Reaction mass of allyl (2-methylbutoxy)acetate and allyl (3-methylbutoxy)acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test): the substance Allyl Amyl Glycolate was not mutagenic in the strains S. typhimurium TA98, TA100, TA1535 and TA1537, and E. coli WP2 uvr A in the presence and absence of Delor 106-induced rat liver S9 metabolic activation. (OECD 471/GLP).

Chromosome aberration (in vitro cytogenicity/micronucleus study): Allyl amyl glycolate did not induce any induce structural and/or numerical chromosomal damage in human peripheral blood lymphocytes in the presence or absence of Delor 106-induced rat liver S9 (OECD 487/GLP).

Gene mutation (mammalian cell gene mutation assay): there was no evidence of induced mutant colonies over background in Chinese hamster V79 lung fibroblasts cells exposed to allyl amyl glycolate in the presence or absence of Delor 106-induced rat liver S9 metabolic activation (OECD 476/GLP).

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:

21st November 2017 - 26th March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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
- Source and lot/batch No.of test material: O’Laughlin (Nantong) Fine Chemicals Co., Ltd.; NTA375
- Expiration date of the lot/batch: Sep 25, 2020
- Purity: CAS No. 67634-00-8: 79.45 %; CAS No.: 67634-01-9 20.28 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store in cool place. Keep container tightly closed in a dry and well-ventilated place.

Species / strain / cell type:

other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537, E. coli WP2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

Delor 106-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary test (plate-incorporation): 10 - 5000 µg/plate
Main test (plate-incorporation + and - S9): 10 - 1000 µg/plate
Main test (plate-incorporation + S9; E coli WP2 uvrA):100 - 1500 µg/plate

Preliminary test (pre-incubation): 250 - 1000 µg/plate
Main test (pre-incubation + and - S9): 10 - 200 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine: TA 98 without metabolic activation; 2-aminoanthracene : TA 1535, TA 1537, E.coli with metabolic activation; N-methyl-N´-nitro-N-nitrosoguanidine: E. coli without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) for first main test; pre-incubation for confirmatory test; in agar (plate incorporation) for E. coli repeat test +S9.

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: changes in bacterial background and/or decreased number of revertants

Evaluation criteria:

The main criterion for evaluation of results was modified two-fold increase rule, which is compatible with the application of statistical methods . After this rule the result is positive, if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached.

An increase is considered as ”biologically relevant“:
-if the number of reversions is at least twice as high as that in the solvent control for the strains having spontaneous reversion >10;
-if the number of reversions is at least three times as high as that in the solvent control for the strains having spontaneous reversion ≤10;

A test item producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system

According to OECD TG 471, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Statistics:

The main criterion for evaluation of results was modified two-fold increase rule, which is compatible with the application of statistical methods.
According to OECD TG 471, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Species / strain:

S. typhimurium TA 98

Remarks:

Plate incorporation

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Remarks:

Pre-incubation

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

200 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Remarks:

Plate incorporation

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

300 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Remarks:

Pre-incubation

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

200 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Remarks:

Plate incorporation

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

300 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Remarks:

Pre-incubation

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Remarks:

Plate incorporation

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not valid

Species / strain:

S. typhimurium TA 1537

Remarks:

Pre-incubation

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not valid

Untreated negative controls validity:

not valid

Positive controls validity:

not valid

Species / strain:

E. coli WP2 uvr A

Remarks:

Plate incorporation

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Remarks:

Plate incorporation

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Remarks:

Plate incorporation (repeat)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Remarks:

Pre-incubation

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The preliminary cytotoxicity test was performed in Salmonella typhimurium TA 98 without metabolic activation. Results of the toxicity testing are given in Table B. In the preliminary cytotoxicity test, signs of cytotoxicity occurred from 1000 µg per plate.

The second experiment was performed with pre-incubation, so a cytotoxicity test with pre-incubation was performed in Salmonella typhimurium TA 100 without metabolic activation. The test item was dosed in the volume of 50 µL. Pre-incubation was performed at 37±1°C and shaking for 30 minutes. Signs of cytotoxicity occurred from 250 µg per plate. Results are given in the Table C.

The additional experiment in E. coli with metabolic activation was performed with doses with expected effect - 100, 250, 500, 1000 and 1500 μg per plate. 1500 μg per plate was used as maximum because no toxicity was observed in this bacterial strain.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
Spontaneous reversions, solvent and positive controls were compared with historical controls in our laboratory. The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. Average revertant colony counts for the vehicle controls were within the current historical control range for the laboratory. The current ranges are given in the Table A.

