Glycollic acid

Endpoint summary

Description of key information

A key repeated dose study is available for glycolic acid. In this 90-day oral toxicity gavage study in rats in which effects were observed at the dose levels of 300 or 600 mg/kg bw/day. These included lower mean body weight, overall body weight gain, food consumption and food efficiency. No adverse clinical signs indicative of systemic toxicity and no test substance-related ophthalmological findings were observed. Toxicologically significant increased neutrophil levels in male rats dosed with 300 mg/kg per day or 600 mg/kg per day and increased urea nitrogen, phosphorus, and creatine and decreased urine concentration in male rats dosed with 300 mg/kg per day or 600 mg/kg per day were observed. Mean kidney weight of male rats in the 300 mg/kg per day and 600 mg/kg per day were significantly higher than that of the control group. Gross findings of renal pelvis dilation were observed and correlated with microscopic findings of oxalate crystal nephrosis and unilateral hydronephrosis in males dosed with 300 or 600 mg/kgper day. The effects were not evident at the 150 mg/kg bw/day level. The NOEL for this study is based on 100% glycolic acid dosed (adjusted for 70% purity of the test substance).

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Guideline compliant Commission Directive 87/302/EEC, U.S. EPA Pesticide Assessment Guidlines Subdivisoin F, 82-1 ( 1982), OECD Test Guideline 407 , Maff Japan NohSan No. 4200 ( 1985). This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Qualifier:

according to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

Deviations:

yes

Remarks:

Ophthalmological findings and gross pathological lesion incidences were not evaluated by statistics (EPA FIFRA).

Qualifier:

according to guideline

Guideline:

other: Commission Directive 87/302/EEC

Qualifier:

according to guideline

Guideline:

other: MAFF Japan NohSan 40 4200

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD (SD) IGS.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: 48 days. Birthdated 18 August 1998. Rats were circa 5 weeks old at time of receipt.
- Group mean bodyweight range at time of allocation to study was 229.5 to 232.6g for males and 163.7 to 165.0g for females.
- Housing: With the exception of some portions of the reproductive study, all rats were housed one per cage, sexes separate, in stainless steel, wire-mesh cages suspended above cage boards.
- Diet (e.g. ad libitum): All rats were fed PMI Nutrition International, Inc. Certified Rodent Checkers LabDiet@ 5002 ad libitum.
- Water (e.g. ad libitum): Tap water was provided ad libitum.
- Acclimation period: Upon arrival at Haskell Laboratory, the rats were quarantined for six days of the 13-day pretest period.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23" +/- 1°C.
- Humidity (%): 50% +/- 10%.
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark, with fluorescent light.
IN-LIFE DATES: From: October 5, 1998 To: March 1, 1999

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Dose solutions were prepared at 15, 30 and 60 mg/mL on a daily basis and administered on same day by gavage in a dose volume of 10 mL/kg bw to achieve dose levels of 150, 300, and 600 mg/kg per day, based on the most recently recorded weight. For pregnant rats, from gestation day 18 until delivery, dose volumes were based on the gestation day 18 body weights. Dosing solutions were stored refrigerated until used. Dosing solutions stored beyond 14 days after preparation were not administered to animals. Control animals were dosed with commercially-supplied water only.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HPLC: Hewlett-Packard 1090; UV 210 nm; column: Zorbax® SB-C18, 4.6 mm x 150 mm; 40°C mobile phase: 2.0% acetonitrile/98.0% 3.1 mM H3P04; 1.0ml/min., Injection volume: 4.0 µl
From each dosing solution sample, 1 mL was aliquoted and diluted to 10 mL with high-performance liquid chromatography (HPLC) grade water, then mixed. The 0 mg/mL and 15 mg/mL solutions were analyzed without further dilution. The 30 mg/mL and 60 mg/mL samples were further diluted with HPLC grade water to an expected concentration of 1.5 mg/mL active ingredient (a.i.) prior to analysis. Samples submitted for analysis were analyzed the day the solutions were prepared by the testing group.

Duration of treatment / exposure:

90 days with continuation of treatment through to test day 131 (males) or day 21 of lactation (females) for animals in reproduction phase.

Frequency of treatment:

daily administration

Remarks:

Doses / Concentrations:
150, 300 or 600 mg/kg bw ; 15, 30 and 60 mg/mL administered in 10 mL/kg bw volume of water

No. of animals per sex per dose:

40 per sex per dose level. Each dosage group was divided into subchronic toxicity, immunotoxicity, neurotoxicity, and reproductive toxicity subsets (10 per sex per subset per concentration).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels for this study were selected based on results from a developmental toxicity study in which glycolic acid was administered by gavage to Crl:CD%R female rats (25 per group) on days 7-21 of gestation at daily dose levels of 0, 75, 150, 300, or 600 mg/kg per day.
- Rationale for animal assignment (if not random): Rats were selected for study use on the bases of adequate body weight gain, freedom from any
clinical signs of disease or injury, and a body weight within 20% of the mean within a sex. In error, two rats with ophthalmological abnormalities were assigned to study groups in the reproduction subset (see Examinations below.) The selected rats were distributed by computerized, stratified randomization so that there were no statistically significant differences among group body weight means within a sex. See study design table in "other information section".

