Allyl (3-methylbutoxy)acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

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:

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

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:

The mammalian liver post-mitochondrial fraction, 10% Mutazyme, is a pre-mixed fraction with all co-factors including glucose-6-phosphate, NADP, MgCl2, KCl and rat liver S-9 MolToxTM S-9 were stored frozen in aliquots at -20 ºC and thawed prior to use.

Test concentrations with justification for top dose:

The test item was tested for mutation in five strains of Salmonella typhimurium of exactly 108 cells per mL (TA98, TA100, TA1535, TA1537 and TA102) and at the concentrations of 0.2, 0.4, 0.6, 0.8, 1, 2 µL/plate using triplicate plates with and without S-9 mix.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

Details on test system and experimental conditions:

All the test item concentrations were administered to the test system within 4 h and 15 mins of preparation. Once set, the plates were inverted and incubated at 37 C for 48 h (15 May 2018, 1:55 p.m. to 17 May 2018, 1:55 p.m.). Plating was achieved as described in the range finder experiment.

Rationale for test conditions:

according to the guideline

Evaluation criteria:

The test item was considered to be mutagenic in this assay if:
1. The assay is valid.
2. A clear increase in mutation frequencies induced by test item compared to the
concurrent solvent controls.
Results which partially satisfy the above criteria was dealt with on a case-by-case basis.
Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.

Statistics:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Based upon the results obtained in this study and in line with OECD Guideline for Testing of Chemicals, 471 (adopted on 21st July 1997) it is concluded that, the
given test item Allyl Amyl Glycolate (Batch No. - AAG-TEST 1) is non-mutagenic.

Executive summary:

The test item, Allyl Amyl Glycolate (Batch No. - AAG-TEST 1) was assayed for its
ability to induce mutation in histidine-requiring five strains ofSalmonella typhimurium
(TA98, TA100, TA1535, TA1537 and TA102) both in the absence and in the presence
of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial
fraction (S-9).
An initial toxicity Range-Finder Experiment was carried out in strain TA100,
both in the absence and presence of S-9, using six different concentrations (0.0016,
0.008, 0.04, 0.2, 1 and 5 µL/plate) of the test item, positive and negative controls
(in triplicates). The test item which was completely solubilized in DMSO (Dimethyl
Sulphoxide) were administered to the test system within 2 h and 49 mins. Evidence of
toxicity with moderately reduced background lawn was observed in 1 µL/plate and
inhibition of background lawn was observed in 5 µL/plate concentration following
treatments. As per OECD 471, the test items that are cytotoxic below 5 µL/plate should
be tested up to a cytotoxic concentration. Hence 2 µL/plate was selected as the top dose
for main experiment.
The main experiment, were performed with the fiveSalmonella typhimuriumtester
strains (TA98, TA100, TA1535, TA1537 and TA102). Based on the results of the range
finding experiment, the test item was tested at concentrations of 0.2, 0.4, 0.6, 0.8, 1,
2 µL/plate along with positive and negative controls (in triplicates) both in the absence
and presence of S-9. All the test item concentrations were administered to the test
system within 4 h and 15 mins. Moderately reduced background lawn was observed in
1 µL/plate concentration and complete inhibition of background lawn was observed in
2 µL/plate concentration as the evidence of cytotoxicity.
The mean numbers of revertant colonies on negative control plates fell within historical
ranges, and an increase in the revertant colonies in the positive control chemicals
confirms the discrimination between different strains, and an active S-9 preparation.
Not more than 5% of the plates were lost through contamination or some other
unforeseen event. Hence, the assay was considered valid.
No dose-related and reproducible increase in revertant colonies were observed in all
strains following test item administration, both in the absence and presence of metabolic
activation, thereby providing evidence that the test item does not mediated mutagenic
activity.
Based upon the results obtained in this study and in line with OECD Guideline
for Testing of Chemicals, 471 (adopted on 21st July 1997) it is concluded that, the
given test item Allyl Amyl Glycolate (Batch No. - AAG-TEST 1), supplied by
MORAYA GLOBAL LIMITED, is non-mutagenic.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human peripheral blood lymphocytes

Remarks:

human primary cultures

Additional strain / cell type characteristics:

other: pooled blood of three male donors

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

1 µg/mL

Vehicle / solvent:

dimethyl sulphoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Appropriate number of whole blood cultures were established as in table.
Ingredients Make and Expiry Volume per tube (mL) Final concentration

RPMI minimal medium (1x): Himedia, Lot No.:0000248711 Expiry Date: 30 November 2018 6.598 90%
Heat inactivated fetal bovine serum (20%): Thermo fisher scientific, Lot No.:42F3575KExpiry Date: 30 October 2022 1.692 90%
L-Glutamine (1%): Thermo fisher scientific, Lot no: 1886876,Expiry Date: 30 May 2019 0.085 90%
Penicillin / streptomycin (1%): Thermo fisher scientific, Lot no.1935446, Expiry Date: 30 November 2018 0.085 90%
Phytohemagglutinin (PHA-M) (stimulate the lymphocytes to divide): Thermo fisher scientific, Lot no. 1950443,Expiry Date: 28 February 2020 0.188 2%
Pooled heparinized blood (Blood from healthy human): Blood from healthy human 0.752 8%

Blood cultures were incubated at 37 °C for approximately 48 h and shaken continuously.

