Dichloroacetic acid

Carcinogenicity

Description of key information

Various carcinogenicity studies were performed with DCA (and other drinkwater chlorinating agents) based on the potential hepatocarcinogenicity.  Two key studies were identified in rats and mice, respectively, where DCA was administered for life time and which revealed hepatocellular carcinomas and adenomas at higher dose levels. Lowest NOEL for carcinogenicity was found in rats at 50 mg/L, corresponding with 3.6 mg/kg bw/day. Two supporting studies in rats and mice, confirmed preterminal mortality and hepatocellular hyperproliferative lestions (hyperplastic nodules) in rats and carcinomas and adenomas in mice.  Additional studies were perfomed in mice (and rats) to clear out the mechanism of hepatocarcinogenicity. DCA was found to be a complete carcinogen, i.e. initiation by genotoxic carcinogen was not needed. A threshold dose response was further examined to take place in mice, therefore DCA is considered to deviate from a linear approach to derive a negligible risk level. The substance is considered to be a non-genotoxic rodent carcinogen.

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

Study period:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Although some details are missing, the study is consdired to be reliable, relevant and adequate.

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

GLP compliance:

not specified

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Portage, MI)
- Age at study initiation: 28-30 days of age at the beginning of treatment
- Weight at study initiation: mean initial body weights ranging from 59-79g
- Housing: 2-3 per polycarbonate cage
- Diet (e.g. ad libitum): Purina Rodent Laboratory Chow (St. Louis, MO) ad libitum
- Water (e.g. ad libitum): water ad libitum
- Acclimation period: 1 week in quarantine

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22°C
- Humidity (%): 40-60%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: drinking water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DCA was dissolved in distilled water to produce nominal (target) concentrations of 2.5 g/L). The pH of the solutions were adjusted to 6.9-7.1 by the addition of an appropriate volume of 10 N sodium hydroxide. Freshly prepared solutions were administered to the animals in brown glass water bottles fitted with Teflon stoppers and stainless steel, double-balled sipper tubes. The drinking water solutions were changed every 5-7 days. The stability of DCA over these time periods was demonstrated by gas liquid capillary chromatography.

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 the actual DCA concentrations. The solutions were pipetted into 7mL liquid scintillation mini-vials which were then tightly capped and stored at 5°C until analysis by UV absorption at 331nm against standard concentrations set by gas-liquid capillary chromatography.

Duration of treatment / exposure:

14, 26, 52, 78 and 103 weeks

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
2500 mg/L
Basis:
nominal in water
The 2.5g/L DCA group had a mean daily concentration (MDC) of 1.6g/L over the course of the study

No. of animals per sex per dose:

78

Control animals:

other: yes: deionized water

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cageside observations were conducted daily for physiological and behavioral responses and overt signs of toxicity. Mortality and morbidity checks were made twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Careful physical examinations were conducted twice weekly to detect any abnormal changes of the skin, eyes or systemic organ systems.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were measured at the start of the studies, twice monthly for the first two months and then monthly.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was measured at the start of the studies, twice monthly for the first two months and then monthly. Water consumption was calculated by dividing the amount of water used over a particular time interval by the whole cage animal weight and is expressed as mL/kg/day.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Sacrifice times for this assay were 14, 26, 52, 78 and 103 weeks. Liver, kidneys, testes, thyroid, stomach, rectum, duodenum, ileum, jejunum, colon, urinary bladder and spleen were examined for gross lesions at all time periods.
The tissues and lesions were harvested, fixed, processed and examined microscopically as described below.
At the interim sacrifices, body, liver, kidneys, testes, and spleen were weighed and examined for gross lesions including discoloration, surface irregularities, nodular changes, and/or tumor masses. Harvested tissue were preserved in 10% neutral buffered formalin for 24hr and then stored in 70% ethanol. The formalin-fixed tissues were trimmed, processed, embedded in paraffin, and sectioned. Slides were prepared and stained with hematoxylin and eosin. The remaining liver and kidneys were placed in foil packs, quick frozen in liquid nitrogen and stored at - 70°C.
At the 100 week sacrifice a complete rodent necropsy was performed on all animals. They were examined for gross lesions, including discolorations, surface irregularities, nodular changes and masses. All gross lesions and representative samples 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, caecum, rectum, kidneys, urinary bladder, prostate, seminal vesicles, testes, preputial gland, mammary gland, femur, nasal cavity, larynx, skin, mesenteric and mandibular lymph nodes were placed in buffered formalin.
Liver, kidneys, spleen and testes were examined microscopically by the same board-certified veterinary pathologist at the interim and final sacrifices.
In addition a complete pathologic examination was performed on five high dose animals from
each of the final sacrifices. Gross observations and microscopic diagnoses were correlated for each animal. Data were tabulated according to individual animal and summarized by group. Labcat® histopathology software (Princeton, NJ) was used for data management.

