Sodium 1-(3,4-dihydro-6-methyl-2,4-dioxo-2H-pyran-3-ylidene)ethanolate

Endpoint summary

Description of key information

Repeat oral toxicity:

In a study in the monkey ( Macaca mulatta) gavage doses 0, 50, 100, 200 or 300 mg/kg bw/day (single animal/group)– DHA equivalent, DHA or DHA-Na dosed. One year study, for doses up to 100 mg/kg bw/day, no adverse toxicity was evident. Adverse toxicity at 200 or 300 mg/kg bw/day. NOAEL 100 mg/kg bw/day.

In a study in the rat gavage doses 10 to 300 mg/kg bw/day, 24 doses in 34 days; treatment related mortality and adverse toxicity at 300 mg/kg bw/day. No adverse effects at 0, 10, 30, 100 mg/kg bw/day. NOAEL 100 mg/kg bw/day.

In a two -year study in the rat by dietary dosing, animals (25/group) were dosed at 0.02, 0.05, and 0.10 % (by weight, DHA – equivalent to 200, 500 or 1000 ppm or 20, 50 or 100 mg/kg bw/day). No adverse toxicity was seen. A NOEL of 1000 ppm (100 mg/kg bw/day) is proposed.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Circa 1950

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

A sub-chronic, one-year, study in primates, dosed by oral gavage, in which key indicators of toxicity and pathology were monitored. Additionally, some TK parameters were assessed.

GLP compliance:

no

Limit test:

no

Species:

monkey

Strain:

other: Macaca mulatta

Sex:

male/female

Details on test animals or test system and environmental conditions:

Imported rhesus monkeys (Macaca mulatta), that had been acclimatised for several months in the laboratory were fully adapted to the laboratory environment, diet, and handling, were used in this work.
The animals were caged separately aud maintained on "Complete Laboratory Chow" (Purina) supplemented with canned tomatoes, fresh fruits and vegetables, and peanuts. At the start of the experiment tho monkeys were apparently in excellent condition and had all gained considerable weight since their arrival in the laboratory.

Route of administration:

oral: gavage

Details on route of administration:

Repeated oral doses of dehydroacetic acid in olive oil solution (5% cent) and of its sodium salt in water solution (10% cent) were administered by stomach tube to these monkeys at dosage levels of 0, 50, 100, 200 or 300 mg/kg bw/day – DHA equivalent, DHA or DHA-Na dosed. All doses of the sodium salt were calculated on the basis of its dehydroacetic acid equivalent.

Vehicle:

water

Details on oral exposure:

See above

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

One year

Frequency of treatment:

Daily - not precisely specified in publication.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

One/group; with the exception of one animal (no. 90) all animals were female

Control animals:

yes, concurrent vehicle

Details on study design:

Monkeys at 200 mg/kg bw/day dosed only 3-4 days a week due to adverse clinical condition.

Positive control:

No

Observations and examinations performed and frequency:

Twice weekly body weights, daily observations.
Blood taken periodically for haematology, plasma N content and DHA analysis. Urine was also examined for DHA analysis.

Sacrifice and pathology:

Sacrificed at one year, heart, liver, kidneys and spleen sampled; blood taken for urea-N analysis an d lung, heart, kidney, spleen, adrenal, pancreas testis, ovary, bone marrow stomach, small intestine, voluntary muscle, bladder, lymph node, thyroid and brain sampled for histopathological examination. Liver and kidney stained with Oil Red O for lipid assessment.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg bw/day inappetence, ataxia, vomiting and clonic and tonic convulsions. Monkeys at this dosage were killed before scheduled termination.
At 200 mg/kg bw/day ataxia, conjvulsions, retching, salivation and vomiting - discontinuation of dosing for up to 3 days lead to a recovery

Mortality:

mortality observed, treatment-related

Description (incidence):

One monkey (no. 95) on day 26 (after the 20th dose)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 200 or 300 mg/kg bw/day body weight loss.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 200 or 300 mg/kg bw/day inappetance recorded.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

effects observed, non-treatment-related

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No effects recorded at 200 mg/kg bw/day or below.

Neuropathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 200 or 300 mg/kg bw/day ataxia, convulsions.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No effects recorded at 200 mg/kg bw/day or below. At 300 mg/kg bw/day one animal show moderate degenerative changes to the tubular epithelium of the kidney. Considerable inflammation in the small intestine was noted for the animal which died.

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

In one monkey treated at 50 mg/kg bw/day the total lipid content of the liver was increased.

Key result

Dose descriptor:

LOAEL

Effect level:

>= 200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

Key result

Dose descriptor:

NOAEL

Effect level:

> 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects observed

Critical effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day (nominal)

System:

central nervous system

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

no

See attached summary of results.

