Pent-1-ene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test on S. typhimurium TA 98, TA 100, TA 1535 and TA 97: not mutagenic

- QSAR prediction (DEREK, 2017): No structural alerts for mutagenicity

- HPRT: not mutagenic in mouse lymphoma (L5178Y) cells

- CAT: not clastogenic in rat lymphocytes (read-across on butene-2)

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

Well documented study but not performed under GLP. Testguideline similar to OECD test guideline, but the organism E.Coli was not tested. Substance purity not reported.

Qualifier:

according to guideline

Guideline:

other: NTP Standard Protocol

Deviations:

yes

Remarks:

The test was not conducted with E. Coli

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

The test was not conducted with E. Coli

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine gene

Species / strain / cell type:

bacteria, other: S. Typhimurium TA97, TA98, TA100, TA1535

Metabolic activation:

with and without

Metabolic activation system:

metabolic activation enzymes and cofactors from Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver S9 (10 and 30%)

Test concentrations with justification for top dose:

0, 100, 333, 1000, 3333, 10000 µg/plate

Vehicle / solvent:

Dimethyl Sulfoxide (DMSO)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

without S9

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (or occasionnally, sterigmatocystin)

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION:
preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 2 days

Species / strain:

S. typhimurium, other: S. typhimurium TA 1535, TA 97, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

only slight cytotoxicity was measured for 10000 ug/plate without HLI

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

See attachment in section "Attached background material".

Conclusions:

Under the test condition,1-Pentene is not mutagenic with and without metabolic activation in S. typhimurium strains (TA1535, TA97, TA98 and TA100) according to the criteria of the Annex I of the of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

In a reverse gene mutation assay performed according to the NTP Standard Protocol (Ames Test), Salmonella typhimurium strains TA 1535, TA 97 and TA 98 and TA 100 were exposed to 1-pentene diluted in DMSO both in the presence and absence of metabolic activation system (S9 fraction) from 100 to 10000 µg/plate using the preincubation method. Vehicle (dimethyl sulphoxide) and positive control groups were also included in mutagenicity tests.

No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA97, TA98 and TA100) according to the criteria of the Annex I of the of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

13 January 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Remarks:

DEREK is a validated (Q)SAR software, widely used to predict genotoxicity properties

Justification for type of information:

1. SOFTWARE
Derek Nexus: 5.0.2, Nexus: 2.1.1, Lhasa Ltd.

2. MODEL (incl. version number)
Derek KB 2015 v2.0

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CCCC=C

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See attached QMRF

5. APPLICABILITY DOMAIN
See attached report

6. ADEQUACY OF THE RESULT
See attached report

Qualifier:

no guideline followed

Principles of method if other than guideline:

Software used: Derek Nexus: 5.0.2, Nexus: 2.1.1, Lhasa Ltd.

GLP compliance:

no

Type of assay:

other: Derek Nexus evaluation for mutagenicity

Target gene:

Not applicable

Species / strain / cell type:

bacteria, other: Salmonella typhimurium and Escherichia coli

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

Not applicable

Vehicle / solvent:

Not applicable

Details on test system and experimental conditions:

Not applicable

Rationale for test conditions:

Not applicable

Evaluation criteria:

Not applicable

Statistics:

Not applicable

Key result

Species / strain:

bacteria, other: Salmonella typhimurium and Escherichia coli

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: not applicable

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Additional information on results:

Mutagenicity in vitro in bacterium is INACTIVE: No misclassified or unclassified features
Mutagenicity in vitro in Escherichia coli is INACTIVE: No misclassified or unclassified features
Mutagenicity in vitro in Salmonella typhimurium is INACTIVE: No misclassified or unclassified features

Details
The query structure does not match any structural alerts or examples for (bacterial in vitro) mutagenicity in Derek. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be inactive in the bacterial in vitro (Ames) mutagenicity test.

None

Conclusions:

DEREK Nexus evaluation showed no alerts for mutagenicity.

Executive summary:

DEREK software (Derek Nexus: 5.0.2, Nexus: 2.1.1, Lhasa Ltd.) was used to predict the mutagenicity of pent-1-ene.

The query structure does not match any structural alerts or examples for (bacterial in vitro) mutagenicity in Derek. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be inactive in the bacterial in vitro (Ames) mutagenicity test.

