (%)	Mean (%) aberrant cells^1	Historical control Mean (%) aberrant cells^1,2
(mM)	(µg/mL)
0	0	100	-	2.0	0 – 3.42%
5	801	74	-	3.3
8	1282	90	11	3.7
10	1603	85	9	2.0
CPA (5 µg/mL)		81	13	18*	5.60 – 25.69
* p<0.05 1 Excluding gaps 2 lower control limit – upper control limit (95%, mean +/-2 S.D collected 2011 - 2016

Overall it can be concluded that 1,9-Nonanediol did not show an increase in the incidence of chromosome aberrations in isolated human lymphocytes. These conditions included treatments up to the maximum recommended concentration (10 mM) in accordance with current in vitro genotoxicity test guidelines in the absence (4 hours [+ 20 hours recovery]) and presence (4 h [+20 h]) of a rat liver metabolic activation system (S9) following a single experiment.

Experiment 2:

In the continuous treatment (24 h) in the absence of S9, toxicity, with relative MIs of 64, 75, 38 and 47% at concentrations of 40, 80, 160 and 321 µg/mL, respectively. The toxicity was considered to be overt at a concentrations of 160  and 321 µg/mL (i.e. cytotoxicity of 62 and 53%, required range 45% ±5%)

Biologically relevant increases in the number of cells carrying structural chromosome aberrations was observed at concentrations of 80 µg/mL and greater. The aberration rates of the cells (excluding gaps) after treatment with the test article at concentrations 40, 80, 160 and 321 µg/mL were 4.7%, 5.0%, 2.5% and 6.0%, respectively. The frequency of aberrant cells (excluding gaps) exceeded the concurrent control value (1.7%) at all concentrations levels, with concentrations at 40, 80 and 321 µg/mL exceeding the range of the laboratory historical solvent control data (0 - 3.16%). Whilst no accompanying trend test confirmed a statistically significant dose response, statistically significant increases in aberrant cells were obtained at concentrations of 80 and 321 µg/mL.

 

Table 7.6.1/03-3:
Experiment 1: 24 h [+24 h]) -S9 cytotoxicity and chromosomal aberration data

Conc		Relative MI (%)	Cytotoxicity (%)	Mean (%) aberrant cells^1	Historical control Mean (%) aberrant cells^1,2
(mM)	(µg/mL)
0	0	100	-	1.7	0 – 3.16%
0.25	40	64	36	4.7
0.5	80	75	25	5.0*
1	160	38	62	2.5
2	321	47	53	6.0*
EMS (600 µg/mL)		9	91	36.0*	1.07 – 28.59
* p<0.05 1   Excluding gaps 2   lower control limit - upper control limit (95%, mean +/-2 S.D. collected 2011 - 2016

In the continuous treatment in the absence of S9 (24 h [+24 h]), 1,9-Nonanediol increased the incidence of chromosome aberrations in isolated human lymphocytes when tested up to the limit of toxicity (relative mitotic index 47%).

No evidence of an increase in polyploid metaphases were noticed after treatment with the test article as compared to the control cultures.

In both experiments, either EMS (660 µg/mL) or CPA (5 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

References:

Hayashi, M. et al (2011). Compilation and use of genetic toxicity historical control data. Mut. Res. 723, pp 87 -90.

Conclusions:

It is concluded that 1,9-Nonanediol did not show an increase in the incidence of chromosome aberrations in isolated human lymphocytes. These conditions included treatments up to the maximum recommended concentration (10 mM) in accordance with current in vitro genotoxicity test guidelines in the absence (4 hours [+ 20 hours recovery]) and presence (4 h [+20 h]) of a rat liver metabolic activation system (S9) following a single experiment.

In a separate experiment, in the absence of S9 (24 h [+24 h]), 1,9-Nonanediol increased the incidence of chromosome aberrations in isolated human lymphocytes when tested up to the limit of toxicity (relative mitotic index 47%).

Executive summary:

In a mammalian chromosomal aberration assay, human lymphocytes, in vitro were exposed to 1,9-Nonanediol using RPMI cell culture media as the solvent in either the presence of metabolic activation (+S9, 4 h + [4 h recovery]) and absence of metabolic activation (-S9, 4 [4 h recovery] and 24 h [24 h recovery]).