Statistics:

See under "Any other information on material and methods"

Clinical signs:

no effects observed

Description (incidence and severity):

There were no significant differences in animal survival between any of the control and treatment groups. The unscheduled deaths were primarily due to mononuclear cell leukemia which occurred in then neoplasm in the male F344/N rat.

Mortality:

no mortality observed

Description (incidence):

There were no significant differences in animal survival between any of the control and treatment groups. The unscheduled deaths were primarily due to mononuclear cell leukemia which occurred in then neoplasm in the male F344/N rat.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

After 103 weeks, the mean body weight of the animals exposed to 1.6g/L DCA was significantly reduced to 73% of the control value (308 ± 9g versus 424 ± 4g; p < 0.05).

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

no effects observed

Description (incidence and severity):

Time-weighted water consumptions were 61.7 ± 0.03mg/kg bw/day (water) and 86.4 ± 0.6mL/kg bw/day (1.6g/L DCA). The water consumptions in the DCA treated groups did not differ significantly from their respective control values.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Relative liver and kidney weights were increased because of the depressed body weights of the treated animals (P < 0.05). The absolute testes weight at 78 weeks (2.85 ± 0.39g versus 5.69 ± 0.49g, P < 0.01) was depressed.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

When compared to the deionized water control group, the animals treated with 1.6g/L DCA showed an increased prevalence of HC (21.4 versus 3.0%; P < 0.05), combined neoplasia (28.6 versus 3.0%; P < 0.01), and total proliferative lesions (32.1 versus 6.1 %)

Details on results:

Ergebnis: Alle Tiere der höchsten Dosisgruppe mußten in der 60. Woche getötet werden aufgrund der hohen Tumorinzidenz und der Entwicklung einer Hinterbeinlähmung, bis zur 45. Woche keine Läsionen in der Leber, in der 45. Woche in der höchsten Dosisgruppe bei 1/7 Tieren 1 Leberadenom, danach bei 500 mg/L nach 104 Wochen bzw. in der höchsten Dosisgruppe nach 60 Wochen signifikante Zunahme an hyperplastischen Knötchen (HN), Leberadenomen (HA) und Leberkarzinomen (HC), NOEL = 50 mg/L (500 mg/L: HN 3/29, HA 6/29, HC 3/29; 2400 mg/L: HN 19/27, HA 7/27, HC 1/27; Kontrolle: HA 1/23), Anzahl der GST–positiven Leberfoci nicht signifikant unterschiedlich zur Kontrolle,
dagegen sind nach Meinung der Autoren die hyperplastischen Knötchen möglicherweise eine präneoplastische hepatokarzinome Läsion.

CLINICAL SIGNS AND MORTALITY
There were no significant differences in animal survival between any of the control and treatment groups. The unscheduled deaths were primarily due to mononuclear cell leukemia which occurred in then neoplasm in the male F344/N rat.

BODY WEIGHT AND WEIGHT GAIN
The initial mean body weights of animals assigned to each treatment group did not differ from one another. After 103 weeks, the mean body weight of the animals exposed to 1.6g/L DCA was significantly reduced to 73% of the control value (308 ± 9g versus 424 ± 4g; p < 0.05).