Conclusions:

At 300 mg/kg bw/day the monkeys were killed (before scheduled termination due to adverse toxicity: inappetence, body weight loss, ataxia, vomiting and clonic and tonic convulsions. One animal showed moderate degenerative changes to the tubular epithelium of the kidney. Considerable inflammation in the small intestine was noted for the animal which died.

At 200 mg/kg bw/day inappetence and body weight loss were seen; discontinuation of dosing for up to 3 days lead to a recovery; however, if this wasn’t the case the animals failed showing signs of ataxia, retching, salivation and vomiting. Force feeding the animals helped to alleviate the symptoms. Because of this animals in this group were dosed 3 or 4 times a week only.

At 50 or 100 mg/kg bw/day no adverse effects were observed. There were no changes in either the blood chemistry of haematological investigations. No adverse histopathological abnormalities were seen
in this study with the exception of a monkey treated at 50 mg/kg bw/day where the total lipid content of the liver was increased.

A NOAEL of 100 mg/kg bw/day was determined.

Executive summary:

Monkeys (Macaca mulatta) -1/group - were treated with DHA or DHA-Na via oral gavage at 0, 50, 100, 200 or 300 mg/kg bw/day.

At 300 mg/kg bw/day the monkeys were killed (before scheduled termination) due to adverse toxicity.

At 200 mg/kg bw/day inappetence and body weight loss were seen.

At 50 or 100 mg/kg bw/day no adverse effects were observed. A NOAEL of 100 mg/kg bw/day was determined.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Circa 1950

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline available

Principles of method if other than guideline:

Essentially an oral gavage four week sub-chronic toxicity study, conducted in the rat (a well-established experimental model), in which key toxicity endpoints were evaluated.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

not specified

Details on species / strain selection:

not specified

Sex:

male

Details on test animals or test system and environmental conditions:

not specified

Route of administration:

oral: gavage

Vehicle:

olive oil

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

24 doses in 34 days

Frequency of treatment:

Daily - presumed not at weekends

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 or 6

Control animals:

yes, concurrent vehicle

Details on study design:

Male rats 10 weeks old at start of study

Positive control:

No

Observations and examinations performed and frequency:

General condition, body weight

Sacrifice and pathology:

Pathology (lung, heart, liver, kidney, spleen, adrenal, pancreas, testis, and stomach), histopathology, blood urea-N

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg bw/day - animals were emaciated, thin and unkempt

Mortality:

mortality observed, treatment-related

Description (incidence):

At 300 mg/kg bw/day - two deaths, one at 7 days the other at 11 days (7 doses). Remaining rats killed after 11 days. No adverse effects at 0, 10, 30, 100 mg/kg bw/day

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg bw/day - 20 to 30% of their body weight loss. No adverse effects at 0, 10, 30, 100 mg/kg bw/day

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 specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg bw/day - contracted stomach with blood tinged contents, a congested mucosa, and a few haemorrhagic areas.
No adverse effects at t 0, 10, 30, 100 mg/kg bw/day

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Key result

Dose descriptor:

LOEL

Effect level:

ca. 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical signs

mortality

Key result

Dose descriptor:

NOAEL

Effect level:

> 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse toxicity at 100 mg/kg bw/day.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

not specified

Conclusions:

General condition and body weight at 0, 10, 30, 100 mg/kg bw/day was unaffected. Additionally, no adverse effects were noted for blood urea-N or after histopathological examination of selected tissues (lung, heart, liver, kidney, spleen, adrenal, pancreas, testis, and stomach). At 300 mg/kg bw/day loss, two deaths occurred (7 & 11 days). The remaining animals at 300 mg/kg bw/day were killed after 11 days (7 doses). These animals lost 20 to 30% of their body weight and were thin and unkempt. Examination of each of these animals revealed marked emaciation and contracted stomach with blood tinged contents, a congested mucosa, and a few haemorrhagic areas. The NOAEL was onsidered to be 100 mg/kg bw/day.

Executive summary:

In a repeat dose toxicity study (24 doses in 34 days) rats were dosed by oral gavage: 2 groups of 5 or 6 (10 weeks old at start), the dosages were: 0, 10, 30, 100 or 300 mg/kg bw/day (vehicle: olive oil).

There were no adverse effects at 10, 30, 100 mg/kg bw/day. At 300 mg/kg bw/day loss, two deaths occurred (7 & 11 days), there was 20 to 30% body weight loss, the animals appeared thin and unkempt and had a contracted stomach with blood tinged contents, a congested mucosa, and a few haemorrhagic areas. A NOAEL of 100 mg/kg bw/day was determined.

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

LOEL

Effect level:

ca. 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical signs

mortality

Key result

Dose descriptor:

NOAEL

Effect level:

> 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse toxicity at 100 mg/kg bw/day.