Conclusions:

In a reverse bacteria mutation study (Ames test), Allyl Amyl Glycolate, was non-mutagenic in S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA with and without metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria (18 -122), strains of S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA were exposed to Allyl Amyl Glycolate in DMSO at concentrations of 10 - 1000 µg/plate (plate incorporation), and 10 - 200 µg/plate (30 minute pre-incubation) in the presence and absence of mammalian metabolic activation (Delor 106-induced rat liver S9). The plate incorporation test was repeated in the E. coli WP2 uvrA strain using 100 - 1500 µg/plate with metabolic activation.

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in the S. typhimurium TA 98, TA 100, TA 1535 and TA 1537 strains using the plate incorporation or pre-incubation methods. An additional experiment with E.coli WP2 uvrA with metabolic activation performed to clarify the result of the first experiment, where Rt/Rc 1.5 and 2.1 were observed at 300 and 1000 μg per plate, respectively. No such increase was observed in the third experiment, and the experiment with pre-incubation was clearly negative, so mutagenicity of the test item was not confirmed in this strain.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12th February 2018 - 30th May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: O’Laughlin (Nantong) Fine Chemicals Co., Ltd.; NTA375
- Expiration date of the lot/batch: Sep 25, 2020
- Purity: CAS No. 67634-00-8: 79.45 %; CAS No.: 67634-01-9 20.28 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store in cool place. Keep container tightly closed in a dry and well-ventilated place.

Species / strain / cell type:

other: human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

CELLS & MEDIA USED
- Source of cells: Young healthy non smoking females (18 - 35 years of age)
Peripheral blood (heparinized) is taken from donors in certified medical laboratory (MeDiLa) in the morning and transported into the test facility as soon as possible. The whole human peripheral blood was transferred to the RPMI 1640 growth medium and mitogenic stimulator (phytohaemagglutinin M) was added. These operations were carried out in a laminar box at room temperature. The cultivation runs without interrupting for 48 hours (37°C ± 1°C; 5% CO2).
The lymphocyte`s cell cycle length during the first experiment was 16 hours. This is suitable to experimental design described above according to OECD TG.

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Delor 106-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary test: 0.125, 0.250, 0.500, 1 and 2 uL/mL
Main test 1 (4 hrs; +/-S9): 0.125, 0.250, 0.500, 1 and 2 uL/mL
Main test 2 (23 hrs, -S9): 2 uL/mL
Main test 3: (23 hrs, -S9): 0.5, 1 and 2 uL/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: -S9; Colchicine (0.007 ug/mL - 0.07 ug/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hrs + and – S9; 23 hrs – S9.
- Expression time (cells in growth medium): 23 hrs in total for all test groups
- Fixation time (start of exposure up to fixation or harvest of cells): Cultures were harvested 23 hours after the beginning of treatment (after about 1.5 to 2 cell cycles)

SPINDLE INHIBITOR (cytogenetic assays): 4.5 ug/mL cytochalasin B (3 hr treatments); 3 ug/mL cytochalasin B (23 hr treatments)

STAIN (for cytogenetic assays): Giemsa Romanowski staining solution

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Cultures were harvested 23 hours after the beginning of treatment (after about 1.5 to 2 cell cycles). Cultures were treated by hypotonic solution (RT, ca 10 min.) and then they were centrifuged (1200 rpm, 10 min.). After removing the hypotonic solution, fixation solution was added to cultures and cultures were centrifuged again (1200 rpm, 10 min.). The addition of fixation solution and centrifugation were repeated three times. Suspensions were then dropped on clear microscopic slides. Preparations were let to dry at laboratory temperature and then slides were stained by Giemsa Romanowski staining solution.

NUMBER OF CELLS EVALUATED: 1000 cells were scored per each concentration (cytotoxicity); 2000 binucleated cells were analysed per each concentration and control (divided equally between the duplicates) (genotoxicity)

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis-Block Proliferation Index (CBPI)

Evaluation criteria:

Genotoxicity is indicated by increasing of number of cells with micronuclei in comparison to the negative control (two-fold increase rule) and/or by dependence of increasing number of cells with micronuclei on dose (dose-response relationship).