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
The 2.5g/L DCA group had a mean daily concentration (MDC) of 1.6g/L over the course of the study. Time-weighted water consumptions were 61.7 ± 0.03mg/kg bw/day (water) and 86.4 ± 0.6mL/kg bw/day (1.6g/L DCA). The water consumptions in the DCA treated groups did not differ significantly from their respective control values. These regimens yielded time-weighted MDD of 139mg/kg/day for 1.6g/L DCA.

ORGAN WEIGHTS
The absolute weights of liver, kidneys and spleen of animals exposed to 1.6g/L DCA did not differ from those of the control group, whereas the weights of the liver and kidney relative to body weight were increased because of the depressed body weights of the treated animals (P < 0.05). The absolute testes weight at 78 weeks (2.85 ± 0.39g versus 5.69 ± 0.49g, P < 0.01) was depressed. The relative testes weight was lower but not significantly.

GROSS PATHOLOGY
Non-neoplastic hepatic changes diagnosed from gross observations were not considered treatment related and were consistent with aging changes previously observed in F344 rats. All of the non-hepatic neoplastic lesions observed would be considered spontaneous for F344 male rats at 24 months of age.

HISTOPATHOLOGY: NON-NEOPLASTIC
Non-neoplastic hepatic changes diagnosed from pathologic examination of tissues were not considered treatment related and were consistent with aging changes previously observed in F344 rats. Hepatocellular cytoplasmic vacuolization was more prominent in the DCA dosed animals and probably represented an increased glycogen deposition (Mather et al., 1990). Several degenerative hepatic changes such as sinusoidal dilation and cystic degeneration were accentuated somewhat in treated animals.
All of the non-hepatic neoplastic lesions observed would be considered spontaneous for F344 male rats at 24 months of age.


HISTOPATHOLOGY: NEOPLASTIC (if applicable)
When compared to the deionized water control group, the animals treated with 1.6g/L DCA showed an increased prevalence of HC (21.4 versus 3.0%; P < 0.05), combined neoplasia (28.6 versus 3.0%; P < 0.01), and total proliferative lesions (32.1 versus 6.1 %; P < 0.01). At 78 weeks, one animal in the deionized water group had an HC, while two animals each in the 1.6g/L DCA treated group had neoplastic lesions (1 HC and 1 HA).
Animals treated with 1.6g/L DCA exhibited an increased multiplicity of HC when compared to those in the control group (0.25 ± 0.10 versus 0.03 ± 0.03; P < 0.05). The multiplicity of combined neoplastic lesions was 0.36 ± 0.12 versus 0.03 ± 0.03 (P < 0.01 ), while that for total hepatic proliferative lesions was 0.39 ± 0.12 versus 0.06 ± 0.04 (P < 0.01).
The various phenotypically altered hepatic foci (AHF; basophilic, eosinophilic, clear cell or mixed cell) were regularly observed. The AHF contained cells that were small and arranged in irregularity formed hepatic plates and were few in number (1-3 AHF/liver section). The prevalence of AHF did not appear to differ between control and DCA treated animals. The immunohistochemical characterization and quantitation of the AHF and the other proliferative lesions observed in the first study have been published (Richmond et al., 1995).

HISTORICAL CONTROL DATA (if applicable)
None of the neoplastic lesions in other organs exceeded the percent incidence when compared to a historical control data base (Haseman et al., 1984; NIEHS, 1995). Testicular interstitial cell tumours were seen in 100% of the animals in both the water and 1.6g/L DCA groups. Kidney neoplasms were not apparent in any group. The incidence of mononuclear cell leukemia was 9%, 11% for the water and 1.6g/L DCA treated animals respectively. One DCA treated animal had a renal tubular cell adenoma.

Dose descriptor:

NOEL

Effect level:

50 mg/L drinking water

Based on:

test mat.

