Reaction mass of 3-O-acetyl-1,5-anhydro-4-butyl-2,4-dideoxy-2-methyl-D-xylitol and (2R,3S)-2-methyloctane-1,3-diyl diacetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (Ames test): read-across from Jasmal: OECD TG 471: negative

Chromosome aberration in mammalian cells (human lymphocytes): read-across from Jasmal: OECD TG 473: negative

Gene mutation in mammalian cells (mouse lymphoma cells): read-across from Jasmal: OECD TG 476: negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-across information.

Justification for type of information:

The read-across justification is presented in the Endpoint summary Genetic toxicity. The accompanying files are also attached there.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: read-across from Jasmal

Conclusions:

Jessemal is not mutagenic based on read across with Jasmal, which was tested in an OECD TG 491 and showed negative results.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 August, 2002 - 23 September, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This information is used for read across to Jessemal.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

(2000)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced with Aroclor 1254

Test concentrations with justification for top dose:

- Dose range finding test:
Direct plate assay and pre-incubation assay: TA 100 (without and with S9): 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate

- Experiment 1 (direct plate):
Due to cytotoxicity, the following dose levels were used:
TA 1535, TA 1537, TA 98 (without and with S9): 10, 33, 100, 333, 1000 and 3330 µg/plate
TA 102 (with S9): 10, 33, 100, 333, 1000 and 3330 µg/plate
TA 102 (without S9): 33, 100, 333, 1000, 3330 and 5000 µg/plate

- Experiment 2 (preincubation):
Due to cytotoxicity, the following dose levels were used:
TA 1535, TA 1537, TA 98 and TA 102 (without and with S9): 3, 10, 33, 100, 333 and 1000 µg/plate

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: the test substance was found to be soluble in DMSO up to 5000 µg/plate.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

(100 µL/plate)

Positive controls:

yes

Positive control substance:

other: see section "Any other information on materials and methods incl. tables"

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Experiment 1: in agar (plate incorporation)
- Experiment 2 (independent repeat): preincubation

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain (in all experiments)

DETERMINATION OF CYTOTOXICITY
- Method: on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one repeated experiment.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at the concentrations of 3330 and 5000 µg/plate in experiment 1 only.

RANGE-FINDING/SCREENING STUDIES:
- In strain TA100 toxicity was observed at 3330 µg/plate and above, both in the absence and presence of S9 in the direct plate assay. In the pre-incubation assay, in strain TA100 toxicity was observed at 333 or 1000 µg/plate and above in the absence or presence of S9 respectively.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment 1 in all strains toxicity was observed, except in tester strain TA1535 (with S9) and TA1537 (without S9). In experiment 2, toxicity was observed in all tester strains.

Conclusions:

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay performed according to OECD 471 (1997) guideline and GLP principles.

Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) guideline and according to GLP principles. The test was performed in two independent experiment. At first a direct plate assay was performed and secondly a pre-incubation assay, both in the absence and presence of S9-mix. The dose levels were selected based on observed cytotoxicity in dose range finding tests (>= 333 ug/plate). Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the five S. typhimurium tester strains (TA1535, TA1537, TA98, TA100 and TA102), both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Reference 3

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-across information.

Justification for type of information:

The information is derived from read across. The read-across justification is presented in the Endpoint summary Genetic toxicity. The accompanying files are also attached there.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

lymphocytes: Cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: read-across from Jasmal

Conclusions:

For Jessemal the negative in vitro cytogenicity toxicity is based on read across from Jasmal, which was tested negative in a chromosome aberration study according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 May, 2015 - 22 July, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This information is used for read across to Jessemal.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(2014)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(2008)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

Blood samples
Blood samples were collected by venapuncture from healthy non-smoking volunteers (20 - 26 years) using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
The Average Generation Time (AGT) of the cells was determined (December 2014) 12.6 - 13.0 hours.
- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.
- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not applicable, immediately after blood collection lymphocyte cultures were started.
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: not applicable, immediately after blood collection lymphocyte cultures were started.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.