Two-fold increase rule:
Ratio of the number of binucleated cells with micronuclei in test concentration to the number of binucleated cells with micronuclei in negative control (Mt/Mc) should be higher than 2. The result will be considered as positive when two-fold increase rule will be met at least in one test concentration.

Test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the following three conditions are met concurrently:
• at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control (two-fold increase rule)
•the dependence of increasing number of cells with micronuclei on concentration (dose-response relationship) is evident
•any of the results are outside the distribution of the historical negative control data
When all of these criteria are met, the test chemical is then considered able to induce chromosome breaks and/or gain or loss in this test system.

Test chemical is considered clearly negative if, in all experimental conditions examined, the following three conditions apply concurrently:
•none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
•there is no concentration-related increase when evaluated with an appropriate trend test,
•all results are inside the distribution of the historical negative control data

In case of equivocal results, further testing with modification of experiment conditions will be used for clarification.

Species / strain:

other: human peripheral blood lymphocytes

Remarks:

Main test 1

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:

valid

Positive controls validity:

valid

Species / strain:

other: human peripheral blood lymphocytes

Remarks:

Main test 2

Metabolic activation:

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:

valid

Positive controls validity:

valid

Species / strain:

other: human peripheral blood lymphocytes

Remarks:

Main test 3

Metabolic activation:

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH of the medium RPMI 1640 without the test item was 7.05. At the highest concentration (2 uL/mL) the pH was measured and it was 7.77. So the test item did not cause a marked change in the pH of the medium (in used concentrations). This minor shift could not lead to artifactual positive results which do not reflect the genotoxicity of the test chemical.
- Precipitation: No precipitation in any tested concentration was observed

RANGE-FINDING/SCREENING STUDIES:
In the first experiment without and with metabolic activation the concentrations of 0.125, 0.25, 0.5, 1 and 2 uL/mL have been analysed for cytotoxic effect. In the second experiment (prolonged exposition without activation) the concentration of 2 uL/mL have been analysed for cytotoxic effect. In the third experiment (prolonged exposition without activation) the concentrations of 0.5, 1 and 2 uL/mL have been analysed for cytotoxic effect. The test item was non-cytotoxic at all tested concentrations.


CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: There was no difference between treated and control groups.

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: The test item did not show evidently the dependence of increasing number of cells with micronuclei on concentration (dose-response relationship) (see Fig. 1)

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Yes; 20.4.2012 – 28.7.2017 (Annex 3)
- Negative (solvent/vehicle) historical control data: Yes; 20.4.2012 – 28.7.2017 (Annex 3)

Conclusions:

In an in vitro mammalian cell micronucleus test in human peripheral blood lymphocytes, allyl amyl glycolate was not mutagenic in the presence or absence of metabolic activation.

Executive summary:

In an in vitro cytogenicity/micronucleus study (18-123), human peripheral blood lymphocytes were exposed to allyl amyl glycolate in DMSO at concentrations of 0.125, 0.250, 0.500, 1 and 2 µL/mL (first experiment; 3 hrs) with and without metabolic activation (Delor 106-induced rat liver S9), 2 µL/mL (second experiment; 23 hrs) without metabolic activation and to 0.5, 1 and 2 µL/mL (third experiment; 23 hrs) without metabolic activation.

Allyl amyl glycolate was tested up to cytotoxic concentrations. Positive controls induced the appropriate response.  There was no evidence of any chromosome damage or damage to the cell division apparatus induced over background.

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 487 for in vitro cytogenetic mutagenicity data.  

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26th February 2018 - 17 September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: O’Laughlin (Nantong) Fine Chemicals Co., Ltd.; NTA375
- Expiration date of the lot/batch: Sep 25, 2020
- Purity: CAS No. 67634-00-8: 79.45 %; CAS No.: 67634-01-9 20.28 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store in cool place. Keep container tightly closed in a dry and well-ventilated place.

Target gene:

Hprt

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: European Collection of Cell Cultures (ECACC); Lot. No.: 15H003
- Number of passages if applicable: Cells underwent maximum 5 passages after thawing the original culture delivered from cell collection before using for mutagenicity testing.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Complete DMEM medium as used for growing of cultures (10 % FBS, an in incubator (5 % CO2, 37±1 °C, moistened)
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically 'cleansed' against high spontaneous background: Yes; Cleansing of cultures was performed 5 days before treatment with complete medium supplemented with HAT supplement to eliminate mutants. Cleansing was not performed before cytotoxicity experiments.

