Glycollic acid

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

S.typhimurium strain TA97a was used as a substitute for the strains TA97 and/or TA1537 normally recommended by EEC and Japan guidelines. THe substitution is acceptable for this design and has no impact on the study validity. The positive control for thi

Qualifier:

according to guideline

Guideline:

other: EPA-OPPTS 40 CFR 799.9510

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium TA97a- hisD6610, hisO1242.
S. typhimurium TA98- hisD3052.
S. typhimurium TA100- hisG46.
S. typhimurium TA1535- hisG46.
E. coli WP2 uvrA (pKM101)- trpE.

Species / strain / cell type:

S. typhimurium TA 97a

Additional strain / cell type characteristics:

other: Histidine mutations: his D6610, his O1242; Additional mutations: rfa uvrB

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: Histidine mutations: his D3052; Additional mutations: rfa uvrB

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: Histidine mutations: his G46; Additional mutations: rfa uvrB

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: Histidine mutations: his G46; Additional mutations: rfa uvrB R-factor (pKM101 plasmid)

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Additional strain / cell type characteristics:

other: Histidine mutations: trpE; Additional mutations: rfa uvrA R-factor (pKM101 plasmid)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Trial 1
S. typhimurium TA100 and E. coli WP2 uvrA (pKM101)- 1, 5, 10, 50, 100, 500, 1000, 2500, 5000 ug/plate.
S. typhimurium TA97a, TA98 and TA1535- 10, 50, 100, 500, 1000, 2500, 5000 ug/plate.

Trial 2 All strains 10, 50, 100, 500, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Test substance solvent/dilutent/negative control: Phosphate buffered saline (PBS.)
- Deionized water was the solvent for NAAZ and ICR 191
- The solvent for 2AA, 2NF, DMBA, and ENNG was dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Based on a solubility test that allowed for the preparation of a soluble or
workable stock concentration up to 50 mg/ml.

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

Remarks:

applies for S. typhimurium TA 97a + S9 mix

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: ICR 192 acridine mutagen (ICR 191)

Remarks:

applies for S. typhimurium TA 97a - S9 mix

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA)

Remarks:

applies for S. typhimurium TA 98 + S9 mix

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

applies for S. typhimurium TA 98 - S9 mix

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA)

Remarks:

applies for S. typhimurium TA 100 + S9 mix

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

applies for S. typhimurium TA 100 - S9 mix

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA)

Remarks:

applies for S. typhimurium TA 1535 + S9 mix

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

applies for S. typhimurium TA 1535 - S9 mix

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA)

Remarks:

applies for E. coli WP2 uvr A pKM 101 + S9 mix

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

applies for E. coli WP2 uvr A pKM 101 - S9 mix

Details on test system and experimental conditions:

After pouring top agar onto minimal glucose agar plates and allowing to solidify, the plates were incubated for 48 hours at 37°C.

METHOD OF APPLICATION: In agar (plate incorporation method.)
DURATION
- Preincubation period: over night
- Exposure duration: 48hr
- Selection time (if incubation with a selection agent): 48 hr
SELECTION AGENT (mutation assays): histidine for S. typhimurium, and tryptophan for E. coli
NUMBER OF CELLS EVALUATED: 1x10^8 cells/plate
DETERMINATION OF CYTOTOXICITY
- Evidence of toxicity was scored relative to the concurrent negative control plates. A minimum of three on-toxic
concentration levels was required to classify the test substance. A concentration level was considered non-toxic if it
caused less than 50% reduction in the mean number of revertants per plate relative to the mean of the concurrent negative
control.

Evaluation criteria:

Bacterial lawns were evaluated for evidence of toxicity or test substance precipitation and scored by comparison with concurrent negative control plates. Revertant colonies were counted uing an automatic colony counter for each tester strain unless prohibited by excessive toxicity. Manual counting was employed, as appropriate, if test substance precipitation interfered with automatic counting.
- Positive (or mutagenic) if the mean number of revertants in any strain at any test substance concentration was at least 2
times greater than the mean of the concurrent vehicle control and there was a concentration related increase in the mean
revertants per plate in that same strain.
- Negative (or not mutagenic) if there was no test substance concentration with a mean number of revertants that was at
least 2 times greater than the mean of concurrent vehicle control. Or, if there was no positive concentration related
increase in the mean revertants per plate in that same strain.
- Equivocal if the results not meeting positive or negative criteria classification were evaluated using scientific judgement
and experience and possibly reported as equivocal.

