Sodium chloroacetate

Carcinogenicity

Description of key information

Key information is based on read-across from monochloroacetic acid. Toxicity of SMCA (the sodium salt of MCA) is identical to that of MCA. Under physiological conditions both substances are completely ionised and the relevant moiety is the monochloroacetate ion. Many studies have used monochloroacetic acid neutralized with sodium hydroxide or its sodium salt, so that data of monochloroacetate can be used to also evaluate the genotoxicity for sodium monochloroacetic acid (and vice versa). In a dermal toxicity study (see section 7.2.3) with rabbit skin it was shown, in addition, that the toxicity of MCA is not caused by a surplus of H+ ions and that the influence of pH appeared to be secondary, because its direct reaction to biomolecules was the primary cause of cytotoxicity.

Four studies of reliability 2 are available. NTP studies in rats and mice were performed; in rats the NOAEL was 15 mg/kg bw in males, and <15 mg/kg bw in females due to a non-dose related decrease in survival. In mice the NOAEL was < 50 mg/kg bw die to local effects in the forestomach. The third study (DeAngelo, 1997) aimed specifically at liver carcinogenicit; the NOAEL was 3.5 mg/kg bw based on systemic effects (growth depression and reduced water intake) at the next higher levels tested. A 2 -year dermal carcinogenicity study in mice with a single, high dose did not show any increase in skin papilomas. No evidence of carcinogenicity was found.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study focussed at the detection of liver carcinogenicity/toxicity, and did not include haematology and clinical chemistry (except for plasma ASAT and ALAT). Only males were included. Due to scheduled interim sacrifices (and unscheduled deaths) only 23-25 animals per group were left after 104 weeks.

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study determined the potential of MCA administered in the drinking water to induce neoplasia in the male F344/N rat using a modification of the standard carcinogenesis bioassay methodology. Groups of animals were sacrificed at 15, 30, 45 or 60 weeks (18-21 in total per group) or after 104 weeks (remaining animals). At sacrifice gross lesions were examined, and organs were fixed in formalin for histopathology.

GLP compliance:

not specified

Species:

rat

Strain:

other: F344/N

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Portage, MI)
Rats were confirmed free of viral antibodies, bacteria and parasitic infections
- Age at study initiation: 28-30 days
- Weight at study initiation: 56-59 grams
- Fasting period before study:
- Housing: two per cage.
- Diet (e.g. ad libitum): ad libitum (purina rodent laboratory rodent chaw)
- Water (e.g. ad libitum): ad libitum (distilled water)
- Acclimation period: held for one week in quarantine.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 40-60
- Air changes (per hr): not indicated
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: not specified

Route of administration:

oral: drinking water

Vehicle:

water

Remarks:

Distilled water for treatment groups, and 2 g/L sodium chloride for control group

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was dissolved in distilled water to produce nominal (taget) concentrations of 0.05, 0.5 and 2.5 g/L. The pH of the solutions was adjusted to 6.9-7.1 by addition of an appropriate volume of 10N sodium hydroxide.
Administration:
The vehicle control group received 2 g/L sodium chloride, approximately isomolar to the neutralized highest dose (31 mM).
Freshly prepared solutions were administered in brown glass water bottles fitted with teflon stoppers and stainless steel, double balled sipper tubes. The drinking bottles were changed every 5-7 days.
The 2.5 g/L MCA concentration was sequentially lowered to 1.5 g/L at 8 weeks and to 1 g/L at 24 weeks due to severe inhibition of body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The drinking water solutions were sampled throughout the study to determine actual concentrations (except the 0.05 g/L solution was prepared by diluting the 0.5 g/L solution 1:10. The solutions were pipetted into 7-mL liquid scintillation mini vials, which were then tightly capped, each with a teflon-lined urea cap. The vials were stored at 5C until analysis by UV absorption at 331 nm against standard concentrations set by gas-liquid capillary chromatography.

Duration of treatment / exposure:

15, 30, 45, 60 or 104 weeks

Frequency of treatment:

Ad libitum.

Post exposure period:

No

Dose / conc.:

50 mg/L drinking water

Remarks:

corresponding to 3.5 mg/kg/day (time weighed mean daily dose)

Dose / conc.:

500 mg/L drinking water

Remarks:

corresponding to 26.1 mg/kg bw/day (time weighed mean daily dose)

Dose / conc.:

1 100 mg/L drinking water

Remarks:

corresponding to 59.9 mg/kg/day (time weighed mean daily dose)
This group was initially exposed to 2000 mg/L but was lowered in stages to 1000 mg/L (corresponding to 1100 mg/L as time-weighted mean daily dose) when the animals began to exhibit signs of toxicity

No. of animals per sex per dose:

50 males

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: not specified in detail but the authors referred to a study in which at 5 g/L hepatic peroxisome proliferation was observed with trichloroacetic acid (but not with monochloroacetic acid) in the rat (duration and exposure details not provided).
- Rationale for animal assignment (if not random): not specified
- Section schedule rationale (if not random): not specified

Positive control:

Not included.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily, including twice daily mortality/morbidity checks.