Metabolic activation:

with and without

Metabolic activation system:

Delor 106-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary test (+/-S9): 0.01, 0.1, 0.2, 0.5, 1.0, 2.0 mg/mL
Preliminary test (+S9): 0.05, 0.1, 0.15, 0.20, 0.30 mg/mL
Main test (-S9): 0.2, 0.5, 1.0, 1.5, 2.0 mg/mL
Main test (+S9): 0.05, 0.1, 0.15, 0.20, 0.25 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hrs + and – S9
- Expression time (cells in growth medium): 10 days
- Selection time (if incubation with a selection agent): Not stated

SELECTION AGENT (mutation assays): 6-thioguanine 98%, diluted in 0.5% Na2CO3,; 5 µg/mL (final concentration) for selection of mutants

NUMBER OF REPLICATIONS: 2

STAINING TECHNIQUE USED: Plates were stained with methylene blue

DETERMINATION OF CYTOTOXICITY
- Method: Relative Survival

Evaluation criteria:

Each experiment is evaluated separately using modified two-fold increase rule..
The mutagenic potential is indicated by increasing number of mutants in treated groups in comparison to the negative solvent control (modified two-fold increase rule and any of the results outside the distribution of the historical negative control data) and/or by dependence of increasing number of mutants on dose (dose-response relationship).
There is no requirement for verification of a clearly positive or negative response.
In cases when the response is neither clearly negative nor clearly positive than a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, other metabolic activation conditions i.e. S9 concentration or S9 origin) could be performed.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

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:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

0.15 mg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Addition of the test item to cultivation medium did not change pH of treatment solutions (Table 8).
- Precipitation: In the preliminary experiment with metabolic activation, no precipitation of the test item was observed at any concentration.

RANGE-FINDING/SCREENING STUDIES:
Concentrations 0.01, 0.1, 0.2, 0.5, 1.0 and 2.0 mg per mL were used for the cytotoxicity experiment with and without metabolic activation.

Cells treated with the test item were observed at the end of treatment. In the experiment without metabolic activation, at concentrations of 1.0 and 2.0 mg per mL, more cells were rounded than in negative control. The cells were adhered in the bottom of Petri dishes. The test item flows on the top of the medium starting from concentration of 1.0 mg per mL. Almost no cytotoxicity was observed at concentrations of 0.01-0.5 mg per mL. Medium cytotoxicity was observed at 1.0 mg per mL (RS 43.2%) and high cytotoxicity at 2.0 mg per mL (RS 11.9%; see Table 2).
At the end of the experiment with metabolic activation at concentrations of 1.0 and 2.0 mg per mL, all cells were rounded. The test item flows on the top of the medium starting from concentration of 0.5 mg per mL. Potential detachment of cells from the bottom of Petri dishes was not possible to evaluate due to presence of S9 which caused turbidity in the medium. Higher cytotoxicity was observed than without metabolic activation at 0.1 mg per mL (RS 88.4%) and at 0.2 mg per mL (RS 8.3%; see Table 3).
Another experiment with metabolic activation was performed for determination of closer doses for the mutagenicity experiment, with concentrations between the cytotoxic and non-cytotoxic effect. The concentrations used were 0.05, 0.10, 0.15 0.20 and 0.30 mg per mL. No separation of media/ test substance was observed at any concentration. Rounded cells were observed starting from concentration of 0.1 mg per mL. For results, see Table 4.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Yes generated from May 2014 to present
- Negative (solvent/vehicle) historical control data: Yes generated from May 2014 to present

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity in Mutagenicity Experiments:
Based on the cytotoxicity experiments, concentrations 0.2, 0.5, 1.0, 1.5 and 2 mg per mL were used in the experiment without metabolic activation. Cytotoxicity results are given in Table 5. In contrast to the preliminary cytotoxicity test, the highest cytotoxicity was observed in the dose of 0.5 mg per mL. It could be connected with maximum solubility - starting at 1.0 mg per mL the application form separates in two phases such that the test item flows on the top of the medium and caused corrosion of plastic on perimeter of Petri dishes on the walls. The corrosion of dishes probably did not affect results - cells were undoubtedly damaged (rounded) similarly to those with metabolic activation and mutagenicity result was negative. Medium cytotoxicity was observed at the highest concentration (RS 51.4%; see Table 5).