Test concentrations with justification for top dose:

Dose range finding test:
With and without S9-mix, 3hr exposure; 24 hr fixation: 5.4, 17, 52, 164 and 512 µg/mL
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 5.4, 17, 52, 164 and 512 µg/mL

First cytogenetic test:
With and without S9-mix, 3 h exposure time, 24 h fixation time: 50, 200, 350, 450, 500 and 550 µg/mL
The following dose levels were selected for scoring of chromosome aberrations:
With and without S9-mix, 3 h exposure, 24 h fixation time: 50, 450 and 550 µg/ mL

Second cytogenetic test:
Without S9-mix, 24 and 48 hr exposure; 24 and 48 hr fixation: 1, 10, 50, 75, 100, 150, 200 and 250 µg/mL
The following dose levels were selected for scoring of chromosome aberrations:
Without S9-mix, 24 hr exposure; 24 hr fixation: 1, 10 and 200 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 1, 100 and 200 µg/mL

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: Test compound was soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: Mitomycin C in Hanks’ Balanced Salt Solution: 0.5 and 0.75 μg/ml for a 3 h exposure period, 0.2 and 0.3 μg/ml for a 24 h exposure period and 0.1 and 0.15 μg/ml for and 0.15 μg/ml for a 48 h exposure period.

Remarks:

without S9

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: cyclophosphamide in Hank's Balanced Salt Solution: 10 µg/mL

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 150 metaphase chromosome spreads per culture.
Chromosomes of metaphase spreads were analysed from those cultures with an inhibition of the mitotic index of 55 ± 5%, whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

Acceptability of the assay:
A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The number of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range.
b) The positive control substances should produce a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
c) A homogeneous response between the replicate cultures is observed.
d) A possible precipitate present on the slides should not interfere with the scoring of chromosome aberrations.

Data evaluation and statistical procedures:
A test substance is considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Key result

Species / strain:

lymphocytes: Cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the solubility test a concentration of 512 μg/mL showed precipitation in the culture medium. Therefore, a concentration of 512 μg/mL was used as the highest concentration of the test substance. In the dose range finding study a concentration of 512 μg/mL showed no precipitation in the culture medium.

RANGE-FINDING/SCREENING STUDIES:
Toxicity was observed at the highest tested dose of 512 µg/mL in the absence and presence of S9, 3 hr treatment/24 hr fixation. Toxicity was observed at dose levels of 52, 164 and 512 µg/mL in the absence of S9 for the continuous treatment of 24 and 48 hr.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide induced appropriate responses.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Appropriate toxicity was reached at the dose levels selected for scoring in the absence and presence of S9.

Conclusions:

A chromosome aberration study with the substance was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that the substance is not clastogenic in human lymphocytes.

Executive summary:

In a chromosome aberration study, cultured peripheral human lymphocytes were exposed to different concentrations of the substance (dissolved in DMSO), in the presence and absence of S9-mix according to OECD 473 guideline and GLP principles. In the first cytogenetic assay, Jasmal was tested up to cytotoxic concentrations of 550 μg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9 -mix. In the second cytogenetic assay, Jasmal was tested up to the cytotoxic concentration of 200 μg/mL for a 24 h and 48 h exposure time with a 24 h and 48 h fixation time in the absence of S9 -mix. Jasmal did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments. No effects of the substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

Therefore it can be concluded that the substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations nor polyploidy. Finally, it is concluded that Jasmal is not clastogenic in human lymphocytes.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-across information.

Justification for type of information:

The information is derived from read across. The read-across justification is presented in the Endpoint summary Genetic toxicity. The accompanying files are also attached there.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

strain/cell type: Test system L5178Y/TK+/-3.7.2C

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: read-across from Jasmal

Conclusions:

The genemutation in mammalian cells (Mouse lymphoma assay (MLA)) information is based on read across from Jasmal. This MLA was conducted according to OECD 476 guideline and GLP principles and is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions of the study.