Positive control:

A positive control study involved collecting sera from animals previously injected with SRBC and the immunosuppressive agent, Cyclophosphamide. Serum was analysed for SRBC-specific IgM antibody.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: Daily.
DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: Cage-site examinations to detect moribund or dead rats and abnormal behavior and appearance among rats were conducted at least once daily throughout the study. Moribund and dead rats were submitted for a gross and microscopic examination. At every weighing, each rat was individually handled and examined for abnormal behavior and appearance. Rats designated for neurotoxicity evaluations had cage-site examinations approximately one to two hours after dosing on test day 1, and approximately one to two hours after dosing on one day during the weeks that the functional observational battery was conducted.

BODY WEIGHT: Yes.
- Time schedule for examinations: All rats were weighed once per week during the 90-day feeding phase of the study. In addition, the neurotoxicity sub study rats were weighed on the days of neurotoxicity evaluation. During the reproduction sub-study, male rats were weighed on a weekly schedule and female rats were weighed during gestation on days 0, 7, 14, 18, and 21 and weighed during lactation on days 0, 7, 14 and 21. Female rats without a known start of gestation and female rats that copulated but did not deliver a litter continued to be weighed weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)/ FOOD EFFICIENCY
The amount of food consumed by each rat over each weighing interval was determined throughout the study. Food consumption was determined for each female rat designated for reproductive assessment on gestation days 0, 7, 14 and 21. From these determinations and body weight data, mean daily food consumption and mean food efficiency were calculated.

OPHTHALMOSCOPIC EXAMINATION: Yes.
- Time schedule for examinations: Two ophthalmological examinations were conducted by a veterinary ophthalmologist. Both eyes were examined by focal illumination and indirect ophthalmoscopy. The examinations were conducted under subdued lighting after mydriasis had been produced with a 1% tropicamide solution. On test day 9, the initial examination was performed on all rats received for the study, prior to selection and grouping. In error, two rats with ophthalmological abnormalities were assigned to study groups in the Reproduction subset. Since animals in the reproductive toxicity subset did not undergo ophthalmological examination during the study, the pre-existing abnormalities had no impact on the study. All surviving rats designated for subchronic toxicity were examined on test day 86 prior to the scheduled sacrifice.

HAEMATOLOGY: Yes. Blood cell counts, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration were determined on a Serono Baker 9000@ hematology analyzer. Differential cell counts were determined on a Hematrak@Automated Differential System cell counter. Absolute values for the various types of leukocytes were calculated from the leukocytic data.
- Time schedule for collection of blood: Test days 46 and 93 for male rats and test days 47 and 94 for female rats.
- Anaesthetic used for blood collection: Carbon dioxide.
- Animals fasted: Yes, for 16 hours.
- How many animals: 10 per sex per dose.
- Parameters examined were: Red blood cell count (RBC), neutrophil count (Neut), white blood cell count (WBC), band neutrophil count (Band), platelet count (PLT), lymphocyte count (Lymph), hemoglobin concentration (HGB), atypical lymphocyte count (Alym), hematocrit (HCT), monocyte count (Mono), mean corpuscular volume (MCV), eosinophil count (Eosin), mean corpuscular hemoglobin (MCH), basophil count (Baso) and mean corpuscular hemoglobin concentration
(MCHC).
CLINICAL CHEMISTRY: Yes.
Clinical chemical parameters were measured on a Boehringer Mannheim Mtachi 717 clinical
chemistry analyzer using Boehringer Mannheim reagents.
- Time schedule for collection of blood: Test days 46 and 93 for male rats and test days 47 and 94 for female rats.
- Animals fasted: Yes, for 16 hours.
- How many animals: 10 per sex per dose.
- Parameters examined were: Alkaline phosphatase activity (ALP), alanine aminotransferase activity (ALT), aspartate aminotransferase activity (AST), sorbitol dehydrogenase activity (SDH), bilirubin concentration (BILRN), cholesterol concentration (CHOL), total protein concentration (TPROT), albumin concentration (ALBMN), globulin concentration (GLOBN), glucose concentration (GLUCO), urea nitrogen concentration (BUN), creatinine concentration (CREAT),
phosphate concentration (PHOS), calcium concentration (CALC), sodium concentration (Na), potassium concentration (K) and chloride concentration (Cl).