Sex:

male

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

DCA concentration, water consumption, dose determination and survival:
The 2.5g/L DCA group had a mean daily concentration (MDC) of 1.6g/L over the course of the study. Time-weighted water consumptions were 61.7 ± 0.03mg/kg bw/day (water) and 86.4 ± 0.6mL/kg bw/day (1.6g/LDCA). The water consumptions in the DCA treated groups did not differ significantly from their respective control values. These regimens yielded time-weighted MDD of 139mg/kg/day for 1.6g/L DCA.

There were no significant differences in animal survival between any of the control and treatment groups. The unscheduled deaths were primarily due to mononuclear cell leukemia which occurred in then neoplasm in the male F344/N rat.

Body and organ weights:

The initial mean body weights of animals assigned to each treatment group did not differ from one another. After 103 weeks, the mean body weight of the animals exposed to 1.6g/L DCA was significantly reduced to 73% of the control value (308 ± 9g versus 424 ± 4g; p < 0.05).

The absolute weights of liver, kidneys and spleen of animals exposed to 1.6g/L DCA did not differ from those of the control group, whereas the weights of the liver and kidney relative to body weight were increased because of the depressed body weights of the treated animals (P < 0.05). The absolute testes weight at 78 weeks (2.85 ± 0.39g versus 5.69 ± 0.49g, P < 0.01) was depressed. The relative testes weight was lower but not significantly.
Histopathologic findings:

When compared to the deionized water control group, the animals treated with 1.6g/L DCA showed an increased prevalence of HC (21.4 versus 3.0%; P < 0.05), combined neoplasia (28.6 versus 3.0%; P < 0.01), and total proliferative lesions (32.1 versus 6.1 %; P < 0.01). At 78 weeks, one animal in the deionized water group had an HC, while two animals each in the 1.6g/L DCA treated group had neoplastic lesions ( 1 HC and 1 HA).

Animals treated with 1.6g/L DCA exhibited an increased multiplicity of HC when compared to those in the control group (0.25 ± 0.10 versus 0.03 ± 0.03; P < 0.05). The multiplicity of combined neoplastic lesions was 0.36 ± 0.12 versus 0.03 ± 0.03 (P < 0.01 ), while that for total hepatic proliferative lesions was 0.39 ± 0.12 versus 0.06 ± 0.04 (P < 0.01).

The various phenotypically altered hepatic foci (AHF; basophilic, eosinophilic, clear cell or mixed cell) were regularly observed. The AHF contained cells that were small and arranged in irregularity formed hepatic plates and were few in number (1-3 AHF/liver section). The prevalence of AHF did not appear to differ between control and DCA treated animals. The immunohistochemical characterization and quantitation of the AHF and the other proliferative lesions observed in the first study have been published (Richmond et al., 1995).

Non-neoplastic hepatic changes diagnosed from gross observations and pathologic examination of tissues were not considered treatment related and were consistent with aging changes previously observed in F344 rats. Hepatocellular cytoplasmic vacuolization was more prominent in the DCA dosed animals and probably represented an increased glycogen deposition (Mather et al., 1990). Several degenerative hepatic changes such as sinusoidal dilation and cystic degeneration were accentuated somewhat in treated animals.

All of the non-hepatic neoplastic lesions observed would be considered spontaneous for F344

male rats at 24 months of age.

None of the neoplastic lesions in other organs exceeded the percent incidence when compared to a historical control data base (Haseman et al., 1984; NIEHS, 1995). Testicular interstitial cell tumours were seen in 100% of the animals in both the water and 1.6g/L DCA groups. The incidence of mononuclear cell leukemia was 9%, 11% for the water and 1.6g/L DCA treated animals respectively. One DCA treated animal had a renal tubular cell adenoma.

Cyanide-insensitive palmitoyl CoA assay:

Hepatocyte peroxisome proliferation (PP) was assessed by measuring the peroxisomal marker enzyme, PCO. 1.6g/L DCA enhanced the PCO activity relative to the water value at 14 weeks (9.00 ± 0.95 versus 4.07±0.75, 221%, p<0.05), 26 weeks (6.78± 0.80 versus 3.02 ± 0.28, 224%, p < 0.001) and 78 weeks (5.65 ± 0.33 versus 2.58 ± 0.69, 219%, P <0.05).