DETAILED CLINICAL OBSERVATIONS: Yes: physical examinations were conducted biweekly to detect any abnormal changes of the skin, eyes, or systemic organ systems. Aspects of these studies were conducted in compliance with the guidelines of the American Association for Accreditation of Laboratory Animal Care.

BODY WEIGHT: Yes
- Time schedule for examinations: at start of exposure, twice monthly for the first two months, and then monthly after wards.

FOOD CONSUMPTION: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: at start of exposure, twice monthly for the first two months, and then monthly after wards.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Yes, after 104 weeks
- Anaesthetic used for blood collection: No data
- Time schedule for collection of blood: at terminal sacrifice (after 104 weeks).
- Animals fasted: No data
- How many animals: all surviving animals at terminal sacrifice (i.e. 23-25 animals per group)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Not examined, but cageside observations were conducted daily for physiological and behavioral responses.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes:
- in week 15, 30, 45 and 60 (total number of animals killed: 21, 18, 18, 21 for groups 1, 2, 3 and 4 respectively): body, liver, kidneys, spleen, urinary bladder and testes
- at terminal sacrifice (week 104): complete rodent necropsy: All surfaces and orifices, the carcass, the external surface, the brain, cervical tissues, internal organs, and the cranial, thoracic, abdominal, and pelvic cavities were examined for gross lesions, including discolorations, surface irregularities, nodular changes, and masses. All gross lesions and representative tissue samples were collected from the brain, sciatic nerve, salivary gland, pancreas, pituitary, adrenals, thymus, thyroid, parathyroids, trachea, esophagus, lungs, liver, spleen, skeletal muscle, tongue, heart and aorta, stomach, duodenum, jejunum, ileum, colon, cecum, rectum, kidneys, uiinary bladder, prostate, seminal vesicles, testes, preputial gland, mammary gland, femur, nasal cavity, larynx, skin, and mesenteric and mandibular lymph nodes.

Organ/tissue samples were fixed in formalin for 24 hours and then stored in 70% ethanol. Fixed organ/tissue samples were further processed, embedded in paraffin wax and stained with haematoxylin and eosin.

Remaining portions of liver and kidneys were placed in foil packs, quick frozen in liquid nitrogen and stored at -70C.

HISTOPATHOLOGY: Yes: liver, kidneys, spleen, testes and all excised lesions in week 15, 30, 45 and 60 (total number of animals killed: 21, 18, 18, 21 for groups 1, 2, 3 and 4 respectively) and at terminal sacrifice (week 104), and all tissues collected from all high dose animals.

Other examinations:

Peroxisome proliferation was measured by a peroxisome marker enzyme (Cyanide-insensitive palmitoyl coenzyme A (PCO) activity) in frozen liver samples obtained at 15, 30, 45, 60 and 104 weeks. Portions of the frozen livers were homogenized (1:10 w/v) in a buffer containing 0.25 M sucrose, 0.05 M sodium ethyienediamine tetraacetic acid (EDTA), and 0.02 M Tris-HCl, pH 7.4., The homogenates were centrifuged at 800 x g for 5 min, the fatty layers removed by aspiration, and the extracts were stored at -:70°C until assayed. Protein concentratlons were measured accordmg to the method of Lowry et al. (1951). Previous work (DeAngelo et al., 1989) has shown that enzyme activities in frozen extracts did not differ significantly from the activities in liver extracts not frozen prior to assay.

Measurements of hepatocyte proliferation: Five days prior to each scheduled sacrifice, Alzet model 2001 osmotic pumps (Alza Corporation, Palo Alto,CA) containing 200 uL [3H]thymidine (62-64Ci/mmol, ICN Radiochemicals, Costa Mesa, CA were implanted subcutaneously., Autoradiography using paraffin-embedded sections from the left liver lobe was performed according to the procedure of Leblond and Percival (1948) as modified by Gride (1968). The slides were coated with Kodak NTB 3 emulsion and stored in a desiccator for 5-8 wk at 4°C. After developing, the slides were counterstained
with hematoxylin. The numbers of hepatocyte nuclei with a grain count greater than 6 were scored in 1000 cells using random numbers to choose the setting of the mechanical stage for field selection. The labeling index (U) was calculated by dividing the number of hepatocyte nuclei in S phase by the total number of hepatocyte nuclei scored.