 

In the 4 h –S9 and +S9 treatment concentrations of 801, 1282 and 1603 µg/mL were examined for structural and numerical aberrations. No precipitation was observed by eye at the end of treatment, with the MI reduced to 91% and 85% at the maximum concentration for the respective treatments.

 

No biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed in the 4 h –S9 treatment. The aberration rates of the cells (excluding gaps) after treatment with the test article at concentrations of 801, 1282 and 1603 µg/mL were 4.3%, 2.7% and 2.0%, respectively. The frequency of aberrant cells (excluding gaps) was consistent with the concurrent control value (3.0%) and generally within the range of the laboratory historical solvent control data (95th percentile). Of note, whilst the frequency of aberrant cells at the lowest concentration exceeded both the concurrent control value and the historical control range (0 – 3.47%); this increase was not considered biologically relevant as it was neither dose dependent, statistically significant nor accompanied by an appropriate trend test.

 

No biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells (excluding gaps) after treatment with the test article at concentrations of 801, 1282 and 1603 µg/mL were 3.3%, 3.7% and 2.0%, respectively. The frequency of aberrant cells (excluding gaps) was consistent with the concurrent control value (3.0%) and generally within the range of the laboratory historical solvent control data. However the historical control range stated was a range covering the minimum and maximum values. It is not appropriate to use the simple observed range of the accumulated historical data for an assessment. Rather, the distribution of the data together with appropriate descriptive statistics should be considered (i.e. confidence intervals, 95-99% percentiles, as per Hayashi et al (2011)). Taking this approach, the 95th percentiles are 0 – 3.42%. Taking this approach the frequency of aberrant cells at the intermediate concentration exceeded both the concurrent control value and the historical control range (0 – 3.42%); this increase was not considered biologically relevant as it was neither dose dependent, statistically significant nor accompanied by an appropriate trend test.

In the 24 h -S9 treatment, concentrations of40, 80, 160 and 321 µg/mL were examined for structural and numerical aberrations. At the maximum dose concentration tested, toxicity as evidenced by a mitotic index of 47% was observed.

 

No evidence of an increase in polyploid metaphases were noticed after treatment with the test article as compared to the control cultures.

 

In both experiments, either EMS (660 µg/mL) or CPA (5 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

 

It is concluded that 1,9-Nonanediol did not show an increase in the incidence of chromosome aberrations in isolated human lymphocytes. These conditions included treatments up to the maximum recommended concentration (10 mM) in accordance with current in vitro genotoxicity test guidelines in the absence (4 hours [+ 20 hours recovery]) and presence (4 h [+20 h]) of a rat liver metabolic activation system (S9) following a single experiment.

 

In a separate experiment, in the absence of S9 (24 h [+24 h]), 1,9-Nonanediol increased the incidence of chromosome aberrations in isolated human lymphocytes when tested up to the limit of toxicity (relative mitotic index 47%).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In order to address the positive results observed in the chromosome aberrations study in isolated human lymphocytes, an in vivo rodent bone marrow micronucleus study has been undertaken, with concurrent bioanalysis undertaken to confirm plasma exposure to the test article. The bone marrow is a well perfused tissue and it can be deduced therefore that levels of test article relate materials in plasma will be similar to those observed in the bone marrow. This is borne out by direct comparisons of drug levels in the two compartments for a large series of different pharmaceuticals (Probst, 1994). Although drug levels are not always the same, there is sufficient correlation for measurements in blood or plasma to be adequate for validating bone marrow exposure.

 

It can be concluded that 1,9-Nonanediol did not show an increase in the incidence of micronuclei in male mouse bone marrow polychromatic erythrocytes following dosing via oral gavage up to a level of 1000 mg/kg bw/day (a dose deemed to be a maximum tolerated dose) under the experimental conditions described. Bioanalysis data confirmed test article exposure to the target organ.

 

In conclusion, ,9-Nonanediol is considered to be non-genotoxic.

