Glycollic acid

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: EPA health Effect Guidelines 40 CFR 799.9539

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Glycollic acid

EC Number:

201-180-5

EC Name:

Glycollic acid

Cas Number:

79-14-1

Molecular formula:

C2H4O3

IUPAC Name:

2-hydroxyacetic acid

Details on test material:

Glycolic acid 70% solution,

Test animals

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

Administration / exposure

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.

Doses / concentrations

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.

Examinations

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

Results and discussion

Test results

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.