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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

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Diss Factsheets

Administrative data

Description of key information

Study 001: 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.

Study 002: rat dietary 0.02, 0.05, and 0.10 % (by weight, DHA – equivalent to 200, 500 or 1000 ppm or 16, 39 or 78 mg/kg bw/day). Two-year study, 25 animals/group. No adverse effects. NOEL 1000 ppm (78 mg/kg bw/day).

Study 003: 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. Adverse toxicity at 200 or 300 mg/kg bw/day. NOAEL 100 mg/kg bw/day.

Study 004: rat dietary 2500 ppm for 350 days -12 male rats treated group. Part of the study was designed investigate the adverse effects of 4-(dimethylamino)azobenzene (DAB) and any effects DHA may have on its own or in combination with DAB. DHA had no adverse toxic effects, NOAEL 2500 ppm (equivalent to 250 mg/kg/bw/day). The data suggested that DHA delayed the induction of hepatomas and cholangiocarcinoma’s in rats fed DAB.

Study 005: rat dietary 20 mg/day/head (dose in mg/kg not stated), 20 male rats treated 15 weeks with DHA. Transient body weight and food consumption reduced, liver weight increased with concomitant enzyme induction. No NOAEL.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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-acute 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
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)
Remarks:
Vehicle control
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:
no effects observed
Description (incidence and severity):
Blood urea-N was determined; the data were inconclusive
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
Description (incidence and severity):
No adverse effects at 0, 10, 30, 100 mg/kg bw/day
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
Remarks on result:
other:
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:
not specified
Relevant for humans:
not specified

Data from the second group of rats treated at 300 mg/kg bw/day were similar to the first group at the same dosage.

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.

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
Details on species / strain selection:
2-3 months old at commencement of study
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 (calculation approx. 16, 39 or 78 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 in diet
Dose / conc.:
0 ppm
Dose / conc.:
200 ppm
Remarks:
0.02% or 16 mg/kg bw/day
Dose / conc.:
500 ppm
Remarks:
0.05% or 39 mg/kg bw/day
Dose / conc.:
1 000 ppm
Remarks:
0.10% or 78 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 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:
no effects observed
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. 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
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:
78 mg/kg bw/day
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
other: Liver lipid analysis
Critical effects observed:
not specified

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 termination, 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 (78 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 (78 mg/kg bw/day) was considered appropriate.

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 toxicokinetic 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 tho sodium salt were calculated on the basis of its dehydroacetic acid equivalent.
Vehicle:
olive oil
Remarks:
Or water
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)
Remarks:
Control
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
DHA or DHA-Na
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
DHA or DHA-Na
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
DHA or DHA-Na
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
DHA or DHA-Na
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 and 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:
no effects observed
Description (incidence and severity):
No effects recorded at 200 mg/kg bw/day or below.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No effects recorded at 200 mg/kg bw/day or below.
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.
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
Critical effects observed:
not specified

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 show 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 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.

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Circa 1968
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 chronic study in the rat, dietary exposure, which was designed to investigate the ameliorative properties of DHA on tumours induced by 4-(dimethylamino)azobenzene (DAB). Key parameters of toxicity were evaluated.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
Male rats weighing 105 to 150 g were placed in experimental groups fed on a powered diet (CE-2, CLEA Japan Inc.) which incorporated the test material.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
350 days exposure for DHA treated rats.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
350 days
Frequency of treatment:
Daily
Dose / conc.:
0 ppm
Remarks:
Control
Dose / conc.:
2 500 ppm
Remarks:
0.25% DHA or 250 mg/kg bw/day
No. of animals per sex per dose:
12 males at 2500 ppm.
Control animals:
yes, concurrent no treatment
Details on study design:
The study was designed to investigte the adverse effects of 4-(dimethylamino)azobenzene (DAB), and any effects DHA may have on its own or in combination with DAB.
The treament groups are given below. Treatment group 1-D is most relevant and only reviewed here.