Statistics:

For each strain, the mean number of revertants and the standard deviation were calculated at each concentration, with or without metabolic activation

Data for each tester strain were evaluated independently. For each tester strain, the mean number of revertants and the
standard deviation at each concentration in the presence of and absence of the exogenous metabolic activation system
were calculated.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: yes, ≥ 1000 µg/plate (generally)

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: yes, ≥ 1000 µg/plate (generally)

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: yes, ≥ 1000 µg/plate (generally)

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: yes, ≥ 1000 µg/plate (generally)

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in bacterial background lawns at 500ug/plate or higher in trial 1 without activation and 1000ug/plate or higher in trial 2 without activation, and 2500ug/plate or higher with activation in both trial 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Under the conditions of this study, no evidence of mutagenic activity was detected in either of two independent trials. Based on these data, the test substance was judged to be negative for mutagenic activity.

Under the conditions of this study, no evidence of mutagenic activity was detected in either of two independent trials. Based on these data, the test substance was judged to be negative for mutagenic activity.

Conclusions:

Glycolic acid showed no mutagenic effect in any of the bacterial tester strains with and without metabolic activation. Under the conditions of this study, no evidence of mutagenic activity was detected in either of two independent trials. Based on these data, the test substance was judged to be negative for mutagenic activity.

Executive summary:

Glycolic acid showed no mutagenic effect in any of the bacterial tester strains with and without metabolic activation.

Under the conditions of this study, no evidence of mutagenic activity was detected in either of two independent trials. Based on these data, the test substance was judged to be negative for mutagenic activity.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 August 1994 to 13 December 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP Compliance. The number of metaphase cells evaluated is stated to be a minimum of 100 per concentration. The current OECD 473 guideline requires a minimum of 200 well spread metaphases to be evaluated. Viable or total cell counts may be a more reliable parameter for assessing toxicity than is the mitotic index for CHO cells. However both methods are included in the test guideline and the use of one method is not considered to have compromised the study integrity.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

The method of assessing toxicity - mitotic index - may have been improved by use of viable or total cell counts, but was considered an acceptable method at the time the study was conducted.
The number of metaphases analysed in this study was also acceptable at the time the study was conducted although current guidelines indicate a higher number should be evaluated for each concentration.
The test material concentration was quite low - at 20% rather than 70% as normally tested for the technical grade material, reflecting the differing intended uses, THe lower concentration used in this test being more appropriate for the cosmetic uses envisaged by the commissioning body. THe study gave a negative result, consistent with all other genotoxicity assessments and so the difference in concentration is not expected to have adversely affected the evaluataion of glycolic acid.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Complete medium (McCoy's 5A medium containing 10% fetal bovine serum,
2mM L-glutamine, 100 units penicillin/mL and 100 ug streptomycin/mL) for each treatment condition at
approximately 5x10^5 cells/25 cm2 flask.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes (tested using the Hoechst staining procedure)
- Periodically checked for karyotype stability: yes (cells were not used beyond passage 20)
- Other: Repository No. CCL 61, obtained from American Type Culture Collection, Rockville, MD

Additional strain / cell type characteristics:

other: K1

Metabolic activation:

with and without

Metabolic activation system:

S9 (Aroclor 1254-induced rat liver), mixed with 2mM magnesium chloride, 6mM potassium chloride, 1mM glucose-6-phosphate, 1mM nicotinamide adenine dinucleotide phosphate (NADP) S9 mix

Test concentrations with justification for top dose:

The following test concentrations were evaluated: 625, 1250, 2500, 5000 in ug/mL active ingredient
There were also positive, negative and untreated controls (consisting of cells in complete medium or S-9 reaction mixture)
used.
pH of test article in treatment was approximately 4 and required adjustment with 1N NaOH to approximately 6.5.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: phosphate buffered saline (PBS)
- Justification for choice of solvent/vehicle: The test article was soluble in PBS - Sponsor indicated this would be the test
vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

phosphate buffered saline (PBS)

True negative controls:

yes

Remarks:

Untreated cells

Positive controls:

yes

Remarks:

mitomycin C without activation

Positive control substance:

mitomycin C

Remarks:

applies for: non-activated test. lot 34H2510 (obtained from Sigma Chemical Company); dissolved and diluted in sterile distilled water to stock concentrations of 0.8 and 1.5 ug/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Phosphate buffered saline (PBS)