 

 

References:

Probst, G. (1994). Validation of target tissue exposure for in vivo test. In: P.F. D'Arcy and D.W.G. Harron (eds. Proceeding of the Second International Conference on Harmonisation (ICH 2), Greystoke Brooks Ltd., N. Ireland, pp 249-252

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-10-17 to 2018-02-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Mammalian bone marrow erythrocyte micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Chemical name: 1,9-Nonanediol
- CAS no.: 3937-56-2
- Source and lot/batch No.of test material: Kuraray / 72732
- Expiration date of the lot/batch: 19 April 2018
- Molecular weight: 160.254 g/mol
- Purity: 99.23%

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

Test animals:
Species: Mouse
Strain: NMRI
Age: 6-10 wks
Weight at dosing: 32.4 - 37g
Source: Charles River, 97633 Sulzfeld, Germany
Acclimation period: 7 days
Diet: Altromin 1324 (Batch: 0426) maintenance diet for rats and mice, ad libitum
Water: Municipal water, ad libitum
Housing: Housed 5 animals of the same sex/cage

Environmental conditions
Temperature: 19-25°C
Humidity: 45-65%
Air changes: 10/hour
Photoperiod: 12 hours light/dark

Route of administration:

intravenous

Vehicle:

Cottonseed oil (dose volume: 10 mL/kg bw)

Details on exposure:

The test article was suspended in cottonseed oil and administered orally via gavage to three groups of male mice, 5/group at dose levels of 200, 500, 1000 mg/kg bw/d. the maximum dose administered was deemed to be a maximum tolerated dose based on a range finder experiment. Two further groups were included, one dosed with the vehicle control using the same dosing regimen as previously described and a further group dose with the positive control, cyclophosphamide (CPA). CPA (40 mg/kg bw) was administered as a single intraperitoneal injection 24 h prior to necropsy.

Duration of treatment / exposure:

Vehicle control and test article: dose orally via gavage at 0, 24 h
Postive control: dose intraperitoneally 24 h before necropsy

Frequency of treatment:

single dose as indicated above

Post exposure period:

24 h post final dose

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle (cottonseed oil)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

40 mg/kg bw (total dose)

Remarks:

Postive control (CPA)

No. of animals per sex per dose:

5 animals/group

Control animals:

yes

Positive control(s):

CPA (40 mg/kg bw) was administered as a single intraperitoneal injection 24 h prior to necropsy

Tissues and cell types examined:

2000 immature erythrocytes/slide (4000/animal) were analysed for micronucleated immature erythrocytes.
Bone marrow toxicity was determined by counting the number of immature and mature erythrocytes (expressed as a ratio). The total number of erythrocytes counted/animal was 500.

Details of tissue and slide preparation:

Bilateral femurs from each mouse were injected with FBS to flush out bone marrow erythrocytes. Following centrifugation the pellet was resuspended in a small amount of FBS and 1 drop of the erythrocyte suspension was placed on to a microscope slide and smeared. Four slides were prepared/animal. After air drying, slides were fixed with methanol. Slides were coded and then stained with May-Grünwald/Giemsa.

Evaluation criteria:

Evaluation criteria:
Providing all acceptability criteria were fulfilled, a test item was considered clearly positive if:
- at least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control,
- this increase is dose-related at least at one sampling time when evaluated with an appropriate trend test, and
- any of these results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).

If only the highest dose is examined at a particular sampling time, a test item is considered clearly positive if there is a statistically significant increase compared with the concurrent negative control and the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits)

Providing that all acceptability criteria are fulfilled, a test item was considered clearly negative if, in all experimental conditions examined:
- none of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control,
- there is no dose-related increase at any sampling time when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits), and
- bone marrow exposure to the test item occurred.

Statistics:

Pairwise comparison of the proportion of PCE among total erythrocytes as well as the proportion of micronucleated polychromatic (immature) erythrocytes (PCE) among total PCE between the control group and each of the treatment groups was performed by means of the non-parametric Mann-Whitney test at a statistical significance level of 5% (p < 0.05, two-tailed).

The Chi-squaresd test for trend at a statistical significance level of 5% (p <0.05, two-tailed) was used to test whether there was a dose-related increase in the micronucleated cells frequency of the dose groups of the 24 h sampling time.

Statistical methods were performed using the software GraphPad Prism version 6.0.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

A. Formulation analysis
Not applicable, not undertaken

B. Range finding test:
In the pre-experiment a concentration of 200 mg/mL of the test item was evaluated. One male and one female mouse received a single dose of 2000 mg/kg bw orally and showed very strong toxicity such as reduction of spontaneous activity, prone position, bradykinesia, ataxia, constricted abdomen, muscle spams (opisthotonos) and fast breathing. Based on animal welfare aspects both animals were euthanized some minutes after application.
The dose was reduced to 500 mg/kg bw and applied to one male and one female mouse twice at a 24 h interval. Both animals showed mild signs of systemic toxicity such as reduction of spontaneous activity, hunched posture, ataxia, bradykinesia, piloerection and half eyelid closure.
The dose was increased to 1000 mg/kg bw and dosed to three male and female mice twice at a 24 h interval. The animals showed moderate toxicity such as reduction of spontaneous activity, prone position, hunched posture, ataxia, bradykinesia, constricted abdomen, abnormal breathing, piloerection and half eyelid closure.