URINALYSIS: Yes.
Osmolality was determined on a Precision Systems model Multi-OsmetteTM2 430 osometer. Urine biochemical constituents were measured on a ClinitekTM2 00 urine chemistry analyzer using Ames MultistixTMurine chemistry dipsticks. Urine appearance (color and transparency) was recorded and the sediment from each specimen was microscopically examined.
- Time schedule for collection of urine: One day prior to each bleeding time, an overnight (approximately 16 hour) urine specimen was collected from each rat to determine:
- Metabolism cages used for collection of urine: Yes.
- Animals fasted: Yes, 16 hrs.
- Parameters examined were volume (VOL), glucose, osmolality (OSMOL), urobilinogen (UROBL), PH, ketone (acetoacetic acid), hemoglobin or blood (BLOOD), protein and bilirubin.

NEUROBEHAVIOURAL EXAMINATION: Yes.
- Time schedule for examinations: Test days 3 and 4 before substance administration and test days 9 and 10, 45 and 46, and 86 and 87 before substance administration.
- Dose groups that were examined: All dose groups.
- Battery of functions tested: Functional observational battery test.

OTHER: Immunotoxicity Evaluation:
- Humoral Immune Fuction and Immune Organ Weights.
- Test day 23, 10 animals per sex per dose were injected IV with Sheep Red Blood Cells.
- Test day 29 animals sacrificed.
- Spleen and Thymus removed and weighed.
- Serum collected and analyzed for IgM antibody.

OTHER: Reproductive Assessment:
- Test day 97, females housed 1:1 ratio with random males of same dose level.
- In post-partum examination live and dead pups were counted, live pups were weighed and each pup was examined for abnormal behavior and appearance.
- Examinations took place on postpartum days 0, 4, 7, 14, and 21.

Sacrifice and pathology:

Ophthalmoscopic examinations: on day -9, surviving rats on day 86
Haematology, clinical chemistry, urinalysis: 10 per sex /group, day 46/47 and 93/94
Sacrifice and pathology: Organ Weights, Gross and histopathology, Immunotoxicology evaluations
GROSS PATHOLOGY: Yes- The liver, kidneys, adrenal glands, testes(male), ovaries(female) and brain were all weighed at sacrifice. Gross lesions were perserved.
HISTOPATHOLOGY: Yes - All collected tissues ( except nasal tissue without gross lesions) from all animals in the control and high concentration groups were processed and received a full histopathological examination.

Other examinations:

Haematology, clinical chemistry, urinalysis: 10 / sex /group; day 46/47 and 93/94
Immunotoxicology Evaluations - Using the Humoral Immune Function and Immune Organ Weights.
Neurotoxicity Evaluations - Functional Observation battery and Motor activity tests were used.
Reproductive Assessment - Breeding, gestation and lactation procedures

Statistics:

The various statistical analyses applied to each of the study phases are fully detailed in the study report and included, as appropriate, one way analysis of variance followed by Dunnett’s test or linear contrasts; Kruskal-Wallis test followed by Dunn’s test; Cochrane-Armitage trend test; Jonckhere-Terpstra trend test; linear contrast by least square means; Levene’s test for homogeneity, Shapiro-Wilk test for normality, Bartlett’s test.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

Samples of glycolic acid 70% solution, as supplied for the study, were analysed near the beginning and end of the study to determine stability over the course of study duration. The analysis results confirmed suitable long term stability. The average percent active ingredient on test day 2 was 69.5% and on test day 143 was 68.1%.

The dosing solutions were analysed on test day 1, the range was 95 to 109.3% of nominal, and the mean values for each test group were 98.7%, 98.3% and 107.5% for low, intermediate and high concentrations. Further analyses were completed for test days 37, 86, 99 and 142. Results were consistently within the acceptable range of nominal (92.0 to 102.7% were recorded values).

Male and female rats given 300 and 600 mg/kg per day had lower mean body weight, overall body weight gain, food consumption and food efficiency. No adverse clinical signs indicative of systemic toxicity and no test substance-related ophthalmological findings were observed. Toxicologically significant increased neutrophil levels in male rats dosed with 300 mg/kg per day or 600 mg/kg per day and increased urea nitrogen, phosphorus, and creatine and decreased urine concentration in male rats dosed with 300 mg/kg per day or 600 mg/kg per day were observed. Mean kidney weight of male rats in the 300 mg/kg per day and 600 mg/kg per day were significantly higher than that of the control group. Gross findings of renal pelvis dilatation were observed and correlated with microscopic findings of oxalate crystal nephrosis and unilateral hydronephrosis in males dosed with 300 or 600 mg/kg per day.