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 August, 2015 - 05 October, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This information is used for read across to Jessemal.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1997)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

(2008)

Deviations:

no

Principles of method if other than guideline:

The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
- RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3 hours treatment: 17, 52, 164, 512 and 1600 µg/mL
Without S9-mix, 24 hours treatment: 17, 52, 164, 512 and 1600 µg/mL

Experiment 1:
With and without S9-mix, 3 hours treatment: 0.5, 1, 5, 10, 50, 100, 200, 250, 300, 350, 400 and 450 µg/mL
The following dose levels were selected to measure mutation frequencies at the TK-locus :
Without S9-mix, 3 hours treatment: 1, 5, 10, 50, 100, 200, 250 and 300 µg/mL
With S9-mix, 3 hours treatment: 10, 50, 100, 200, 250, 300, 350 and 400 µg/mL

Experiment 2
Without S9-mix, 24 hours treatment: 1, 5, 10, 50, 75, 100, 125, 150, 175, 200, 225 and 250 µg/mL
The following dose levels were selected to measure mutation frequencies at the TK-locus:
Without S9-mix, 24 hours treatment: 5, 10, 50, 75, 100, 125, 150 and 175 µg/mL

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: The solubility test was performed in the Chromosome aberration test. The test item was dissolved in dimethyl sulfoxide. DMSO has been accepted and approved by authorities and international guidelines. Jasmal concentrations were used within 1 hour of preparation.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: Methyl methanesulfonate in DMSO at concentrations of 15 and 5 μg/mL for the 3 and 24 hours treatment periods, respectively.

Remarks:

without S9

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: Cyclophosphamide in HBSS at a final concentration of 7.5 μg/mL.

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and 32-180 (24 hours treatment).
d) The positive control should demonstrate an absolute increase in the total mutation frequency above the spontaneous background MF (an induced MF (IMF) of at least 300 x 10^-6). Furthermore, the positive control should have an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent/control (a small colony IMF of at least 150 x 10^-6).

DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

Statistics:

The global evaluation factor (GEF) has been defined by the IWTGP as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: No data.
- Effects of osmolality: No data.
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 512 µg/mL and above.

RANGE-FINDING/SCREENING STUDIES:
Jasmal was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test item concentration for the dose range finding test was 1600 μg/mL.
In the absence of S9-mix after 3 hours treatment, the relative suspension growth was 62% at the test item concentration of 164 μg/mL compared to the relative suspension growth of the solvent control. Hardly any or cell survival was observed at test item concentrations of 512 μg/mL and above.
In the presence of S9-mix after 3 hours treatment, the relative suspension growth was 59% at the test item concentration of 164 μg/mL compared to the relative suspension growth of the solvent control. Hardly any or no cell survival was observed at test item concentrations of 512 μg/mL and above.
In the absence of S9-mix after 24 hours treatment, the relative suspension growth was 28% at the test item concentration of 164 μg/mL compared to the relative suspension growth of the solvent control. Hardly any or cell survival was observed at test item concentrations of 512 μg/mL and above.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the first experiment, Jasmal was tested up to and including concentrations of 300 and 400 μg/mL in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. The relative total growth (RTG) of the highest test item concentration was 4% and 41% compared to the total growth of the solvent controls in the absence and presence of S9-mix, respectively.
In the second experiment, Jasmal was tested up to and including concentrations of 175 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The relative total growth (RTG) of the highest test item concentration was 14% compared to the total growth of the solvent controls.

Remarks on result:

other: strain/cell type: Test system L5178Y/TK+/-3.7.2C

Remarks:

Migrated from field 'Test system'.

The survival of the highest tested dose levels (Relative total growth) in the absence of S9-mix in the first experiment (4 and 34%) were not within the range mentioned in the study plan.

Evaluation: The dose range used was very narrow and no increase in the mutation frequency of more than two-fold was observed compared to the solvent control; therefore the testing of dose levels with a RTG of 10-20% would have given limited additional information.

 

In the presence of S9-mix, no dose level was tested with a relative total growth between ranges of 10 -20%.

Evaluation: The dose range used was very narrow. The Relative suspension growth (RSG) of the highest selected dose level of 400 µg/mL was 40%, whereas the next dose level of 450 µg/mL showed a RSG of 4%. No increase in the mutation frequency of more than two-fold was observed compared to the solvent controls; therefore the testing of dose levels with a RTG of 10-20% would have given limited additional information.

Conclusions:

A mouse lymphoma assay with Jasmal was conducted according to OECD 476 guideline and GLP principles. It is concluded that Jasmal is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions of the study.

Executive summary:

The mouse lymphoma assay with Jasmal was conducted according to OECD 476 guideline and GLP principles.

In the first experiment, Jasmal was tested up to and including concentrations of 300 and 400 μg/mL in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. The relative total growth (RTG) of the highest test item concentration was 4% and 41% compared to the total growth of the solvent controls in the absence and presence of S9-mix, respectively.