Hepatocyte proliferation:

At 14 weeks, 1.6g/L DCA treatment depressed the BRDU labelling index (LI) of hepatocytes to 41% of the control group value (1.04 ± 0.12 versus 2.53 ± 0.19; P < 0.05). The LI measured at the other time periods, while depressed, did not differ significantly from, the control group value: 26 weeks (28% of control): 52 weeks (9% of control) and 78 weeks (57% of control).

 

Table1. Dosing and survival data for rats exposed to DCA

Target concentration	Water	2.5g/L DCA^a
Measured concentration	-	1.61 ± 0.02^c
Number of measurements		132
Water consumption^c(mL/kg/day)	61.7 ± 0.3	86.4 ± 0.6
Mean daily dose^c(mg/kg/day)		139.1
Number of animals started on study	78	78
Number of unscheduled deaths	17	23
Number of animals at interim sacrifice	28	27
Number of animals at final sacrifice	33	28

^a2.5g/L DCA lowered to 2g/L at five weeks, to 1.5g/L at eight weeks and to 1g/L at 26 weeks
^cTime-weighted mean ± SEM over exposure period.

Table2. Body and organ weights for rats exposed to DCA

Treatment group	Water	2.5g/L DCA^a
Number of animals	28	27
Initial body weight (g)	76 ± 8	72 ± 8
Final body weight (g)	424 ± 4	308 ± 9^c*
Liver weight (g)	15.9 ± 0.3	15.8 ± 0.6
Percent of body weight	3.7 ± 0.1	5.2 ± 0.5*
Kidney weight (g)	3.01 ± 0.04	3.13 ± 0.07
Percent of body weight	0.71 ± 0.01	1.03 ± 0.02*
Spleen weight (g)	2.01 ± 0.16	2.04 ± 0.53
Percent of body weight	0.48 ± 0.05	0.07 ± 0.02
Testes weight (g)^d	5.69 ± 0.49	2.85 ± 0.39**
Percent of body weight	1.24 ± 0.11	0.81 ± 0.14

^aMean daily concentration
^bMean ± SEM
^cStatistically significant at *p ≤ 0.05, **p ≤ 0.01 when compared to the water control
^dPhase two tested weights taken at 78 weeks (n=7)

Table3. Prevalence of hepatocellular lesions^a

Treatment group	Water	1.6/L DCA^b
Number examined^c	33	28
Hyperplastic nodule	3.0	3.6
Adenoma	0	10.7
Carcinoma	3.0	21.4^d*
Neoplasia^e	3.0	28.6**
Proliferative lesions^f	6.1	32.1**

^aPercent of animals with a              t least one lesion
^bMean daily concentration (g/L)
^cAnimals that survived > 78 weeks.
^dStatistically significant at *p ≤ 0.05, ** p ≤ 0.01 when compared to water control
^eCombined adenoma and carcinoma
^fCombined hyperplastic nodules, adenoma and carcinoma.

Table4. Multiplicity of "hepatocellular lesions"^a

Treatmcnt group	Water	1.6g/L DCA^b
No. examined^c	33	28
Hyperplastic nodule	0.03 ± 0.03	0.04 ± 0.04
Adenoma		0.11 ± 0.06
Carcinoma	0.03 ± 0.03	0.25 ± 0.14^e *
Neoplasia^f	0.03 ± 0.03	0.36 ± 0.12**
Proliferative lesions^g	0.06 ± 0.04	0.39 ± 0.12**

^aNumber of lesions/animals
^bMean daily concentration (g/L).
^cAnimals that survived > 78 weeks.
^dMean ± SEM.
^eStatistically significant at *p ≤ 0.05, **p ≤ 0.01 when compared to water control.
^fCombined adenoma and carcinoma.
^gCombined adenoma, carcinoma and hyperplastic nodules.

Conclusions:

DCA is an hepatocarcinogen to the male F344 rat.