No toxicologically important changes in immunotoxicity parameters were measured. No toxicologically important changes in the behavourial parameters of neurotoxicity were measured. No compound-related gross lesions or microscopic findings observed in tissues of the nervous system or skeletal muscle examined. No toxicologically important changes in the measures of reproductive function. No compound-related gross observations were noted in the P1 females or the F1 weanlings. Compound-related gross lesions in the kidneys of male rats in the 600 mg/kg per day group similar to those found in subchronic toxicity evaluation were noted.

Dose descriptor:

NOEL

Remarks:

systemic toxicity

Effect level:

150 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

NOEL

Remarks:

systemic toxicity

Effect level:

600 mg/kg bw/day (actual dose received)

Sex:

female

Basis for effect level:

other: highest dose tested

Dose descriptor:

NOEL

Remarks:

neurotoxicity

Effect level:

600 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Neurotoxicity The FOB evaluations showed no treatment-related effects in either males or females dosed at 150, 300 or 600 mg/kg bw/day.

Dose descriptor:

NOEL

Remarks:

immunotoxicity

Effect level:

600 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: There were no treatment-related adverse effects on spleen or thymus and glycolic acid did not affect the primary humoral immune response to SRBC.

Dose descriptor:

LOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

NOAEL

Remarks:

overall

Effect level:

150 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: effect observed in males at ≥300 mg/kg

Critical effects observed:

not specified

Any changes observed in rats treated with 150 mg glycolic acid/kg per day were either considered not to be compound related or not adverse. Under the conditions of this study, the overall NOEL for both male and female rats was 150 mg/kg per day.

The NOELs for this study are based on 100% glycolic acid dosed (adjusted for 70% purity of the test substance).

Conclusions:

In the subchronic phase of the study, administration of the higher dose levels (300 and 600 mg/kg bw/day) were associated in males with reduced bodyweight gain, reduced food conversion efficiency and neutrophilia (various lesions consistent with an inflammatory response to irritation of the lungs or trachea were considered secondary to aspiration of the acidic test material and not resulting from systemic exposure to glycolic acid). The primary target organ was the kidney. Renal effects included weight changes, clinical pathology consistent with reduced glomerular filtration rate and reduced ability to concentrate urine, and microscopic evidence for oxalate crystal nephropathy for males dosed at 300 and 600 mg/kg bw/day. Similar effects were not present among the treated females.

The NOEL for male rats exposed to glycolic acid 70% solution was found to be 150 mg/kg bw/day based on renal oxalate crystal nephropathy observed at higher doses. The NOEL for females in this study was 600 mg/kg bw/day, the highest dose tested, based on the absence of primary findings in the target organ.

Neurotoxicity: The FOB evaluations showed no treatment-related effects in either males or females dosed at 150, 300 or 600 mg/kg bw/day. The NOEL for neurobehavioural effects was 600 mg/kg bw/day.

Immunotoxicity: There were no treatment-related adverse effects on spleen or thymus and glycolic acid 70% solution did not affect the primary humoral immune response to SRBC. The immune system does not appear to be a primary target for glycolic acid activity. The NOEL for immunotoxicological endpoints in male and female rats was 600 mg/kg bw/day.

Reproductive toxicity: The NOEL for reproductive toxicity was 600 mg/kg bw/day, based on the absence of treatment related effects on reproductive function. The NOEL for reproductive organ pathology in both the P1 generation and the F1 weanlings was 600 mg/kg bw/day, based on the absence of gross pathological changes.

The NOELs for this study are based on 100% glycolic acid dosed (adjusted for 70% purity of the test substance).

Executive summary:

In the subchronic phase of the study, administration of the higher dose levels (300 and 600 mg/kg bw/day) were associated in males with reduced bodyweight gain, reduced food conversion efficiency and neutrophilia (various lesions consistent with an inflammatory response to irritation of the lungs or trachea were considered secondary to aspiration of the acidic test material and not resulting from systemic exposure to glycolic acid). The primary target organ was the kidney. Renal effects included weight changes, clinical pathology consistent with reduced glomerular filtration rate and reduced ability to concentrate urine, and microscopic evidence for oxalate crystal nephropathy for males dosed at 300 and 600 mg/kg bw/day. Similar effects were not present among the treated females.

 

The NOEL for male rats exposed to glycolic acid 70% solution was found to be 150 mg/kg bw/day based on renal oxalate crystal nephropathy observed at higher doses. The NOEL for females in this study was 600 mg/kg bw/day, the highest dose tested, based on the absence of primary findings in the target organ.

 

Neurotoxicity:

The FOB evaluations showed no treatment-related effects in either males or females dosed at 150, 300 or 600 mg/kg bw/day. The NOEL for neurobehavioural effects was 600 mg/kg bw/day

 

Immunotoxicity:

There were no treatment-related adverse effects on spleen or thymus and glycolic acid 70% solution did not affect the primary humoral immune response to SRBC. The immune system does not appear to be a primary target for glycolic acid activity. The NOEL for immunotoxicological endpoints in male and female rats was 600 mg/kg bw/day.