Positive controls:

yes

Remarks:

cyclophosphamide with activation

Positive control substance:

cyclophosphamide

Remarks:

applies for: activated test. lot 72H0088, obtained from Sigma Chemical Company; dissolved and diluted in sterile distilled water to stock concentrations of 0.25 and 0.5 mg/mL

Details on test system and experimental conditions:

In the definitive assay CHO cells were seeded at 5 x 10000 cells per 25 cm2 flask and incubated at 37°C for 16-24 hours. Flasks were re-fed with medium, S9 where appropriate and the cells were exposed to various test substance concentrations for 12 hours, without metabolic activation, or 4 hours, with S9 activation. For the non-activated system, two hours prior to scheduled harvest the cells were treated with Colcemid. For the activated system, after six hours exposure the treatment medium was removed, cells washed and returned to incubator for 8 hours, two hours prior to scheduled harvest the cells were treated with Colcemid. Cells were harvested by trypsinisation approximately 12 hours after initiation of treatment. Cells were collected by centrifugation. The cell pellet was re-suspended, re-centrifuged, the supernatant was aspirated and cells fixed in Carnoy’s fixative overnight. Slides were prepared, the air-dried cells were stained with Giemsa, air dried and permanently mounted.

METHOD OF APPLICATION: in medium - treatment was carried out by refeeding the flasks with complete medium or S-9
reaction mixture and into this the dosing solution was added.
DURATION- Preincubation period: of the CHO cells was 16-24 hours
- Exposure duration: 12 hours for the non-activated study and 4 hours for the activated S9 study
Non-activated study: 2 hours prior to the scheduled cell harvest, Colcemid was added to the treatment flasks at a final
concentration of 0.1 ug/mL and the cells were returned to the incubator until cell collection.
Activated S-9 study: After exposure, the treatment medium was removed, the cells were washed in CMF-PBS, refed with
complete medium and returned to the incubator for 6 hours. At this time, Colcemid was added to each treatment flask at a
final concentration of 0.1 ug/mL and the flasks were incubated for 2 more hours.
- Harvesting of cells: occurred 12 hours after treatment initiation
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: minimum of 100 metaphase spreads were examined and scored (50 per duplicate
flask)
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

The test material was considered to give a positive result if a dose-related increase in percent aberrant cells was observed with statistical significance achieved in comparison with controls.

Toxic effects were based upon mitotic inhibition relative to the solvent control (as the % change in mitotic index).
Aberration effects were calculated as follows:
- number and types of aberrations found,
- the percent structurally damaged cells (% aberrant cells),
- frequency of structural aberrations per cell (mean cell aberrations)
The test article would be considered to induce a positive response when the % of cells with aberrations increased in a
dose-response manner with 1 or more concentrations being statistically elevated relative to the solvent control group. A
significant increase at the high dose with no dose response would be considered suspect. A significant increase at 1 dose
level other than the high dose with no dose response would be considered equivocal.
Criteria for a Valid Test:
The frequency of cells with structural chromosome aberrations in either the untreated or solvent control must be no greater
than 6%.
The percentage of cells with chromosome aberrations in the positive control must be statistically increased relative to the
solvent control or to the untreated control (if a solvent other than water was used).

Statistics:

Statistical analyses of percent aberrant cells were performed using Fischer’s exact test. For the test to be valid the frequency of cells with structural chromosome aberrations in the untreated or solvent controls should be no greater than 6%. The percentage in the positive control group must be statistically significantly increased relative to the solvent or untreated controls.
In the event of a positive Fisher's test at any test article dose level, the Cochran-Armitage test was used to measure doseresponsiveness

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxicity ca. 43% at 5000 µg/mL without metabolic activation; minimal cytotoxicity at 5000 µg/mL with metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No increase in chromosome aberrations was observed in the nonactivated or S-9 activated test systems relative to the
solvent control group.
The percentage of aberrant cells in the MMC and CP groups respectively, was 12% and 30%.

Conclusions:

RI 3343 was negative in the chromosome aberration assay using CHO cells. Glycolic acid did not induce chromosomal aberrations under conditions of metabolic activation or non-activation in Chinese hamster ovary cells. Glycolic acid was considered negative for mutagenicity in this assay. The positive and negative controls fulfilled the criteria for completion of a valid test.