Statistics:

See "Any other information on materials and methods incl. tables.

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):

not specified

Food efficiency:

not examined

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

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Only plasma ALAT and ASAT measured.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No effect on survival was found after 104 weeks

BODY WEIGHT AND WEIGHT GAIN
Body weight was significantly depressed at 26.1 and 59.9 mg/kg/day (13 and 38% respectively)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Water consumption was significantly depressed at 26.1 and 59.9 mg/kg/day (27% at both dosages).

CLINICAL CHEMISTRY
No treatment-related effects on plasma ALAT and ASAT were found at 104 weeks.

ORGAN WEIGHTS
Decreased absolute and relative liver weight, and absolute kidney weights at 26.1 and 59.9 mg/kg/day. Relative testes weight was significantly increased at 26.1 and 59.9 mg/kg/day, while the absolute testes weights in these groups did not differ statistically significantly from controls. The changes in the weights of the liver, kidneys and testes were not accompanied by treatment-related histopathological changes and are considered secondary to the growth depression in these groups. Mean relative and absolute spleen weight were statistically significantly increased in the 3.5 mg/kg/day group whereas spleen weights at 26.1 and 59.9 mg/kg/day were decreased (only statistically significant at 59.9 mg/kg/day). The variation in spleen weight amongst and within the groups parallelled the variation in the incidence of mononuclear cell leukaemia in these groups (i.e. the incidence of mononuclear cell leukaemia was 24, 48, 17 and 4% at 0, 3.5, 26.1 and 59.9 mg/kg/day respectively. This suggests that the increase in mean spleen weight at 3.5 mg/kg bw/day reflected the increase in the weight of the spleen of animals affected by leukaemia rather than a direct toxic effect of MCA on the spleen. The considerable decrease in spleen weight at 59.9 mg/kg/day can be ascribed to the marked growth depression at this level.

HISTOPATHOLOGY: NON-NEOPLASTIC
Non-neoplastic changes were for the most part spontaneous and age-related, except for an increased incidence of myocardial degeneration and chronic inflammation of the nasal cavities at 59.9 mg/kg/day at week 104.

The slight increase in chronic inflammation observed in at 59.9 mg/kg/day and the decreased incidence and severity of cytoplasmic vacuolisation and altered cellular foci (eosinophilic and clear cell) were not consistent with a direct hepatotoxic effect (data not shown in the publication).

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No significant increases in the prevalence of neoplastic lesions (including hepatic neoplasia) were observed. All of the observed neoplastic lesions would be considered spontaneous for F-344 male rats at 24 mo of age (data not shown in the publication). None of the neoplastic lesions exceeded the percent incidence when compared to a historical control database.

Tables with all observed neoplastic lesions were not included in the publication.

HISTORICAL CONTROL DATA (if applicable): For neoplastic lesions the authors refer to a historical control database of Hasemann et al, 1984 and NIEHS, 1995).

OTHER FINDINGS
No treatment-related effects were found on peroxisome and hepatocyte proliferation (measured at the interim and final sacrifice periods).

See Tables attached

Relevance of carcinogenic effects / potential:

No significant increases in the prevalence of neoplastic lesions were observed.
A severe depression of body weight gain and of liver and spleen weights was observed at 59.9 mg/kg/day, which could modify a tumorigenic response (Warner et al, 1995, cited by the authors).

Dose descriptor:

NOEL

Effect level:

>= 59.9 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Absence of carcinogenic effects

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Growth depression and reduced water consumption at 26.1 and 59.9 mg/kg/day

Conclusions:

MCA did not induce carcinogenic effects when administered for 2 years in drinking water to male F344/N rats. The overall NOAEL was 3.5 mg/kg bw/day based on growth depression and decreased water intake at the higher levels tested (26.1 and 59.9 mg/kg bw/day).

Executive summary:

Male Fisher F344/N rats were exposed to MCA via drinking water for 104 weeks at 0, 3.5, 26.1 or 59.9 mg/kg bw/day.

MCA did not induce cancer when administered for 2 years in drinking water. At 26.1 and 59.9 mg/kg/day, a reduced body weight gain and water intake was observed, and at 59.9 mg/kg/day an increased incidence of myocardial degeneration and chronic inflammation of the nasal cavities was noted at week 104.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

3.5 mg/kg bw/day

Study duration:

chronic

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: dermal

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No detailed description of materials and methods employed. Non-GLP. No guidelne followed. Detailed results of histopathology performed were not presented.