 

Reproductive toxicity:

The NOEL for reproductive toxicity was 600 mg/kg bw/day, based on the absence of treatment related effects on reproductive function. The NOEL for reproductive organ pathology in both the P1 generation and the F1 weanlings was 600 mg/kg bw/day, based on the absence of gross pathological changes. 

The NOELs for this study are based on 100% glycolic acid dosed (adjusted for 70% purity of the test substance).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Organ:

kidney

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Principles of method if other than guideline:

Groups of 10 rats were exposed to 0, 0.23, 0.72, and 2.0 mg/L in air for 6 hrs/day, 5 days/week for 2 weeks. At the end of the exposure period and after a 14-day recovery period, blood and urine samples were collected for clinical analysis and rats were sacrificed for pathological examination.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: Crl:CD®

Sex:

male

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Exposure was for 6 hours per day, 5 days per week for 2 weeks. Due to deteriorating condition in the high dose group, these rats were only dosed on eight occasions.

Frequency of treatment:

five days/week for two weeks followed by a 14 day observation period.

Remarks:

Doses / Concentrations:
0.20, 0.70 and 2.0 mg/L
Basis:
nominal conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Dose descriptor:

NOAEL

Effect level:

0.23 mg/L air (nominal)

Sex:

male

Critical effects observed:

not specified

Mortality: Exposure of the 2.0 mg/L treatment was stopped after the 8th exposure. Seven of the rats at this treatment were sacrificed in extremis: one after the 8th exposure, four the following day, and one each on the 11th and 12th days after the eighth exposure. A single rat died in the 0.72 mg/L treatment 13 days after the 10th exposure.

Clinical signs: No clinical signs or weight gain differences were seen between the control and 0.23 mg/L animals. Rats in the 0.72 and 2.0 mg/L treatments experienced significant and severe weight loss throughout the exposure and recovery periods. Symptoms of toxicity included laboured breathing, lung noise, ruffled and discoloured fur, red and clear nasal and ocular discharges.

Clinical chemistry: Measurements made at the end of the exposure period showed no compound-related changes in rats exposed to 0.23 mg/L. Changes were noted in the 0.72 and 2.0 mg/L treatments. Rats exposed to 2.0 mg/L had decreased concentrations of serum protein, increased serum activities of glutamic-pyruvic transaminase and glutamic-oxaloacetic transaminase, and decreases in urine volume and pH. These changes were interpreted as evidence of compound related effects on the integrity and function of hepatic tissue. However the effects were all reversible following the 14 day recovery period.

Gross necropsy: Gross pathological examination following the exposure period revealed no compound-related changes in rats exposed to 0.23 and 0.72 mg/L. Rats exposed to 2.0 mg/L showed distended G. I. tract, small spleen and small thymus. Similar effects were seen in rats at all test levels 14 days after exposure. Microscopic examination showed mild, diffuse hepatocellular degeneration in one rat in the 0.23 mg/L treatment, 9 rats in the 0.72 and 7 rats at 2.0 mg/L. A comparison of organ and body weights between control and rats at 0.243 mg/L showed no changes. Effects were seen in rats exposed to 0.72 and 2.0 mg/L. Fourteen days later, rats exposed to 0.72 mg/L had significantly lower lung, kidney, liver and thymus weights than control rats. Rats exposed to 2.0 mg/L had significantly lower kidney weights. On an organ/body weight basis, rats exposed to 0.72 mg/L had elevated testes weights.

Conclusions:

Based on parameters evaluated in this study, the 0.72 and 2.0 mg/l treatments are definite and dose-dependent effect levels with the primary effects on survival and on hepatic integrity. The only effect seen at 0.23 mg/L was very mild diffuse hepatocellular degeneration in one of ten rats, 14 days post exposure. Based on the steepness of the dose-response curve (disappearance of mortality, weight loss and clinical signs with concentration) this level is practically a no-effect level. NOAEL = 0.23 mg/L for repeated administration by inhalation exposure.

Executive summary:

Groups of 10 rats were exposed to 0, 0.23, 0.72, and 2.0 mg/L in air for 6 hrs/day, 5 days/week for 2 weeks. At the end of the exposure period and after a 14-day recovery period, blook and urine samples were collected for clinical analysis and rats were sacrificed for patological examination. Based on parameters evaluated in this study, the 0.72 and 2.0 mg/l treatments are definite and dose-dependent effect levels with the primary effects on survival and on hepatic integrity. The only effect seen at 0.23 mg/L was very mild diffuse hepatocellular degeneration in one of ten rats, 14 days post exposure. Based on the steepness of the dose-response curve (disappearance of mortality, weight loss and clinical signs with concentration) this level is practically a no-effect level. NOAEL = 0.23 mg/L for repeated administration by inhalation exposure.