Executive summary:

The chlorinated acetic acids, in particular dichloroacetic acid (DCA), are found as chlorine disinfection by-products in finished drinking water supplies. DCA has previously been demonstrated to be a mouse liver carcinogen. Chronic studies are described in which male Fischer (F344) rats were exposed to DCA in their drinking water. In the first study, 28 day old rats were exposed to a regimen of 0.05, 0.5 and 5.0 g/L DCA. When animals in the high dose group began to exhibit peripheral hind leg neuropathy, the dose was lowered in stages to 1 g/L. These animals were sacrificed at 60 weeks due to the severe, irreversible neuropathy and were not included in this analysis. The remaining groups of animals were treated for 100 weeks. In the second study (this endpoint), rats were initially exposed to 2.5 g/L DCA which was lowered to 1 g/L after 18 weeks. The mean daily concentration (MDC) of 1.6 g/L was calculated over the 103 week exposure period. Time-weighted mean daily doses (MDD) based on measured water consumption were 3.6, 40.2 and 139 mg/kg bw/day for the 0.05, 0.5 and 1.6 g/L DCA respectively. Based upon the pathologic examination, DCA induced observable signs of toxicity in the nervous system, liver and myocardium. However, treatment related neoplastic lesions were observed only in the liver. A statistically significant increase of carcinogenicity (hepatocellular carcinoma) was noted at 1.6 g/L DCA. Exposure to 0.5 g/L DCA increased hepatocellullar neoplasia, (carcinoma and adenoma) at 100 weeks. These data demonstrate that DCA is an hepatocarcinogen to the male F344 rat. Calculation of the MDD at which 50% of the animals exhibited liver neoplasia indicated that the F344 male rat (approximately 10 mg/kg bw/day) is ten times more sensitive than the B6C3F1 male mouse (approximately 100mg/kg bw/day). A 'no observed effects level' (NOEL) of 0.05g/L (3.6 mg/kg/day) was the same as for the mouse (3-8 mg/kg/day).

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

3.6 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

reliable

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance was demonstrated to be a rodent carcinogen at high doses after long-term administration in the drinking water; a threshold NOEL for hepatic tumors could be demonstrated both in rats and mice. Evaluation of genotoxicity and carcinogenicity was done by previous IARC Working Groups in February 1995 and October 2004 (IARC, 1995, 2004). New data have since then become available, and these have been taken into consideration. There is inadequate evidence in humans for the carcinogenicity of dichloroacetic acid. There is sufficient evidence in experimental animals for the carcinogenicity of dichloroacetic acid. In conclusion, Dichloroacetic acid is possibly carcinogenic to humans (Group 2B) (IARC MONOGRAPHS VOLUME 84 and 106). This classification corresponds to GHS/CLP Category 2 classification according to CLP regulation (No. 1272/2008 of 16 December 2008) with signal word 'Warning' and hazard statement: H351 – Suspected of causing cancer (oral route). According to the Directive 67/548/EEC, Annex VI, DCA is classified Category 3 Xn R40, Limited evidence of a carcinogenic effect.

Additional information

The following two studies were considered to be key studies, as they comply with the classic bioassay in 2 species and delivered the lowest NOELs as threshold levels:

•       DeAngelo et al, 1996 (Endpoints 07.07.00_03 and 07.07.00_04) treated rats in a first study with DCA in the drinking water at 0.05, 0.5 and 5.0 g/L DCA. When animals in the high dose group began to exhibit peripheral hind leg neuropathy, the dose was lowered in stages to 1 g/L. These animals were sacrificed at 60 weeks due to the severe, irreversible neuropathy and were not included in this analysis. The remaining groups of animals were treated for 100 weeks. In a second study, rats were initially exposed to 2.5 g/L DCA which was lowered to 1 g/L after 18 weeks. The mean daily concentration (MDC) of 1.6 g/L was calculated over the 103 week exposure period. Time-weighted mean daily doses (MDD) based on measured water consumption were 3.6, 40.2 and 139 mg/kg bw/day for the 0.05, 0.5 and 1.6 g/L DCA respectively. DCA induced observable toxicity in the nervous system, liver and myocardium. However, treatment related neoplastic lesions were observed only in the liver. A statistically significant increase of carcinogenicity (hepatocellular carcinoma) was noted at 1.6 g/L DCA. Exposure to 0.5 g/L DCA increased hepatocellullar neoplasia, (carcinoma and adenoma) at 100 weeks. These data demonstrate that DCA is an hepatocarcinogen to the male F344 rat. Calculation of the MDD at which 50% of the animals exhibited liver neoplasia indicated that the F344 male rat (approximately 10 mg/kg bw/day) is ten times more sensitive than the B6C3F1 male mouse (approximately 100 mg/kg bw/day). A NOEL of 50 mg/L (3.6 mg/kg bw/day) was the same as for the mouse (3-8 mg/kg/day).

•       DeAngelo et al. 1991 (Endpoint 07.07.00_06), treated male B6C3F1 mice (N = 50) with DCA via the drinking water at 0.05, 0.5, and 5 g/liter DCA. Treatment of 30 animals n each group was carried out to 60 or 75 weeks. In a separate experiment, mice exposed to 3.5 g/liter DCA and the corresponding acetic acid control group were killed at 60 weeks. Groups of 5 mice were killed at 4, 15, 30, and 45 weeks. Time-weighted mean daily doses of 7.6, 77, 410, and 486 mg/kg/day were calculated for 0.05, 0.5, 3.5, and 5 g/liter DCA treatments. Animals exposed to 3.5 and 5 g/liter DCA had final body weights that were 87 and 83%, respectively, of the control value. Relative liver weights of 136, 230, and 351% of the control value were measured for 0.5. 3 5, and 5 g/liter, respectively. At 60 weeks mice receiving 5.0 g/liter DCA had a 90% prevalence of liver neoplasia with a mean multiplicity of 4.50 tumors/animal. Exposure to 3.5 g/ liter DCA for 60 weeks resulted in a 100% tumor prevalence with an average of 4.0 tumors/ animal. The prevalence of liver neoplasia and tumor multiplicity at 60 and 75 weeks in the 0.05 g/liter DCA (24.1 %; 0.31 tumors/animal) and in the 0.5 g/liter group (11.1%; 0.11 tumors/animal) did not differ significantly from the control value (7.1% and 0.07 tumors/animal). No liver tumors were found in the group treated with acetic acid. Hyperplastic nodules were seen in the 3.5 (58%; 0.92/animal) and 5 g/liter DCA groups (83%; 1.27/animal). There was a significant positive dose-related trend in the age-adjusted prevalence of liver tumors. These data confirm the hepatocarcinogenicity of DCA administered in the drinking water to male B6C3F1 mice for 60 weeks. The results together with those in an earlier report from this laboratory suggest, for the conditions under which these assays were conducted, a threshold concentration of at 500 mg/liter followed by a steep rise to a maximum tumor incidence at 2 g/liter DCA.

The following studies were considered to be supporting studies for the hepatic carcinogenicity potential:

•       DeAngelo et al, 1992 (Endpoint 07.07.00_05) treated groups of male F344 rats to 0.05, 0.5 and 5 g/L DCA in the drinking water for 100-104 weeks. The 5 g/L DCA treatment was progressively lowered until termination at 60 weeks due to severe toxicity. Time-weighted mean daily doses (mg/kg/day) of 4.3, 48 and 295 were calculated for DCA. High dose DCA treatment resulted in mortality and a decreased body weight gain. There was no significant hepatic neoplasia, but a significant increase in altered foci and hyperplastic nodules was observed. At 0.5 g/L, a DCA enhanced prevalence of hepatic proliferative lesions was not statistically significant. The 0.05 g/L DCA treatment was without effect.