Due to the results obtained in the pre-experiment a dose of 1000 mg/kg bw/day was selected as the maximum tolerated dose (1 MTD). In the absence of substantial inter-sex differences in toxicity (a difference in MTD of 2-fold or greater), or likely sex-specific human exposure, only males were used for the main experiment

C. Micronucleus assay:
1. Observations: All animals survived to the scheduled necropsy.
200 mg/kg bw/d: toxicity limited to half eyelid closure 30 minutes after the first application.
500 mg/kg bw/d: mild signs of toxicity such as reduction of spontaneous activity, hunched posture and half eyelid closure up to 2 h after application.
1000 mg/kg bw/d: moderate toxic effects after both applications .Two hours post the final application no toxic symptoms were observed.
2. Bodyweight: No test article related effects were observed, generally all animals gained weight with the exception of 3 animals from the high dose group which had minimally lost weight (up to 2%) (refer to Table CA 7.6.2/01-1).
3. Immature erythrocyte ratio (toxicity) [PCE:NCE]: Refer to Table CA 7.6.2/01-2
The group mean negative control data was within the historical control limits of the negative control (95% confidence limit: 0.45 - 0.80). The mean value of the relative PCE noted for the negative control was 0.45.
200 mg/kg bw/d: relative PCE of 0.37. The value observed in this group was decreased compared to the concurrent negative control but this decrease was not statistically significant.
500 mg/kg bw/d relative PCE of 0.40. The value observed in this group was within the range of the concurrent negative control.
1000 mg/kg bw/d: relative PCE of 0.43. The value observed was within the range of the concurrent negative control.
A marginal dose related increase in group mean %PCE frequency was observed in treated animals compared to the concurrent vehicle control. Whilst this could be considered evidence of mild erythropoiesis, individual animal values were variable, no significant increase in %PCE was observed. Therefore it is concluded that the marginal increase in %PCE is considered unrelated to treatment and consistent to with the laboratory historical control range.
Animals treated with the positive control, CPA showed neither an increase (evidence of erythropoiesis) or decrease (evidence of bone marrow toxicity) in PCE ratio.
4. Micronucleated polychromatic erythrocytes (MN PCE): Refer to Table CA 7.6.2/01-2
All animals treated with 1,9-Nonanediol exhibited both group mean and individual MN PCE which were comparable with both the concurrent vehicle control group and the laboratory’s historical solvent control data. The incidence of MN PCE in the bone marrow of all negative control animals were within the historical solvent control data range (mean ±2 S.D.) and positive control animals gave the expected response.
The criteria for an acceptable assay were met and the assay was therefore considered sensitive.

D. Bioanalysis:
The test item 1,9-Nonanediol could be detected in plasma of one animal where blood was obtained 2 h after final application and in one animal where blood was obtained 3 h after the final application. For all other animals the quantification showed values below the lower limit of detection.
However, the detectability of the test item in blood plasma of the animals demonstrates systemic bioavailability after oral administration which is considered as evidence for exposure of bone marrow to the test item.

E. Deficiencies
None.

Table CA 7.6.2/01-1:
Overview of bone marrow micronucleus study in mice treated orally via gavage with 1,9-Nonanediol: terminal bodyweight (g)

Parameter	Vehicle control: cottonseed oil
Animal no.	V1	V2	V3	V4	V5
Individual bwt	37.5	34.0	35.8	36.5	35.3
Group mean (±SD)	35.8 ±4.9
Parameter	Low dose group: 200 mg/kg bw/d
Animal no.	L1	L2	L3	L4	L5
Individual bwt	32.4	34.8	32.8	34.6	34.2
Group mean (±SD)	33.8 ±1.1
Parameter	Mid dose group: 500 mg/kg bw/d
Animal no.	M1	M2	M3	M4	M5
Individual bwt	37.2	34.1	36.4	32.6	33.6
Group mean (±SD)	34.8 ±1.9
Parameter	High dose group: 1000 mg/kg bw/d
Animal no.	H1	H2	H3	H4	H5
Individual bwt	33.4	36.8	35.9	36.1	36.6
Group mean (±SD)	35.8 ±1.4
Parameter	Positive control: CPA 40 mg/kg bw
Animal no.	P1	P2	P3	P4	P5
Individual bwt	33.2	36.9	33.8	35.4	37.0
Group mean (±SD)	35.3 ±1.7