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Article submitted in 2019.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Aerosol inhalation.
- Short description of test conditions: Aerosols were generated by using an atomizer with a clean air flow of 250 L/min. Rats were exposed to several glycolic acid concentrations for 28 days. In each concentration group, half of the rats were sacrificed 1 day following exposure; the recovery of the remaining rats was monitored for 7 days following exposure and they were subsequently euthanized.
- Parameters analysed / observed: Hematology, Serum biochemistry, Analysis of bronchoalveolar lavage fluid (BALF), Histopathological analysis.

GLP compliance:

not specified

Remarks:

GLP compliance was not reported in the publication

Limit test:

no

Specific details on test material used for the study:

Glycolic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Orient Bio Inc. (Sungnam, South Korea).
- Age at study initiation: 6-week old
- Fasting period before study: No.
- Diet (e.g. ad libitum): Laboratory diet (LabDiet 5053, Orient Bio Inc., South Korea), ad libitum.
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: 1 week. Following the acclimatization period the rats were randomly divided into four groups (20 rats/group).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3ºC.
- Humidity (%): 50 +/- 20%.
- Photoperiod (hrs dark / hrs light): 12:12 hour light/dark cycle.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

not specified

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 0.87 - <= 1.82 µm

Geometric standard deviation (GSD):

2.57

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- System of generating particulates/aerosols: atomizer.
- Temperature, humidity, pressure in air chamber: Temperature, humidity, air flow, and air pressure were monitored during exposure by using a Model VT3-X15 environmental controller (Sibata Scientific Technology Ltd., Saitama, Japan).
- Air flow rate: 250 L/min.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of glycolic acid was monitored by sampling the chamber air using a SIP-32L (SIBATA) and comparing the weight of the membranes (T60A20, Φ55).
The mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD) of glycolic acid in the exposure chamber were measured using an Andersen cascade impactor (AN-200 Sibata Science Technology, Japan).

Duration of treatment / exposure:

28 days

Frequency of treatment:

6 hours/day, 5 days/week.

Dose / conc.:

50 mg/m³ air (nominal)

Remarks:

Actual dose 49.5 +/- 8.5 mg/m3

Dose / conc.:

10 mg/m³ air (nominal)

Remarks:

Actual dose 9.9 +/- 1.79 mg/m3

Dose / conc.:

2 mg/m³ air (nominal)

Remarks:

Actual dose 2.35 +/- 0.46 mg/m3

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

In each group, half of the rats were sacrificed 1 day following exposure; the recovery of the remaining rats was monitored for 7 days following exposure and they were subsequently euthanized.

Positive control:

N/A

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No.

DETAILED CLINICAL OBSERVATIONS: No.

BODY WEIGHT: Yes.

OPHTHALMOSCOPIC EXAMINATION: No.

HAEMATOLOGY: Yes.
- Time schedule for collection of blood: N/A.
- Blood samples were collected through the abdominal aorta, and were transferred to tubes containing trisodium EDTA as anticoagulant. Hematological analyses were performed using an automatic hematological analyzer (LC-662G, Kyoto, Japan).
- Anaesthetic used for blood collection: Yes (identity not specified).
- Animals fasted: Not specified.
- How many animals: N/A.
- Parameters checked: i) white blood cells (WBCs), ii) red blood cells (RBCs), iii) hemoglobin (Hgb), hematocrit (Hct), iv) mean corpuscular volume (MCV), v) mean corpuscular hemoglobin (MCH), vi) mean corpuscular hemoglobin concentration (MCHC), vii) red blood cell volume distribution width (RDW), viii) platelets (PLT), ix) mean platelet volume (MPV), x) plateletcrit (PCT), and xi) plate volume distribution width (PDW).

CLINICAL CHEMISTRY: Yes.
- Time schedule for collection of blood: N/A.
- Blood samples were centrifuged at 2000 rpm for 10 minutes. An autoanalyzer (FUJI DRI-CHEM 4000ie, Tokyo, Japan) was used.
- Animals fasted: Not specified
- How many animals: N/A
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes / No / Not specified
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked: i) triglyceride (TG), ii) total cholesterol (T-CHO), iii) glutamic-oxaloacetic
transaminase (GOT), iv) lactate dehydrogenase (LDH), v) glucose (GLU), vi) alkaline phosphatase (ALP), vii) glutamic-pyruvic transaminase (GPT), viii) total bilirubin (T-BIL), ix) albumin (ALB), x) creatinine (CRE), and xi) blood urea nitrogen (BUN).

NEUROBEHAVIOURAL EXAMINATION: No.

IMMUNOLOGY: No.