•       Daniel et al, 1992 (Endpoint 07.07.00_02) treated male B6C3F1 mice for 104 weeks to DCA (0.5 g/L) next to other water chlorination products. The mean daily ingested dose was approximately 93 mg/kg/day. 63% of the 104-week survivors had hepatocellular carcinomas (carcinomas) and 42% possessed hepatocellular adenomas (adenomas) and the combined prevalence for carcinomas plus adenoma was 75%. The corresponding prevalence rate for carcinomas, adenomas, and combined tumors were 10, 5, and 15% for H2O.

 

Finally, following studies were considered to be more mechanistic, therefore they were disregarded for hazard potential:

•       Herren-Freund, 1987 (Endpoints 07.07.00_07 and 07.07.00_08) tested DCA to act as tumor promoter in mouse liver. In the first endpoint, male B6C3F1 mice given drinking water containing 5g/liter DCA at 28 days of age without prior initiator were evaluated after 61 weeks of exposure. DCA at a concentration of 5 g/liter was carcinogenic without prior initiation with ENU, resulting in hepatocellular carcinomas in 81% of the animals. DCA also increased the incidence of animals with adenomas and the number of adenomas/animal in those animals that were not initiated with ENU. In the second endpoint, the mice were placed on drinking water DCA at 2 or 5 g/liter (corresponding with 400 and 1000 mg/kg bw) at 28 days of age with prior initiaton by 2.5 µg/g body wt ethylnitrosourea (ENU) on Day 15 of ag . DCA at a concentration of 2 and 5 g/liter was carcinogenic witg prior initiation by ENU, resulting in hepatocellular carcinomas and adenomas. None of the untreated animals had hepatocellular carcinomas. The results demonstrate that DCA is complete hepatocarcinogen in B6C3F1 mice.

•       Bull et al, 1990 (Endpoint 07.07.00_01) treated male and female B6C3Fl mice with DCA (and TCA) in the drinking water at concentrations of 1 or 2 g/L for up to 52 weeks. Hepatoproliferative lesions (HPL) in male mice ( hepatocellular nodules, adenomas and hepatocellular carcinomas) were observed within 12 months. The induction of HPL by TCA was linear with dose. In contrast, the response to DCA increased sharply with the increase in concentration from 1 to 2 g/L. Suspension of DCA treatment at 37 weeks resulted in the same number of HPL at 52 weeks that would have been predicted on the basis of the total dose administered. Throughout active treatment DCA-treated mice displayed greatly enlarged livers characterized by a marked cytomegaly and massive accumulations of glycogen in hepatocytes throughout the liver. Areas of focal necrosis were seen throughout the liver.

•       Pereira, 1996 (Endpoint 07.07.00_09) tested concentration-response relationships for the hepatocarcinogenic activity of DCA (and TCA) in female B6C3F1 mice. DCA at 2.0, 6.67, or 20.0 mmol/liter was administered to the mice in the drinking water starting at 7 to 8 weeks of age and until sacrifice after 360 or 576 days of exposure. The liver-to-body weight ratio increased linearly, as did the vacuolization of the liver induced by DCA. The foci of altered hepatocytes and tumors in the animals treated with DCA were predominantly eosinophilic and contained glutathione S-transferase-π (GST- π, over 80% of the lesions).

•       Richmond et al, 1995 (Endpoint 07.07.00_10) examined the incidence of proliferative lesions, hyperplastic nodules and altered hepatic foci, in male F344 rat liver, to determine their preneoplastic potential during DCA)induced hepatocarcinogenesis. Immunohistochemical and image analysis methods were used to detect the expression of 6 histochemical markers of neoplastic cells; p21ras, p39 c-jun, p55 c-fos, aldehyde dehydrogenase (ALDH), glutathione s-transferase (GST-p), and alpha fetoprotein (AFP) during DCA-induced hepatocarcinogenesis. Results indicated that the hyperplastic nodules, rather than altered hepatic foci, is a putative preneoplastic lesion during DCA-induced hepatocarcinogenesis in the male F344 rat.

Justification for selection of carcinogenicity via oral route endpoint:

Lowest NOEL.

Carcinogenicity: via oral route (target organ): digestive: liver