Concentrations 0.05, 0.1, 0.15, 0.20 and 0.25 mg per mL were used in experiment with metabolic activation. Cytotoxicity is given in Table 6. No cytotoxicity was observed at concentrations 0.05 and 0.1 mg per mL and high cytotoxicity was observed at 0.15 mg per mL. Therefore an additional experiment was performed with one extra concentration of 0.125 mg per mL; this concentration was also quite cytotoxic (RS 16.9%; see Table 7). No separation to two phases was observed in experiment with metabolic activation.

Conclusions:

In an in vitro gene mutation study (HPRT) in Chinese hamster V79 mammalian cells, allyl amyl glycolate is not mutagenic in the presence or absence of metabolic activation.

Executive summary:

In an in vitro gene mutation study (HPRT) in mammalian cells (18-323), Chinese hamster V79 lung fibroblasts cells were exposed to allyl amyl glycolate in DMSO at concentrations of 0.2, 0.5, 1.0, 1.5, 2.0 mg/mL for 3 hrs without Delor 106-induced rat liver S9 metabolic activation and 0.05, 0.1, 0.15, 0.20, 0.25 mg/mL for 3 hours with metabolic activation.

Allyl amyl glycolate was tested up to the limits of cytotoxicity in the absence of metabolic acitvation and cytotoxicity was noted at 0.15 mg/mL in the presence of metabolic acitvation. The positive controls induced the appropriate response. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro gene mutation data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test)

There is one gene mutation study (Bacterial Reverse Mutation Assay/Ames test) with Allyl Amyl Glycolate available.

In a reverse gene mutation assay in bacteria (OECD 471/GLP), strains of S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA were exposed to Allyl Amyl Glycolate in DMSO at concentrations of 10 - 1000 µg/plate (plate incorporation), and 10 - 200 µg/plate (30 minute pre-incubation) in the presence and absence of mammalian metabolic activation (Delor 106-induced rat liver S9). The plate incorporation test was repeated in the E. coli WP2 uvrA strain using 100 - 1500 µg/plate with metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in the S. typhimurium TA 98, TA 100, TA 1535 and TA 1537 strains using the plate incorporation or pre-incubation methods. An additional experiment with E.coli WP2 uvrA with metabolic activation performed to clarify the result of the first experiment, where Rt/Rc 1.5 and 2.1 were observed at 300 and 1000 μg per plate, respectively. No such increase was observed in the third experiment, and the experiment with pre-incubation was clearly negative, so mutagenicity of the test item was not confirmed in this strain.

Chromosome aberration (in vitro cytogenicity/micronucleus study):

There is one in vitro cytogenicity/micronucleus study available.

In an in vitro cytogenicity/micronucleus study (OECD 487/GLP), human peripheral blood lymphocytes were exposed to allyl amyl glycolate in DMSO at concentrations of 0.125, 0.250, 0.500, 1 and 2 µL/mL (first experiment; 3 hrs) with and without metabolic activation (Delor 106-induced rat liver S9), 2 µL/mL (second experiment; 23 hrs) without metabolic activation and to 0.5, 1 and 2 µL/mL (third experiment; 23 hrs) without metabolic activation. Allyl amyl glycolate was tested up to cytotoxic concentrations. Positive controls induced the appropriate response.  There was no evidence of any chromosome damage or damage to the cell division apparatus induced over background.

Gene mutation (mammalian cell gene mutation assay):

There is one gene mutation (mammalian cell gene mutation assay) available.

In an in vitro gene mutation study (HPRT) in mammalian cells (OECD 476/GLP), Chinese hamster V79 lung fibroblasts cells were exposed to allyl amyl glycolate in DMSO at concentrations of 0.2, 0.5, 1.0, 1.5, 2.0 mg/mL  for 3 hrs without Delor 106-induced rat liver S9 metabolic activation and 0.05, 0.1, 0.15, 0.20, 0.25 mg/mL for 3 hours with metabolic activation. Allyl amyl glycolate was tested up to the limits of cytotoxicity in the absence of metabolic acitvation and  cytotoxicity was noted at 0.15 mg/mL in the presence of metabolic acitvation. The positive controls induced the appropriate response. There was no evidence of induced mutant colonies over background.

Justification for classification or non-classification

Based on the available information in the dossier, the substance allyl amyl glycolate (EC No. 916-328-0) does not need to be classified for germ cell mutagenicity when the criteria outlined in Annex I of 1272/2008/EC are applied.