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes.
- Time schedule for analysis: N/A.
- Dose groups that were examined: All doses.
- Number of animals: 5 animals/group.
- The rat lungs were lavaged three times with 4 mL calcium and magnesium-free phosphate-buffered saline (PBS, pH 7.4). The BALF was centrifuged at 1,500 rpm for 10 minutes in a Hanil Union 32R centrifuge (Incheon, Korea) to separate the supernatant from the cells. The supernatants were stored at –80˚C prior to the assay. The cell pellets were counted by using a Vi-Cell® XR analyzer (Beckman Coulter, Brea, CA, USA) and diluted to 2×105 cells/mL; 50 μL was
placed on a slide by using Shandon Cytospin (Shandon, Pittsburgh, PA, USA) and the slides were stained with Diff-Quik (International Reagents, Kobe, Japan).
- Parameters checked: The supernatant was used for the analysis of total protein, LDH, and inflammatory cytokine levels. The total protein (TP) and LDH levels were determined using a bicinchoninic acid (BCA) protein assay kit (Intron Biotechnology, Korea) and an EZ-LDH Cell Cytotoxicity Assay Kit (Daeil Lab Service, Korea), respectively. To evaluate the inflammatory cytokine levels, ELISA kits for tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1) were purchased from R&D systems (Minneapolis, MN, USA), and for macrophage inflammatory protein-2 (MIP-2) from
Invitrogen (Carlsbad, CA, USA).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, lung and nasal cavity.

HISTOPATHOLOGY: Yes. Following the inhalation of glycolic acid for 4 weeks, the animals were euthanized. After gross examination, the lung and nasal cavity were fixed in 10% neutral buffered formalin. The nasal cavity was decalcified with a decalcification solution and then cross-sectioned into four section-levels: i) level 1 (posterior part of the upper incisors), ii) level 2 (incisive papilla), iii) level 3 (second palatine crest) and iv) level 4 (first upper molar teeth). Following the routine tissue processing, the tissues were embedded in paraffin and 3 μm slices were cut. The sections were stained with hematoxylin and eosin (H&E) for histological examination under a light microscope (Olympus BX41, Tokyo, Japan). The lesions were graded,
depending on severity by pathologists.

Statistics:

Statistical analyses were computed by using GraphPad Prism version 5.01 (GraphPad Software Inc., USA). The values were expressed as the mean ± standard error (SE). A one-way multiple variance of analysis (ANOVA) test, followed by Student’s t-test was used to compare the exposure groups with the control group and to observe whether the p-Value was less than 0.05, or 0.01.

Clinical signs:

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no significant changes in the lung, liver and kidney weights of rats in the exposed groups.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No effects related to glycolic acid were observed in the nasal cavity and the lung. Microcysts of the respiratory epithelium in level 1 sections were found in the low exposure and high-recovery groups, but also in the control group, thus they were not considered to be the result of
glycolic acid treatment. Alveolar macrophage aggregation and the perivascular infiltration of mononuclear cells were often observed in the lungs of rats in the exposure and recovery groups. However, it was minimal in severity and also occurred in the control group. Vascular
mineralization found in the lungs is a finding that is often observed in normal rats.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The LDH levels in BALF followed an increasing trend in the exposure groups (autopsied 1 day after the final exposure) and the recovery groups (autopsied 7 days after the final exposure), but the changes were not statistically significant.
The TP and PMNs significantly increased in the high-exposure group and returned to normal
during the recovery period.
No significant difference among groups was found for IL-6 and MCP-1 in BALF in the exposure and recovery groups, but slight increases in IL-1β, TNF-α, and MIP-2 were detected in medium-exposure, high-recovery, and low- and high recovery groups, respectively.

Key result

Dose descriptor:

NOAEC

Effect level:

> 50 mg/m³ air

Sex:

male

Basis for effect level:

clinical biochemistry

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

serum/plasma biochemistry

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Conclusions:

There were no deaths in any of the glycolic acid-treated groups; moreover, no changes occurred in body weight, organ weight, hematology, and serum biochemistry. The results of histopathologic analysis of the nasal cavities and lungs of the rats also showed no toxic effects of glycolic acid, although microcysts in the nasal cavity, alveolar macrophage aggregation, perivascular cell infiltration, pulmonary fibrosis, and vascular mineralization were often found, irrespective of the exposure to glycolic acid.

The increased TP and PMNs in BALF returned to normal during the recovery period. Changes in cytokines (IL-1β, MIP-2, and TNF-α) were observed in the exposure and recovery groups, but the extent of changes was considered minor and they were not significant.

The NOAEC for nasal and pulmonary toxicity of glycolic acid is over 50 mg/m3, the highest
concentration in this 28-day inhalation study.