DEREK Nexus evaluation showed no alerts for mutagenicity.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 08 June to 19 December, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP, guideline study.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

hemizygous hypoxanthine phosphoribosyl transferase (HPRT) gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr Donald Clive, Burroughs Wellcome Co. Cells
- Suitability of cells: Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated at 37±1ºC. When the cells were growing well, subcultures were established in an appropriate number of flasks.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction (2% v/v); S9 fraction was prepared from male Sprague Dawley rats induced with Aroclor 1254.

Test concentrations with justification for top dose:

Cytotoxicity Range-Finder Experiment: 6 concentrations were tested in the absence and presence of S-9 ranging from 21.94 to 702 μg/mL
The upper concentration level was selected as being equivalent to 10mM, which is the maximum recommended level to be used in mammalian cells assays.

Mutation tests: The upper concentration levels were selected based on cytotoxicity.
-S9 mix Test (3 hours) 25, 50, 100, 125, 150, 160, 170, 180, 190, 200 and 250 μg/mL.
+S9 mix Test (3 hours) 25, 50, 100, 150, 175, 200, 225, 250, 275, 300325 and 350 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Formulation preparation: Test article stock solutions were prepared by formulating Pentene 1 under subdued lighting in Acetone to give the maximum required concentration. Subsequent dilutions were made using Acetone. The test article solutions were protected from light and used within approximately two hours of initial formulation.

Untreated negative controls:

yes

Remarks:

culture medium alone

Negative solvent / vehicle controls:

yes

Remarks:

Acetone diluted in the treatment medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- RPMI 1640 media supplied containing L-glutamine and HEPES

DURATION
- Exposure duration: 3 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days
- The plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air.

SELECTION AGENT (mutation assays): Selective medium, RPMI 1640 media supplied containing L-glutamine and HEPES.

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single culture/dose for test item and vehicle controls.
- Main test: 1 culture for positive control substances, 2 cultures/dose for test item and negative controls.

NUMBER OF CELLS EVALUATED: 2 x 10^5 cells were analyzed for mutant frequencies.

DETERMINATION OF CYTOTOXICITY
- Method: Cloning Efficiency, Relative Survival
Cloning Efficiency (CE): CE= P/No of cells plated per well
Percentage Relative Survival (%RS): %RS = [CE (test)/CE (control)] x 100
At the end of the expression period, cloning efficiency and relative survival were calculated.


OTHER:
Mutant Frequency (MF) is usually expressed as "mutants per 10^6 viable cells": MF = [CE (mutant)/CE (viable)] x 10^6

Rationale for test conditions:

Mutation tests: The upper concentration levels were selected based on cytotoxicity.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic in this assay if:
1. The MF at one or more concentrations was significantly greater than that of the negative control (p≤0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p≤0.05)
3. If both of the above criteria were fulfilled, the results should exceed the upper limit of the last 20 studies in the historical negative control database (mean MF +/ 2 standard deviations.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. Positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological relevance. Extreme caution was exercised with positive results obtained at levels of RS lower than 10%.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No fluctuations in pH of the medium of more than 1.0 unit compared with the vehicle control were observed at 702 μg/mL in the Range-Finder experiment.
- Effects of osmolality: No fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed when compared with the vehicle control at 702 µg/mL in the Range-Finder experiment.
- Precipitation: no precipitate was observed in the main test.

PRELIMINARY TOXICITY TEST:
- In the preliminary toxicity test, a three-hour exposure at concentrations from 21.94 to 702 µg/mL of test item, in the absence or the presence of S9 mix, resulted in Day 1 relative survivals of 100 to 24 % up to 175.5 µg/mL (not plated at 351 and 702 µg/mL) without S9 mix and 95 to 0 % with S9 mix. Precipitate was seen at the highest three concentrations (175.5 to 702 μg/mL).
Concentrations for the main test were based upon these data and toxicity was the primary determinant for the dose selection.