Executive summary:

Glycolic acid did not induce chromosomal aberrations under conditions of metabolic activation or non-activation in Chinese hamster ovary cells. Glycolic acid was considered negative for mutagenicity in this assay. The positive and negative controls fulfilled the criteria for completion of a valid test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP Compliance. Equivocal results. Negative without metabolic activation, positive with metabolic activation at high concentrations (32.9 mM to 65.8 mM). Negative at or below the recommended limit dose as per OECD 476 .

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

yes

Remarks:

Tested concentrations above the 10mM concentration of concern defined by the EPA guideline for this assay

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase (TK) locus in the mouse lymphoma L5178Y cell line

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Initial mutation assay with and without activation-39.3, 78.5, 157, 313, 625, 1250, 2500, 5000 ug/ml.
Confirmatory mutation assay with and without activation-250, 500, 1000, 2000, 2500, 3000, 4000, 5000ug/ml.

6.25; 125; 250; 500; 1000; 2000; 2500, 3000; 4000 and 5000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Fisher's medium.
- Justification for choice of solvent/vehicle: To aid in pH adjustment.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

non-activated mutation assay

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

activated mutation assay

Details on test system and experimental conditions:

Stock cultures were obtained from Dr. Donald Clive and stored in liquid nitrogen. All laboratory cultures were maintained in logarithmic growth by serial subculturing for up to 4 months and were replaced by cells from the frozen stock.
Cultures were grwon in a shaker incubator at approximately 37°C.

METHOD OF APPLICATION: In medium.
DURATION
- Preincubation period: no data
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2 days
SELECTION AGENT (mutation assays): 5-trifluorothymidine
NUMBER OF CELLS EVALUATED:6x10^6 cells per 10ml treatment medium for exposure treatment. During expression
period on day 1 cells were adjusted to 3x10^5 cells/ml. At day 2 after cytotoxicity determined, the selected were seeded at
3x10^6 cells in soft agar to recover mutants. For cloning assay 600 cells in agar were used.
DETERMINATION OF CYTOTOXICITY
- Method- relative total growth compared to vehicle control cultures
Other
-Sizing analysis-quantified large mutant colonies, which are associated with changes within the gene (point mutations),
and small mutant colonies, which are associated with gross, chromosomal damage (Chromosome changes). Bimodal
curve was generated, and colonies quantified by area under the curve.

Evaluation criteria:

The mutant frequency was calculated as a ratio of the total number of mutant colonies to the total number of cells seeded (adjusted by the absolute selection cloning efficiency). Cytotoxicity of each treatment was determined as the relative suspension growth of the cells over the two day expression period multiplied by the relative cloning efficiency at time of selection.

Positive (mutagenic)- Defined as if dose-dependent increases of 2-fold or greater in mutant frequency is obtained over the
concurrent background mutant frequency. The background mutant frequency is defined as the average mutant frequency
of the vehicle control cultures. The 2-fold or greater increase is based on extensive experience which indicates such
responses are repeatable in additional trials. It is desirable to obtain this relationship for at least 3 doses, but this goal
depends on the dose steps chosen for the assay and toxicity at which mutagenic activity appears. The dose-dependent
requirement is waived if there is a large increase in mutant frequency, defined as 4-fold or higher, and is obtained for a
single dose at or near the highest testable toxicity. However, for the test article to be evaluated as positive increases must
be repeatable in the second trial.
Negative (nonmutagenic)- In a single trial if a 2-fold increase in mutant frequency was not observed for (1) a range of
doses that extended to toxicities causing 10-20% relative total growth, or (2) for relatively nontoxic test articles, a range of
doses extending to the maximum concentration of 5mg/ml, or (3) a range of doses that extended to a level of
approximately twice the solubility limit in the culture medium, or (4) the increase(s) are not in a confirmatory trial.
Equivocal-Defined as if there was no consistent evidence for either a positive or negative evaluation.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

not at concentrations used for mutagenic study, based on dose range finding assay

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

at 5000ug/ml in initial assay, and at 2500 and 5000 ug/ml in confirmatory assay

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

not at concentrations used for the mutagenic study, based on dose range finding assay

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The activated system had a positive mutagenic response, but all concentrations that showed this response were above the
10mM concentration of concern defined in the EPA guideline for the mouse lymphoma L5178Y forward mutation assay.
Study also looked at a sizing analysis of mutant colonies. For the non-activated assay there was a good recovery of both
small and large colonies, but no definitive increase in either small or large colonies was observed. For the activated assay,
preferential increases in small colonies was observed,indicating a possible clastogenic response (chromosomal change.)