Executive summary:

A sub-acute inhalation test was conducted to identify the potential toxicities of glycolic acid. Inhalation exposure to glycolic acid for 28 days did not cause any specific changes in clinical examinations, including body weight, organ weight, hematology, serum biochemistry, and histopathology. The polymorphonuclear neutrophils (PMNs) and inflammatory cytokines in Bronchoalveolar lavage fluid (BALF) increased in rats exposed to repeated inhalations. The changes induced by glycolic acid were minor or returned to normal during the recovery period. The No Observed Adverse Effect Concentration (NOAEC) for the nasal and pulmonary toxicity of glycolic acid was determined to be over 50 mg/m3 at the end of a 28-day inhalation test in male rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

230 mg/m³

Study duration:

subacute

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral

In the subchronic phase of a 90-day study, administration of the higher dose levels (300 and 600 mg/kg bw/day) were associated in males with reduced bodyweight gain, reduced food conversion efficiency and neutrophilia (various lesions consistent with an inflammatory response to irritation of the lungs or trachea were considered secondary to aspiration of the acidic test material and not resulting from systemic exposure to glycolic acid). The primary target organ was the kidney. Renal effects included weight changes, clinical pathology consistent with reduced glomerular filtration rate and reduced ability to concentrate urine, and microscopic evidence for oxalate crystal nephropathy for males dosed at 300 and 600 mg/kg bw/day. Similar effects were not present among the treated females. The NOEL for male rats exposed to glycolic acid 70% solution was found to be 150 mg/kg bw/day based on renal oxalate crystal nephropathy observed at higher doses. The NOEL for females in this study was 600 mg/kg bw/day, the highest dose tested, based on the absence of primary findings in the target organ. The NOEL for this study is based on 100% glycolic acid dosed (adjusted for 70% purity of the test substance).

  

Neurotoxicity:

The FOB evaluations showed no treatment-related effects in either males or females dosed at 150, 300 or 600 mg/kg bw/day. The NOEL for neurobehavioural effects was 600 mg/kg bw/day.

  

Immunotoxicity:

There were no treatment-related adverse effects on spleen or thymus and glycolic acid did not affect the primary humoral immune response to SRBC. The immune system does not appear to be a primary target for glycolic acid activity. The NOEL for immunotoxicological endpoints in male and female rats was 600 mg/kg bw/day.

  

Reproductive toxicity:

The NOEL for reproductive toxicity was 600 mg/kg bw/day, based on the absence of treatment related effects on reproductive function. The NOEL for reproductive organ pathology in both the P1 generation and the F1 weanlings was 600 mg/kg bw/day, based on the absence of gross pathological changes.

  

Dermal

A 90-day oral study in rats was conducted. As the outcome of such a study can be predicted for dermal exposure, performing a repeat administration study would contravene Directive 86/609/EC, which militates against unnecessary testing using animals. 

 

Inhalation

Two studies were identified using the inhalation route of exposure, none of them being described in sufficient details to be used as key study to assess repeated dose toxicity via inhalation.

In the first one, a 14-day repeated exposure study via inhalation, Crl:CD rats were exposed to 230, 720 and 2000 mg/m³ air for 6 hrs/day, 5 days/week. Definite and dose-dependent effect levels were seen at 720 and 2000 mg/m³ with the primary effects on survival and on hepatic integrity. The only effect seen at 230 mg/m³ was very mild diffuse hepatocellular degeneration in one of ten rats, 14 days post exposure. Based on the steepness of the dose-response curve (disappearance of mortality, weight loss and clinical signs with concentration) this level is practically a no-effect level. NOAEL = 230 mg/m³ for repeated administration by inhalation exposure.

In the second study, a 28-day repeated dose study via inhalation, Sprague-Dawley rats were exposed to 2 mg/m³, 10 mg/m³ and 50 mg/m³ air (nominal doses). There were no deaths with no changes in body weight, organ weight, haematology and serum biochemistry. The results of the histopathological analysis of the nasal cavities and lungs of the rats indicated no toxic effects of dosing. It was noted the presence of microcysts in the nasal cavity, alveolar macrophage aggregation, perivascular cell infiltration, pulmonary fibrosis and vascular mineralisation were found irrespective of exposure to the test substance. Increased levels of TP and PMNs in BALF were noted as returning to normal during the recovery period. Changes in cytokines were observed in both the exposure and recovery groups but the extend of the changes were considered minor and not significant. The NOAEC for nasal and pulmonary toxicity of glycolic acid is over 50 mg/m³ (highest concentration tested).

Justification for classification or non-classification

No serious damage was observed in the animals such as functional disturbances or morphological change as a result of prolonged exposure. On this basis, according to Regulation (EC) 1272/2008, glycolic acid does not warrant any classification as "Danger for serious damage to health by prolonged exposure" or "specific target organ toxicity - repeated exposure".