Table CA 7.6.2/01-2:
Overview of bone marrow micronucleus study in mice treated orally via gavage with 1,9-Nonanediol: individual and group mean MN PCE and PCE:NCE

MN PCE/4000 PCE

%MN PCE

PCE:NCE

Parameter

Vehicle control: cottonseed oil

Animal no.

V1

V2

V3

V4

V5

V1

V2

V3

V4

V5

V1

V2

V3

V4

V5

Individual score

4

6

6

14

13

0.1

0.15

0.15

0.35

0.33

0.45

0.49

0.36

0.47

0.48

Group mean (±SD)

8.6 ±4.6

0.22 ±0.11

0.45 ±0.05

Parameter

Low dose group: 200 mg/kg bw/d

Animal no.

L1

L2

L3

L4

L5

L1

L2

L3

L4

L5

L1

L2

L3

L4

L5

Individual score

8

6

6

3

3

0.20

0.15

0.15

0.08

0.08

0.27

0.42

0.27

0.55

0.36

Group mean (±SD)

5.2 ±2.2

0.13 ±0.05

0.37 ±0.12

Parameter

Mid dose group: 500 mg/kg bw/d

Animal no.

M1

M2

M3

M4

M5

M1

M2

M3

M4

M5

M1

M2

M3

M4

M5

Individual score

8

10

6

10

14

0.20

0.25

0.15

0.25

0.35

0.47

0.40

0.36

0.38

0.37

Group mean (±SD)

9.6 ±3.0

0.24 ±0.07

0.40 ±0.05

Parameter

High dose group: 1000 mg/kg bw/d

Animal no.

H1

H2

H3

H4

H5

H1

H2

H3

H4

H5

H1

H2

H3

H4

H5

Individual score

4

7

5

25

14

0.10

0.18

0.13

0.63

0.35

0.48

0.41

0.42

0.45

0.39

Group mean (±SD)

11 ±8.7

0.28 ±0.22

0.43 ±0.04

Parameter

Positive control: CPA 40 mg/kg bw

Animal no.

P1

P2

P3

P4

P5

P1

P2

P3

P4

P5

P1

P2

P3

P4

P5

Individual score

92

157

81

100

153

2.30

3.92

2.03

2.50

3.83

0.44

0.45

0.40

0.46

0.45

Group mean (±SD)

117 ±35.7

2.92 ±0.89**

0.44 ±0.02

Historical control data (2012-2013)

Vehicle control

Positive control

PCE:NCE

%MN PCE

PCE:NCE

%MN PCE

No. of studies.: Total animal no.:

6 30

6 30

Group mean

Mean ±S.D: Range (min – max): Control range (95%):

0.62 ±0.09 0.44 – 0.70 0.45 – 0.80

0.19 ±0.06 0.12 – 0.27 0.07 – 0.31

0.64 ±0.05 0.53 – 0.69 0.53 – 0.74

2.36 ±0.78 1.31 – 3.50 0.81 – 3.91

Individual values

Mean ±S.D: Range (min – max): Control range (95%):

0.62 ±0.14 0.31 – 0.90 0.34 – 0.90

0.19 ±0.12 0.00 – 0.50 0.00 – 0.43

0.64 ±0.13 0.37 – 0.91 0.38 – 0.89

2.36 ±1.21 0.40 – 4.65 0.00 – 4.48

**p= 0.01 (p 0.0079) Trend test: Not significant (p 0.1491)

PCE:NCE: polychromatic erythrocyte : normochromatic erythrocyte MN PCE: Micronucelated polychromatic erythrocyte CPA: cyclophosphamide

Conclusions:

It can be concluded that 1,9-Nonanediol did not show an increase in the incidence of micronuclei in male mouse bone marrow polychromatic erythrocytes following dosing via oral gavage up to a level of 1000 mg/kg bw/day (a dose deemed to be a maximum tolerated dose) under the experimental conditions described. Bioanalysis data confirmed test article exposure to the target organ.