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Reference 4

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Circa 1950

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

Primarily a chronic dietary, two-year, study in rats in which key indicators of toxicity and pathology were monitored. Additionally, some TK parameters were assessed.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

Details not specified
Diet was a modified Sherman diet

Route of administration:

oral: feed

Details on route of administration:

Note: Diets containing 0.02, 0.05, and 0.10 % (by weight) DHA – equivalent to 200, 500 or 1000 ppm or 20, 50 or 100 mg/kg bw/day).

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Two year feeding study

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Two years

Frequency of treatment:

Daily

Dose / conc.:

0 ppm

Dose / conc.:

200 ppm

Remarks:

0.02% or 20 mg/kg bw/day

Dose / conc.:

500 ppm

Remarks:

0.05% or 50 mg/kg bw/day

Dose / conc.:

1 000 ppm

Remarks:

0.10% or 100 mg/kg bw/day

No. of animals per sex per dose:

25

Control animals:

yes, concurrent no treatment

Details on study design:

Rats 2-3 months old; 25 each sex/group; 5 same sex/cage, food & water ad lib. A group of ten female rats, receiving the diet containing 0.05% DHA, and a similar group of controls, were selected for periodic hematological studies during the course of the experiment

Positive control:

No

Observations and examinations performed and frequency:

Clinical condition, body weight and food consumption (twice/week) , haematology (erythrocyte count, haemoglobin concentration, total leucocyte count, and differential count) - frequency not specified

Sacrifice and pathology:

After 237 days on the experimental diets, two female rats from each of the groups were sacrificed in order to obtain plasma for DHA analyses. After two years on the diets containing DHA all of the surviving rats were starved overnight, weighed, killed by decapitation, aud examined. The.heart, liver, kidneys, spleen, and testes from,each rat were weighed.

Other examinations:

Oxalated blood was taken for urea-N determinations and a portion of the liver from each animal was frozen with dry ice for subsequent analysis of the total lipid content. Total lipids were determined gravimetrically.

Statistics:

The t-test was used in comparing the mean values obtained on the experimental groups with those of the controls; probability values (P) of 0.05 or less indicated a signiûcant difference.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Considered to be age-related and due to infection

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At termination, the total liver lipid values were significantly higher in the 1000 ppm dose group.

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Mild fatty changes in the liver were observed in most of the animals in each of the groups, control as well as experimental groups. These changes were usually very mild in degree (as substantiated by the liver weights and liver lipid values) and were observed in Oil Red O stained sections as small, brilliantly stained globules diffuse in distribution, although usually most numerous in the periportal areas.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Mild fatty changes in the liver were observed in most of the animals in each of the groups, control as well as experimental groups.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 ppm

Based on:

test mat.

Remarks:

100 mg/kg bw/day

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

other: Liver lipid analysis

Critical effects observed:

no

System:

hepatobiliary

Organ:

liver

See attached results summary from paper

Conclusions:

General condition and body weight during the in-life phase at all doses were essentially similar. At termination, the body weights of the males were higher than the controls. Haematological parameters were unaffected. No adverse treatment related pathological changes at any dosage were reported. Changes seen after terminal necropsy were considered to be related to the age of the rats. At term ination, the total liver lipid values were significantly higher in the top dose group. No histopathological changes were seen at any dosage, however lipid staining of the livers revealed staining particularly in the periportal areas. There were also some incidences of hyaline casts in the renal tubules but there was no apparent relationship to treatment. The NOAEL was consider to be 1000 ppm (100 mg/kg bw/day).

Executive summary:

Male and female rats treated via the diet for 2 years with DHA were not adversely affected by treatment at any dosage. Lipid staining of the livers revealed some staining aprticularly in the periportal areas,

additionally the lipid content of the liver was increased at 1000 ppm, an adverse relationship to treatment was not concluded. A NOAEL of 1000 ppm (100 mg/kg bw/day) was considered appropriate.

Reference 5

Endpoint:

chronic toxicity: oral

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

other: Liver lipid analysis

Remarks on result:

other: Equivalent to 100 mg/kg bw/day

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subchronic

Species:

rat

System:

other: liver, stomach

Organ:

liver

stomach

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

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

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Endpoint conclusion

Endpoint conclusion:

no study available

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

Mode of Action Analysis / Human Relevance Framework

These data suggest that the liver may be a target, e.g. changes in liver lipid content/distribution. This may be an adaptive response, no overt adverse pathology of the liver was stated. Some effects were also noted on the stomach but the significance of systemic toxicity here was considered equivocal.

In the study in the monkey,only a single monkey/dose group was used, the biological significance of any findings therefore may be open to interpretation.

Justification for classification or non-classification

No significant adverse toxicity/pathology on the organs and tissues examined was noted . Changes in the liver (weight, pathology, lipid content/distribution) were considered to be in relation to xenobiotic induced changes in metabolism and therefore an adaptive response. No classification is proposed.