At very high concentrations a positive response was induced in the presence of S9 but at dose concentrations of concern (at or below the limit level of 10 mM, set by EPA) there were no increases in mutant frequency. The result, under the conditions of the test was positive for mutagenicity but at levels relevant for human risk assessment glycolic acid is not considered a positive mutagen. Both non-activated and both activated assays showed no or only weak cytotoxic effects.

 

Initial positive results achieved in the study when dosed at 65.8 mM (without activation) were not reproducible. But the results were positive when tested with S9 metabolic activation at the two highest dose levels, 2500 or 5000 µg/ml.

 

The conclusion of the report author is that the study result was overall negative, with a comment that the OECD 476 maximum testing limit for the assay is 5 µl/mL, 5 mg/mL or 10 mM, whichever is less, and that concentrations over six times higher than the recommended molar limit were tested. It should be noted that positive results were generated at ≥ 32.9 mM and with 65.8 mM (or 5000 µg/mL) as the top dose. 

 

The results indicating an overall negative response were consistent with all other genotoxicity results, and also in accord with human exposure responses indicating an absence of genotoxic carcinogenicity and so the report conclusion is adopted despite use of variant target dose concentrations and some indications of cytotoxicity at very high doses.

Conclusions:

At very high concentrations a positive response was induced in the presence of S9 but at dose concentrations of concern (at or below the limit level of 10 mM, set by EPA) there were no increases in mutant frequency. The result, under the conditions of the test was positive for mutagenicity but at levels relevant for human risk assessment glycolic acid is not considered a positive mutagen. Both non-activated and both activated assays showed no or only weak cytotoxic effects.

At levels relevant for human risk assessment, glycolic acid is not considered a positive mutagen, neither as cytotoxic.

Executive summary:

Glycolic acid was negative without S9 metabolic activation and positive with metabolic activation under the conditions used in the L5178Y TK +/- Mouse Lymphoma Forward Mutation Assay. However, the EPA Guideline for this assay recommends a maximum testing limit of 5 µl/mL, 5 mg/mL or 10 mM, whichever is less. In the present study, concentrations over six times higher than the recommended molar limit was tested. No mutant frequencies that were considered elevated were observed at concentrations less than 10 mM in the mouse lymphoma L5178Y forward mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 July 1998 to 20 October 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA health Effect Guidelines 40 CFR 799.9539

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: Crl:CD-1 (ICR)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Inc., Raleigh, North Carolina.
- Age at study initiation: 43 days, 51 days when treated.
- Weight at study initiation: Males weighed 29.4-33.3g, females weighed 22.2-25.3g.
- Assigned to test groups randomly: yes, by computer generated random numbers
- Fasting period before study: no data
- Housing: 2-3 per cage during the quarantine period and individually during the study phase in standard wire mesh cages
- Diet: ad libitum
- Water : ad libitum
- Acclimation period: 8 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 degrees +or -1 degree
- Humidity (%): 50 +or - 10%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12-hr light/dark cycle

Approximately 7 weeks old at time of dosing in the bodyweight range 29.4 to 33.3 g for males and 22.2 to 25.3 g for females

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: HPLC grade water.
- Justification for choice of solvent/vehicle: No data.
- Concentration of test material in vehicle: 42.5, 56.7, 85.0, 113.3, 170.0, 226.7 mg/ml administered by oral intubation in a
dose volume of 10 ml/kg.
- Amount of vehicle (if gavage or dermal): 10ml/kg.

Details on exposure:

Acute exposure by oral intubation.

Duration of treatment / exposure:

single dosage

Frequency of treatment:

Single oral gavage administration

Post exposure period:

Negative controls and low/intermediate test groups: 5 male and 5 female animals were terminated 24 or 48 hours after dosing. Positive control animals were terminated 24 hours after dosing.
High dose treatment group: 5 male and 5 female animals were terminated 24 hours after dosing, the remaining animals were terminated after 48 hours.

Remarks:

Doses / Concentrations:
0, 300 / 400, 600 / 800 and 1200 / 1600 mg/kg (males / females )
Basis:
actual ingested
male / female achieved dose levels

No. of animals per sex per dose:

5 males and 5 females in low and intermediate dose groups (300/400 mg/kg bw and 600/800 mg/kg bw for males/females); 10 mice per sex in the control group and 15 males and 15 females in the high dose group (1200 or 1600 mg/kg bw for males and females respectively).