Executive summary:

In the preliminary dose range finding study male mice were dose via oral gavage of1,9-Nonanediol suspended in cottonseed oilvehicle at either 2000, 1000 and 500 mg/kg bw/d (employing a dose volume of 10 mL/kg). Mice received a dual dose of the test article formulation, with a 24 hour period between doses.Mice dosed at 2000 mg/kg bw/day showed very strong toxicity such as reduction of spontaneous activity, prone position, bradykinesia, ataxia, constricted abdomen, muscle spasms and fast breathing. Based on animal welfare aspects both animals were euthanized some minutes after application. The dose was reduced to 500 mg/kg bw/d and applied to 1 male and 1 female mice twice. Both animals showed mild signs of systemic toxicity such as reduction of spontaneous activity, hunched posture, ataxia, bradykinesia, piloerection and half eyelid closure.

The dose was increased to 1000 mg/kg bw and dosed to 3 male and 3 mice. The animals showed moderate toxicity such as reduction of spontaneous activity, prone position, hunched posture, ataxia, bradykinesia, constricted abdomen, abnormal breathing, piloerection and half eyelid closure.The maximum tolerated dose was deemed to be 1000 mg/kg bw/day.

In the micronucleus experiment groups of 5 male mice were dosed with vehicle (cottonseed oil), 1,9 -Nonanediol (200, 500, 1000mg/kg bw/d) using the same dosing regimen, dose volume and route as previously stated. A positive control (cyclophosphamide, CPA: 40 mg/kg bw) was included, with mice receiving a single dose via intraperitoneal injection (dose volume 10 mL/kg) on day 2. All mice were killed 24 hours post the final dose, bone marrow harvested and slides prepared for micronucleated polychromatic erythrocyte (MN PCE) and polychromatic erythrocyte (PCE) : normochromatic erythrocyte (NCE) frequency were analysed on 5 mice/group.

For analytical purposes blood samples of additional two animals/time point were obtained two hours, three hours and four hours after the final application of the maximum tolerated dose of the test item that were stored at -80°C as plasma sample

All animals treated with1,9-Nonanediolexhibited both group mean and individual MN PCE which were comparable with both the concurrent vehicle control and the laboratory’s historical solvent control data. All animals treated with the positive control exhibited marked increases in MN PCE such that the frequency of MN PCE in the positive control group was significantly (p< 0.01) greater than the observed frequency in the concurrent vehicle control group. The frequency of MN PCE in the concurrent negative control group was within the distribution (mean ±2SD) of laboratory’s historical solvent control data. The criteria for an acceptable assay were met and the assay was therefore considered sensitive.

The groups that were treated with 1,9-Nonanediolshowed no test article related increase or decrease in the ratio of PCE:NCE compared to the concurrent vehicle control group. The positive control group showed neither an increase (evidence of erythropoiesis) or decrease (evidence of bone marrow toxicity) in PCE ratio.

The test item 1,9-Nonanediol could be detected in plasma of one animal where blood was obtained 2 h after final application and in one animal where blood was obtained 3 h after the final application. For all other animals the quantification showed values below the lower limit of detection. However, the detectability of the test item in blood plasma of the animals demonstrates systemic bioavailability after oral administration which is considered as evidence for exposure of bone marrow to the test item.

It can be concluded that1,9-Nonanedioldid not show an increase in the incidence ofmicronuclei in male mouse bone marrow polychromatic erythrocytesfollowing dosing via oral gavage up to a level of 1000 mg/kg bw/day (a dose deemed to be a maximum tolerated dose) under the experimental conditions described. Bioanalysis data confirmed test article exposure to the target organ.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Comparison with the CLP criteria

There was no indication that 1,9-Nonanediol has a mutagenic effect on somatic cells in the Ames and mammalian gene mutation in vitro assays. An increase in the incidence of chromosome aberrations in isolated human lymphocytes following a continuous exposure was observed. The biological relevance of this result has been addressed in an in vivo rodent bone marrow micronucleus study, with concurrent assessment of target organ exposure. The in vivo micronucleus test confirmed that 1,9-Nonanediol was devoid of genotoxic potential, in vivo. 

 

The criteria for classification for mutagenicity were not met, therefore no mutagenicity classification is proposed.