Qualifier:

no guideline followed

Principles of method if other than guideline:

A single dose of MCA was administered dermally (3 times per week) to female mice during 580 days. Complete necropsies were conducted, with histopathology on all abnormal appearing tissues/organs. No haematology/clinical biochemistry was conducted.
In the same study a separate group of mice was treated subcutaneously with MCA. This separate experiment is not considered relevant for risk characterisation, and is therefore not included in this endpoint study record.

GLP compliance:

no

Species:

mouse

Strain:

ICR

Sex:

female

Details on test animals or test system and environmental conditions:

Source: A. R.· Schmidt-Sprague-Dawley, Madison, Wise.
Mice were vaccinated against ectromelia, and treatments were begun when they were 6-8 weeks old. The mice were housed 10 to a cage, on sterile, hardwood chips (Iso-Dry, Fisher & Son, Bound Brook, N.].) in stainless-steel cages 11 X 7 X 6 inches high, fed Purina Laboratory Chow and water ad libitum, and weighed monthly. The animal rooms were maintained at 22-24 deg C.
Mice were house in well ventilated hoods for at least 3 hours after treatment.

Route of administration:

dermal

Vehicle:

acetone

Details on exposure:

Mice were shaved initially, and whenever necessary throughout the test.
2.0 mg MCA was applied in 0.1 mL acetone on the interscapular region, 3 times a week, with a micropipette.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

No data provided.

Duration of treatment / exposure:

580 days

Frequency of treatment:

3 times a week

Post exposure period:

No

Remarks:

Amount is applied in 0.1 ml acetone. Assuming a mouse weighs 25 grams, this would correspond to 80 mg/kg bw

No. of animals per sex per dose:

50 (MCA group)
50 (acetone only)
100 (untreated)

Control animals:

other: vehicle control and untreated control used.

Details on study design:

- Dose selection rationale: The dosage was based on short·term toxicity evaluations (for 4 wk), and the highest possible doses that gave minimal cytotoxic effects were used.
Complete necropsies were conducted, with histopathology on all abnormal appearing tissues/organs. No haematology/clinical bochemistry was conducted.

Positive control:

No

Observations and examinations performed and frequency:

All animals were weighed and examined regularly, and the findings were recorded monthly. Animals in poor health or with large tumor masses were killed. Except for the cranial region, animals were completely autopsied at the end of the experiment or at death. Although samples of all tissues and organs were not taken from each animal on test, the autopsies were done carefully and samples of all abnormal appearing tissues and organs were excised for histopathologic diagnosis. All tissue sections were processed routinely for histopathologic examination.

Sacrifice and pathology:

Complete necropsies were conducted, with histopathology on all abnormal appearing tissues/organs.

Other examinations:

None

Statistics:

The cut-off point for significance was P=0.05. If a compound had a P value of 0.05, it was considered borderline. P values >0.05 were considered not
significant. Any compound with a P value <0.05 was considered to have significant tumorigenic activity. All statistical data were computed based on 1
degree of freedom. Type of statistical test not specified.

Clinical signs:

not specified

Description (incidence and severity):

Only median survivial time specified.

Mortality:

not specified

Description (incidence):

Only median survivial time specified.

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

No skin papillomas or carcinomas were detected.
The median survival time was 506 days.

Relevance of carcinogenic effects / potential:

Not applicable; no tumors were detected.

Dose descriptor:

NOEL

Effect level:

>= 80 mg/kg bw (total dose)

Based on:

other: 2 mg MCA per day assuming mice are weighing 25 g

Sex:

female

Basis for effect level:

other: Absence of skin papillomas and carcinomas.

Detailed results of histopathology performed were not presented.

Conclusions:

No evidence for carcinogenic activity was found in female mice following dermal application of~80 mg/kg bw MCA for 580 days.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Justification for classification or non-classification

No carcinogenicity was found in two 2 -year carcinogenicity studies in rats and one in mice, nor in a 2 -year dermal carcinogencity study in mice. SMCA does not need to be classified for carcinogenicitiy. The overal NOAEL was 3.5 mg/kg bw/day.

Additional information

Although all three oral studies cited above have deficiencies, taken together these are adequate and sufficient to conclude that MCA has no carcinogenic activity. The dermal study did not show any neoplastic activity on the skin at a dose that was established to be the maximum tolerated does in terms of causing minimal cytotoxic effects in the skin. Taken together it can be concluded the MCA is not carcinogenic. This conclusion is in line with the EU RAR for MCAA (2005). Also According to MAK (2019): Since there is no evidence of carcinogenic effects in rats and mice, monochloroacetic acid and its sodium salt are not classified in a carcinogen category.