In the second experiment, Jasmal was tested up to and including concentrations of 175 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The relative total growth (RTG) of the highest test item concentration was 14% compared to the total growth of the solvent controls. Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses. In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency. It is concluded that Jasmal is not mutagenic in the mouse lymphoma L5178Y test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity is assessed based on read-across from Jasmal to Jessemal. The executive summary of the source information on the substance is presented below, followed by the read-across rationale.

Jasmal Ames test information

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) guideline and according to GLP principles. The test was performed in two independent experiment. At first a direct plate assay was performed and secondly a pre-incubation assay, both in the absence and presence of S9-mix. The dose levels were selected based on observed cytotoxicity in dose range finding tests (>= 333 µg/plate). Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the five S. typhimurium tester strains (TA1535, TA1537, TA98, TA100 and TA102), both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Jasmal chromosome aberration information

In a chromosome aberration study, cultured peripheral human lymphocytes were exposed to different concentrations of the substance (dissolved in DMSO), in the presence and absence of S9-mix according to OECD 473 guideline and GLP principles. In the first cytogenetic assay, Jasmal was tested up to cytotoxic concentrations of 550 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9 -mix. In the second cytogenetic assay, Jasmal was tested up to the cytotoxic concentration of 200 µg/mL for a 24 h and 48 h exposure time with a 24 h and 48 h fixation time in the absence of S9 -mix. Jasmal did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments. No effects of the substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations nor polyploidy. Finally, it is concluded that Jasmal is not clastogenic in human lymphocytes.

Jasmal genemutation in mammalian cells information

The mouse lymphoma assay with Jasmal was conducted according to OECD 476 guideline and GLP principles. In the first experiment, Jasmal was tested up to and including concentrations of 300 and 400 μg/mL in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. The relative total growth (RTG) of the highest test item concentration was 4% and 41% compared to the total growth of the solvent controls in the absence and presence of S9-mix, respectively. In the second experiment, Jasmal was tested up to and including concentrations of 175 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The relative total growth (RTG) of the highest test item concentration was 14% compared to the total growth of the solvent controls. Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses. In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency. It is concluded that Jasmal is not mutagenic in the mouse lymphoma L5178Y test system.

Genotoxicity of Jessemal based on read across from data available for Jasmal (CAS #18871-14-2).

 

Introduction and hypothesis for the read across

For the multi-constituent substance Jessemal all constituents > 1% are identified. The substance consists mainly of two sub-groups of constituents, Tetrahydropyran acetate -like constituents, and Branched alkyl diacetates-type.For Jessemal no experimentalgenotoxicity information is available.In accordance with Article 13 of REACH, lacking information can be generated by means other than experimental testing, i.e. applying alternative methods such as, QSARs, grouping and read-across. For assessing the genotoxic potential of Jessemalthe analogue approach is selected because for one of the constituents, Jasmal, data on genotoxic potential is available which can be used for read across.

Hypothesis: Jessemal has the same genotoxicity as Jasmal.

Available information: For Jasmal, the following data is available: Ames test (OECD TG 471), in vitro chromosome aberrations test (OECD TG 473) and in vitromutagenicity assay in mammalian cells (mouse lymphoma, OECD TG 476). All tests were conducted according to OECD guidelines and performed in compliance with GLP principles (Klimisch 1). Jasmal was negative in all these tests and is therefore not genotoxic.

Target chemical and source chemical(s)

Chemical structures of the target chemical and the source chemical(s) are shown in the data matrix, including physico-chemical properties.

Purity / Impurities

The major and minor constituents of Jessemal are presented in the Data matrix. The impurities < 10% are grouped based on their resemblance with Tetrahydropyran acetate -like constituents, and Branched alkyl diacetates-type.

Analogue justification

According to REACH Annex XI 1.5 read across can be used to replace testing when the similarity can be based on a common backbone and a common functional group. When using read across the result derived should be applicable for C&L and/or risk assessment and it should be presented with adequate and reliable documentation, which is presented below.

Analogue selection:For Jessemal the substance Jasmal was selected as source chemical for read-across because Jessemal‘s constituents are the same and/or very similar to Jasmal’s and for Jasmal experimental genotoxicity toxicity information is available.