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide in Milli-Q water as solvent.
- Justification for choice of positive control(s): No data.
- Route of administration: Oral intubation.
- Doses / concentrations: 40mg/kg.

Tissues and cell types examined:

Micronucleated polychromatic erythrocytes in mouse bone marrow and normochromatic erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on a rangefinding study that identified clinical signs of toxicity.
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): treatment was a one time oral
intubation following an eight day acclimation period. Clinical signs of toxicity- before dosing, 2hr post dosing, and daily
thereafter for two days. Body weights recorded prior to dosing and sacrifice. preparation of bone marrow smears made at
24 hr and 48 hr.

Immediately after sacrifice, marrow from both femurs of each animal was aspirated into a syringe of foetal bovine serum (FBS) then transferred to a test tube containing FBS. Marrow samples were collected by centrifugation. The supernatant was removed before re-suspending the cell pellet in the remaining 1-2 drops of FBS. A small drop was spread onto an appropriately labelled slide. Three slides per animal were prepared and fixed in absolute methanol. The slides were then stained in acridine orange in phosphate buffer for 3 minutes.

METHOD OF ANALYSIS: using incident light fluorescence microscopy. Only cells with good morphology and staining
were counted. Color used to distinguish PCEs (reddish orange) from NCEs (dark green). PCEs (2000/animal) were
evaluated for the presence of micronuclei. Unit of scoring was the micronucleated cell; therefore, PCEs with more than 1
micronucleus wre scored as a single MNPCE.

Evaluation criteria:

Bone marrow smears were examined using incident light fluorescence microscopy. Only cells with good morphology and staining were counted. Colour was used to distinguish PCEs (reddish orange) from NCEs (dark green). PCEs (2000/animal) were evaluated for the presence of micronuclei (round, bright yellow fluorescing bodies). The unit of scoring was the micronucleated cell; therefore, PCEs with more than 1 micronucleus were scored as a single MNPCE. Micronucleated NCEs were counted in each optic field while scoring the 2000 PCEs. Additionally, the number of PCEs among 1000 erythrocytes was recorded for each animal.

Statistics:

For a test substance to be judged negative, no statistically significant increases in MNPCEs above the concurrent vehicle control values can occur at any dose level or sampling time.
In order for a test substance to be judged positive, statistically significant increases in MNPCEs above concurrent vehicle control values must be observed at more than one sampling time, or in both sexes.

Data for the proportion of MNPCEs among 2000 PCEs and the proportion of PCEs among 1000 erythrocytes (MNPCE and
PCE frequency, respectively)were transformed prior to analysis using the arcsine square root function. This transformation
is appropriate for proportions since the distributionof the transformed data more closely approximates a normal distribution
than does the non-transformed proportion. Transformed data for PCE andMNPCE frequencies were analyzed separately
for normality of distribution and/or equality of variance using the Shapiro-Wilk and Bartlett's or variance ratio tests,
respectively. Results indicated that the transformed values for PCE frequency were not normally distributed. Therefore,
nonparametric statistics were performed using nontransformed data (Kruskal-Wallis, Dunn's and Mann-Whitney U tests).
The transformed values for MNPCE frequency were normally distributed and had equal variance. Therefore, parametric
statistics were performed using transformed data ( ANOVA and Dunnett's test). Weight change data were assumed to be
normally distributed and were analyzed by ANOVA. Data from each sex and sacrifice time were analyzed separately, and
individual group comparisons to the controls were made. The animal was considered the experimenta unit. All analyses
were conducted at a significance level of 5%. Positive indicator data were analyzed separately

Sex:

male/female

Genotoxicity:

negative

Remarks:

no statistically significant increases in micronucleated PCEs

Toxicity:

yes

Remarks:

systemic toxicity (lethargy, moribundity, abnormal gut) in highest dose groups (5/15 males and 2/13 females), and 1 female and 1 male died, no significant decreases in body weight

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range:382-3147 mg/kg.
- Solubility: No data.
- Clinical signs of toxicity in test animals included abnormal gait, lethargy, ruffled fur, gasping, moribundity, pallor and/or
vocalization in most mice receiving 1588mg/kg or higher.
- Evidence of cytotoxicity in tissue analyzed: No data.
- High dose with and without activation: No activation used. 3147mg/kg.
- Other: 3 mice died following a 3147mg/kg dose, 3 were dead after a dose of 2353mg/kg, and one was dead after a dose
of 1588mg/kg.
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No statistically significant increases.
- Ratio of PCE/NCE (for Micronucleus assay): 0.78-1.45 for female and 0.86-1.24 for males.
- Appropriateness of dose levels and route: Based on rangefinding study in male and female mice that looked at clinical
toxicity. Picked doses in which clinical toxicity would not be expected.