Evaluation criteria:

The test item will be considered to have the potential to induce chromosome aberrations in this assay if:
1. The assay is valid
2. A proportion of cells with structural aberrations at one or more concentrations that exceeds the historical negative control (normal) range is observed in both replicate cultures
3. A significant increase in the proportion of cells with structural aberrations (excluding gaps) is observed.
4. There is a concentration-related trend in the proportion of cells with structural aberrations (excluding gaps).
Results which only partially satisfy the above criteria was dealt with on a case-by-case basis. Evidence of a concentration-related effect is considered useful but not essential in the evaluation of a positive result. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments, or effects occurring only at high or very toxic concentrations, and the types and distribution of aberrations. Analysis of additional cells from vehicle / or treated cultures or further experimental work may be deemed necessary to aid evaluation of the data.

Statistics:

not specified

Key result

Species / strain:

lymphocytes: human primary culture

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Analysis of Chromosomal aberration data

The details of chromosomal aberrations are presented inAppendix3,Appendix4andAppendix5. Positivecontrolsfrom both experiments induced a clear increase in chromosomal aberrations compared to the concurrent solvent control cultures. The chromosomal aberration frequencies in the solvent controls fell within the historical negative control ranges (Appendix6). A total of 300 cells were scored from each culture. The assay was therefore considered valid.

Structural aberrations

Treatment of cultures with the test item in the absence and presence of S-9 (both experiments) resulted in frequencies of cells with structural aberrations which were similar to those in concurrent negative controls. Numbers of aberrant cells (excluding gaps) in all treated cultures fell within historical negative control ranges.

Numerical aberrations

No increases in the frequency of cells with numerical aberrations, which exceeded the historical negative control range, were observed in cultures treated with the test item in the absence and presence of S-9 (both experiments).

Conclusions:

Based upon the results obtained in this study and in line with OECD Guideline for Testing of Chemicals, 473 (adopted on 29 July 2016) it is concluded that, the given test item Allyl Amyl Glycolate, supplied by Moraya Global Ltd., did not induce chromosome aberrations in cultured human peripheral blood lymphocytes.

Executive summary:

Allyl Amyl Glycolate was tested for its ability to induce structural chromosomal aberrations in cultured human lymphocytes. Blood was obtained from three healthy, male donors and pooled prior to culturing. Duplicate cultures prepared from the pooled blood of three healthy male donors in two independent experiments. The test item was dissolved in dimethyl sulphoxide (DMSO) and the highest dose level used in the main experiment,
1 µg/mL, was determined following a solubility trial and subsequent preliminary cytotoxicity range-finding experiment. Treatments covering a broad range of doses were performed both in the absence and presence of metabolic activation (S-9).

A dose range-finding experiment was conducted which included a short-term treatment of 3-h followed with 17-h recovery in the absence and presence of the exogenous metabolic activation system (S9 from Aroclor 1254-induced rats was used) and a continuous 20-h exposure in the absence of S-9 was also conducted. The concentrations of the test item used were: 2, 1, 0.5, 0.25, 0.125, and 0.0625 µL/mL. Appropriate solvent and negative controls were included. Mitotic index was used as the measure of cytotoxicity.

As the maximum concentration is based on cytotoxicity, the highest concentration should aim to achieve 55 ± 5% cytotoxicity using the recommended cytotoxicity parameter
i.e. reduction inmitotic index(MI).

Based on the range finding study, a main experiment was performed with the same study design (3-h + 17-h recovery with and without S9; 20-h continuous exposure without S9). Six concentrations of the test item (1, 0.75, 0.50, 0.250, 0.125 and 0.0625 µL/mL) was selected for testing in the main study. Appropriate negative (solvent) control cultures were included in the test system in experiment under each treatment condition.

The dose levels for chromosome analysis were selected by evaluating the effect of test item on mitotic index. Chromosomal aberrations were analysed at three dose levels (see below). The highest concentrations chosen for analysis, 1 µL/mL both in the presence and absence of S-9, induced approximately 58.2% and 51.2% cytotoxicity (reduction in mitotic index), respectively and absence (20+0) h of S-9, induced approximately 62.5% cytotoxicity (reduction in mitotic index).

S-9	Treatment + recovery (h)	Solvent control	Concentration of test item (µL/mL)	Positive control
-	3+17	0^a	1,0.25, 0.0625	Mitomycin - C: 0.40, 0.80 µg/mL
+	3+17	0^a	1,0.25, 0.0625	Cyclophosphamide: 6.25,12.5 µg/mL
-	20+0	0^a	1,0.25, 0.0625	Mitomycin - C: 0.40, 0.80 µg/mL
^aSolvent control comprised of DMSO

Treatment of cultures with test item in the absence and the presence of S-9 resulted in frequencies of cells with structural aberrations which were similar to those in concurrent negative controls. Numbers of aberrant cells (excluding gaps) in all treated cultures fell within historical negative control ranges.

The proportion of cells with structural aberrations in these cultures fell within historical solvent control ranges. Mitomycin C and Cyclophosphamide were employed as positive control chemicals in the absence and presence of S-9, respectively. Cells receiving these were sampled in experiment, 20-h after the start of treatment; both compounds shows increase in the proportion of cells with structural aberrations.

No increases in the frequency of cells with numerical aberrations, which exceeded the historical negative control range, were observed in cultures treated with the test item in the absence and presence of S-9.

Based upon the results obtained in this study and in line with OECD Guideline for Testing of Chemicals, 473 (adopted on 29 July 2016) it is concluded that, the given test itemAllylAmyl Glycolate, supplied byMoraya Global Ltd., did not induce chromosome aberrations in cultured human peripheral blood lymphocytes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)