Structural similarities and differences: Jessemal andJasmal are both reaction masses containing similar Tetrahydropyran acetate-like constituents with molecular weight of 214. Jessemal also contains constituent of Branched alkyl diacetates with molecular weights of 244. These diacetates have one additional acetic ester and have an open ring structure not present in Jasmal. Jessemal has two minor impurities just exceeding the 1% level, one being the alcohol derivative of the Tetrahydropyran acetates and the other one of the Branched alkyl diacetates, which will not be addressed further being only very minor constituents.

Toxico-kinetic: Jessemal and Jasmalare expected to have similar absorption via all routes based on the similarity in chemical structure and physico-chemical properties. Jessemal and Jasmal both have molecular weights, water solubility and log Kow values favourable for uptake. All constituents contain acetic ester groups which will be cleaved by carboxyl esterases and the corresponding alcohols and acetic esters will be the key metabolites (Toxicological Handbooks, Belsito et al., 2008).The Branched diacetates will be present twice as much acetic acid than Jasmal.

Genotoxic reactivity: The similar constituents present in Jessemal and Jasmal will present similar reactivity and therefore similar genotoxicity toxicity.

Uncertainty of the prediction: There are no uncertainties other than those already addressed above.

Data matrix

The relevant information on physico-chemical properties and toxicological characteristics are presented in the data matrix below.

Conclusions on genotoxicity for hazard and risk assessment

For Jessemal no data on genotoxicity is available. For Jasmal, a close analogue, such information is present and can be used for read-across. When using read-across the result derived should be applicable for C&L and/or risk assessment. This documentation is presented in the current document. For Jasmal, an Ames test (OECD TG 471), an in vitro chromosome aberrations test (OECD TG 473) and an in vitro mutagenicity assay in mammalian cells (mouse lymphoma, OECD TG 476) are available with negative results (not genotoxic). This information can be used for Jessemal.

Final conclusion:Jessemal is not genotoxic.

 

Data matrix for supporting the read across to Jessemal from Jasmal for genetic toxicity.

Naming	Jessemal	Tetrahydropyran acetates	Jasmal + 3 other similar impurities	Branched alkyl diacetates	Other branched alkyl diacetates (4 impurities)
Read-across	Target	Target Major constituent	Source (and impurities)	Target Minor constituent:	Target Impurities
Chemical structures	Not applicable
Typical conc. (%)	Not applicable	20-40	<23	10-20	<22
CAS #	Not applicable	38285-49-3	18871-14-2	67634-09-7	--
Einecs	945-946-3		242-640-5
REACH registration	2018		Registered
MW	Not applicable	214	214	244	244
Phys-chem*
Log Kow	3.5 (exp.)*	3.2 (est.)	3.2 - 3.7 (exp.)	3.7 (est.)	3.7 (est.)
Ws (mg/L)	714.1 (exp.)	67.8 (est.)	214.9 (exp.)	17.8 (est.)	17.8 (est.)
Human health
Ames (Genemutations in bacterial cells)	Negative (Read across)	Negative (Read across)	Negative (OECD TG 471)	Negative (Read across)	Negative (Read across)
Chrom. Aberr. In vitro cytogenicity	Negative (Read across)	Negative (Read across)	Negative (OECD TG 473)	Negative (Read across)	Negative (Read across)
MLA (Genemutations in mammalian cells	Negative (Read across)	Negative (Read across)	Negative (OECD TG 476)	Negative (Read across)	Negative (Read across)

Log Kow value is based on the major constituent, the log Kow range is 3.5 to 4.2; Chrom. Aberr.= Chromosomal aberrations.

 

References:

- Belsito, D., Bickers, D., Bruze, M., Calow, P., Greim, H., Hanifin, J.M., Rogers, A.E., Saurat, J.H., Sipes, I.G., Tagami, H., 2008, A toxicologic and dermatologic assessment of cyclic acetates when used as fragrance ingredients, Food and Chemical Toxicology 46, Suppl 12:S1-27.

Justification for classification or non-classification

Based on the negative results in an Ames test, in an in vitro chromosome aberration study, and in an in vitro genemutation study in mammalian cells, the substance does not need to be classified for genotoxicity according to EU CLP (EC No. 1272/2008, and its amendments).