In the high dose group, clinical signs of reaction to treatment were apparent within 2 hours of dosing.  Signs of lethargy, moribundity and/or abnormal gait were recorded for several males and females.  Signs of lethargy and moribundity persisted for up to 48 hours after dosing.  On the day following dosing 4 males and 2 females in the high dose group were found dead and a further one male and one female died on the following day.  Mortalities were considered to be treatment-related.

 

There were no effects on bodyweight gains for any of the surviving mice dosed with glycolic acid.  No clinical signs were noted for the positive control group. There were no statistically significant increases in MNPCE frequency in either male or female mice treated with glycolic acid.

Conclusions:

Glycolic acid was non-mutagenic in the micronucleus test in vivo. The study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).

Executive summary:

Glycolic acid was non-mutagenic in the micronucleus test in vivo. The GA 70% solution was found negative in the mouse micronucleus assay, showing no evidence of test compound induced clastogenicity or aneugenicity. The study was dosed up to the maximum feasible dose level (1600 mg/kg bodyweight) considering test substance related adverse toxic signs and mortality. The negative results are accompanied by an observable albeit not statistically significant depression in the proportion of PCEs/total erythrocytes in both male and male animals at 48 hrs, indicative of some marrow toxicity and thus showing that the test compound reach the target cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Based on the information available from existing studies, glycolic acid does not appear to have any mutagenic potential in vitro at concentrations relevant for human risk assessment.

  

There were no clearly positive responses in the in vitro battery of tests. The in vitro gene mutation study (Ames test) gave a negative response with and without metabolic activation. At concentrations varying from one to 10000 µg/plate, glycolic acid was not mutagenic, with or without metabolic activation, in Salmonella typhimurium strains TA97a, TA98, TA100, TA1535, TA1537, and TA1538 or in Escherichia coli strain WP2uvrA (pKM101). The in vitro cytogenicity test (chromosomal aberration test) was negative with and without metabolic activation. The gene mutation assay in mammalian cells (TK locus in L5178Y mouse lymphoma cells), gave a negative response without S9. However, in the metabolically activated, replicate tests, a positive response was obtained but only at very high concentrations (3-7 fold the EPA limit test threshold of 10 mM). At the limit concentration there were no indications of mutagenic potential. At levels relevant for human risk assessment glycolic acid is not considered a positive mutagen.

  

From the weight of evidence from the standard in vitro battery of tests completed for glycolic acid, the test substance should not be considered a potential mutagenic material. Although additional in vivo testing is not required given the negative in vitro results, a mouse micronucleus study was completed and results were available and therefore are resented in the dossier. The in vivo study gave a negative response confirming the previous results.

Short description of key information:
A standard battery of in vitro genotoxicity tests were completed giving negative responses. The absence of mutagenicity potential was confirmed by a negative in vivo micronucleus assay. Glycolic acid (GA) was found to be negative in the Ames assay, did not induce in vitro structural or numerical chromosome aberrations in Chinese hamster ovary cells, or mutations in the mouse lymphoma forward mutation assay. GA was also negative in the mouse micronucleus assay. The GA 70% solution was found negative in the mouse micronucleus assay, showing no evidence of test compound-induced clastogenicity or aneugenicity. The study was dosed up to the maximum feasible dose level (1600 mg/kg bodyweight) considering test substance related adverse toxic signs and mortality. The negative results are accompanied by an observable albeit not statistically significant depression in the proportion of PCEs/total erythrocytes in both male and male animals at 48 hrs, indicative of some marrow toxicity and thus showing that the test compound reached the target cells.

Justification for classification or non-classification

Glycolic acid shows no evidence of genotoxic or mutagenic potential in a battery of in vitro tests, substantiated by a mouse micronucleus test in vivo. According to Regulation (EC) No 1272/2008, glycolic acid does not warrant any classification for mutagenicity.