MAIN MUTATION TEST
Eleven concentrations, ranging from 25 to 250 μg/mL, were tested in the absence of S-9 and twelve concentrations, ranging from 25 to 350 μg/mL were tested in the presence of S-9. Upon addition of the test article to the cultures, precipitate was observed at 160 μg/mL and above in the absence of S-9 and at 175 μg/mL and above in the presence of S-9. However, following the 3 hour treatment incubation period, no precipitate was observed. Seven days after treatment, the highest five concentrations (170 to 250 μg/mL) in the absence of S-9 and the highest six concentrations (225 to 350 μg/mL) in the presence of S-9 were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 160 μg/mL in the absence of S-9 and 200 μg/mL in the presence of S-9, which gave 12% and 16% RS, respectively.
In the absence of S-9, when tested up to toxic concentrations no statistically significant increases in MF, compared to the vehicle control MF, were observed at any concentration analysed and there were no statistically significant linear trends, indicating a clear negative result.
In the presence of S-9, there was a statistically significant (P≤0.05) increase in MF over the concurrent control observed at 175 μg/mL. However, there was no accompanying significant linear trend. Although the increase in MF at 175 μg/mL (9.39 per 10E6 viable cells) did exceed the mean historical vehicle control MF for the last 20 studies +2SD (9.22 per 10E6 viable cells), the increase was marked in a single culture replicate only (11.57 versus 7.14 per 10E6 viable cells). In addition, the untreated control displayed a similar MF (8.14 per 10E6 viable cells). Therefore, the increase in MF at 175 μg/mL was considered to be of no biological relevance.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with the historical negative control ranges, based on the last 20 experiments performed in this laboratory.

Table 7.6.1/1: Summary results

3 Hour Treatment –S-9			3 Hour Treatment +S-9
Concentration	%RS	MF §	Concentration	%RS	MF §
µg/mL			µg/mL
0	100	5.11	0	100	4.86
UTC	109	6.54	UTC	90	8.14
25	87	8.39 NS	25	75	3.87 NS
50	92	5.42 NS	50	88	7.69 NS
100	46	6.92 NS	100	64	4.83 NS
125	16	6.48 NS	150	41	4.80 NS
150	15	7.69 NS	175 P	22	9.39 *
160 P	12	5.77 NS	200 P	16	7.64 NS
NQO 0.15	58	39.06	B[a]P 2	66	33.50
NQO 0.2	52	51.73	B[a]P 3	49	58.45

Linear trend: Not Significant - 3 hour absence and presence of S-9

Conclusions:

Under the test conditions, test item did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay, when tested up to toxic concentrations.

Executive summary:

 In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, mouse lymphoma (L5178Y) cells were exposed to test item for 3-h, with and without metabolic activation (S9 fraction) at the following concentrations: 

 

Preliminary toxicity test: 21.94, 43.86, 87.75, 175.5, 351 and 702 μg/mL

 

Mutation test:

-S9 mix (3 hours): 25, 50, 100, 125, 150, 160, 170, 180, 190, 200 and 250 μg/mL

+S9 mix (3 hours): 25, 50, 100, 150, 175, 200, 225, 250, 275, 300, 325 and 350 μg/mL

Seven days after treatment the highest concentrations analysed to determine viability and 6TG resistance were 160 μg/mL in the absence of S9 mix and 200 μg/mL in the presence of S9 mix, which gave 12% and 16% RS, respectively.

Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid.

In the absence of S9 mix, no statistically significant increases in MF were observed up to toxic concentrations, compared to the vehicle controle MF.

In the presence of S9 mix, there was a statistically significant (P≤0.05) increase in MF over the concurrent control observed at 175 μg/mL. However, there was no accompanying significant linear trend, the increase did exceed the mean historical vehicle control MF for the last 20 studies and this increase was marked in a single culture replicate only. In addition, the untreated control displayed a similar MF. Therefore, the increase in MF at 175 μg/mL was considered to be of no biological relevance.

Under the test conditions, test item did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay, when tested up to toxic concentrations.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

RAAF document will be attached.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

lymphocytes: Rat

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At a target concentration of 80% for 20-hour harvest group

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Butene-2 induced a very steep dose-related decrease in the mitotic index both with and without S9. Butene-2 was therefore considered to be toxic to rat lymphocytes in vitro at the target concentration of 80% for the 20-hour harvest group.

Butene-2 produced no significant increases in the frequency of chromosome aberrations either in the presence or absence of a liver enzyme metabolising system. Butene-2 is, therefore, considered to be non-clastogenic to rat lymphocytes in vitro.

Conclusions:

Under the test conditions, and based on the read-across approach, pent-1-ene do not induce structural chromosomal aberrations with and without metabolic activation and was therefore considered to be non-clastogenic to rat lymphocytes in vitro.

Executive summary:

In an in vitro chromosome aberration study performed according to the OECD guideline 473 and in compliance with GLP, rat lymphocytes were exposed to the gas butene-2. Three treatment conditions were used, i.e. 4 hours exposure with the addition of an induced rat liver homogenate metabolising system (S9 mix) at 20% in standard co-factors with cell harvest after 16 and 26 hour expression periods and a 20 hour continuous exposure in the absence of metabolic activation. The dose range was selected from a series of 7 dose levels and was 40-60%.

No significant increases were seen in the frequency of chromosome aberrations either in the presence or absence of a liver enzyme metabolising system. Positive control compounds gave the expected results demonstrating the validity of the test.

Under the test conditions and based on the read-across approach, pent-1-ene do not induce structural chromosomal aberrations with and without metabolic activation and was therefore considered to be non-clastogenic to rat lymphocytes in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames Test

In a reverse gene mutation assay performed according to the NTP Standard Protocol (Ames Test), Salmonella typhimurium strains TA 1535, TA 97 and TA 98 and TA 100 were exposed to 1-pentene diluted in DMSO both in the presence and absence of metabolic activation system (S9 fraction) from 100 to 10000 µg/plate using the preincubation method.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA97, TA98 and TA100).

However, as the S. typhimurium strain TA 1537 and E. coli strain WP2 uvrA was not tested in this study, the mutagenic potential of 1-pentene was evaluated with Derek software.

The query structure does not match any structural alerts or examples for (bacterial in vitro) mutagenicity in Derek. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be inactive in the bacterial in vitro (Ames) mutagenicity test.

HPRT

 In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, mouse lymphoma (L5178Y) cells were exposed to test item for 3-h, with and without metabolic activation (S9 fraction) up to 702 μg/mL in the preliminary test and up to 350 or 250 µg/mL with or without metabolic activation, respectively, in the mutation test.

Seven days after treatment the highest concentrations analysed to determine viability and 6TG resistance were 160 μg/mL in the absence of S9 mix and 200 μg/mL in the presence of S9 mix, which gave 12% and 16% RS, respectively.

Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid.

In the absence of S9 mix, no statistically significant increases in MF were observed up to toxic concentrations, compared to the vehicle controle MF.

In the presence of S9 mix, there was a statistically significant (P≤0.05) increase in MF over the concurrent control observed at 175 μg/mL. However, there was no accompanying significant linear trend, the increase did exceed the mean historical vehicle control MF for the last 20 studies and this increase was marked in a single culture replicate only. In addition, the untreated control displayed a similar MF. Therefore, the increase in MF at 175 μg/mL was considered to be of no biological relevance.

Under the test conditions, test item did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay, when tested up to toxic concentrations.

Chromosome Aberration Test

In an in vitro chromosome aberration study performed according to the OECD guideline 473 and in compliance with GLP, rat lymphocytes were exposed to the gas butene-2. Three treatment conditions were used, i.e. 4 hours exposure with the addition of an induced rat liver homogenate metabolising system (S9 mix) at 20% in standard co-factors with cell harvest after 16 and 26 hour expression periods and a 20 hour continuous exposure in the absence of metabolic activation. The dose range was selected from a series of 7 dose levels and was 40-60%.

No significant increases were seen in the frequency of chromosome aberrations either in the presence or absence of a liver enzyme metabolising system. Positive control compounds gave the expected results demonstrating the validity of the test.

Under the test conditions, and based on the read-across approach, butene-2 and Pent-1-ene did not induce structural chromosomal aberrations with and without metabolic activation and was therefore considered to be non-clastogenic to rat lymphocytes in vitro.

Justification for classification or non-classification

Harmonised classification

The test material has no harmonised classification for human health according to the Regulation (EC) No. 1272/2008 (CLP).

Self classification

No additional classification is proposed regarding in vitro genetic toxicity according to the Annex I of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.