Group 1-A: 22 rats were fed 0.06% DAB diet for 126 days and then basal diet for 162 days.
Group 1-B: 21 rats, fed 0.06% DAB diet for 126 days and then 0.25% dehydroacetic acid diet for 162 days.
Group 1-C: 16 rats, fed 0.06% DAB plus 0.25% dehydroacetic acid diet for 173 days and then basal diet for 77 days.
Group 1-D: 12 rats, fed 0.25% dehydroacetic acid diet for 350 days.
Observations and examinations performed and frequency:
Not precisely specified.
Sacrifice and pathology:
After 350 days on study for DHA treated rats
Clinical signs:
no effects observed
Description (incidence and severity):
No adverse findings were observed in DHA treated rats
Mortality:
no mortality observed
Description (incidence):
No adverse findings were observed in DHA treated rats
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No adverse findings were observed in DHA treated rats
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:
no effects observed
Description (incidence and severity):
No adverse findings were observed in DHA treated rats
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No adverse findings were observed in DHA treated rats
Gross pathological findings:
no effects observed
Description (incidence and severity):
No adverse findings were observed in DHA treated rats.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No adverse histopathological findings were observed in DHA treated rats.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Administration of dehydroacetic acid increased the liver activity of metabolizing azo dyes in both demethylation and reductive cleavage.
Dose descriptor:
NOAEL
Effect level:
>= 2 500 ppm
Based on:
test mat.
Remarks:
250 mg/kg bw/day
Sex:
male
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
not specified

The results suggests that dehydroacetic acid delayed the induction of hepatomas and cholangiocarcinomas in rats fed DAB if dehydroacetic acid was administered simultaneously.

Conclusions:
DHA fed to Wistar rats for up to 350 days at 2500 ppm had no adverse toxicity in this study.
Executive summary:

DHA fed to Wistar rats for up to 350 days at 2500 ppm (250 mg/kg bw/day) had no adverse toxicity in this study. The data suggested that DHA delayed the induction of hepatomas and cholangiocarcinomas in rats fed DAB.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
other information
Study period:
Circa 1975
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:
Essentially a sub-chronic oral toxicity study, dietary, using a single treatment level. Key parameters of toxicity were assessed with some toxicokinetic analyses also.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
Feed (described as basic feed) and water were provided in fixed quantities at regular intervals, and the mass of any remaining food was recorded. Additionally, the body weight of each animal was measured twice a week and the environment was maintained at a temperature of 22°C and humidity of approximately 65%.
Route of administration:
oral: feed
Details on route of administration:
To accommodate animal growth, animals were initially given 15 g of feed (dry weight), which was later increased to 20 g. Dehydrated acetic acid (and tobacco extract) were prepared as solutions at a pre-determined concentration and mixed into the hot water used to form animals' feed into spherical dumplings.
Vehicle:
water
Remarks:
Mixed into diet.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
15 weeks.
Frequency of treatment:
Daily in diet
Dose / conc.:
0 other:
Remarks:
Control
Dose / conc.:
20 other: mg/day/head
Remarks:
DHA
No. of animals per sex per dose:
20/group
Control animals:
yes, concurrent vehicle
Positive control:
No
Observations and examinations performed and frequency:
Body weights twice a week; feed intake, observations - assumed at least weekly.
Sacrifice and pathology:
At the end of the study, animals were euthanised and autopsies were performed. Organ weights were measured and the liver was immediately frozen, after which enzyme activity was examined. Furthermore, urinary DHA was assayed in the DHA administration group and a phenol sulfophthalein test (PSP-test) was performed in the final phase of the study to evaluate whether changes in renal tubule function occurred after long-term administration, as DHA imay be a renal tubule inhibitor.
Statistics:
Yes, but methods not specified.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight was reduced in the DHA treated group during the first half of the trial, while in the latter half of the trial body weight growth was actually higher that the control group, though only by a very slight amount.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Results showed that overall feed uptake was lower in the DHA group than the control group. The calculated weight gain per unit of feed consumed and this ratio was significantly lower in the DHA group for the first half of the trial (at a significance level of 0.5%), before increasing to greater values than in the control group in the latter half of the trial. The above data suggest that reduced feed uptake was not the only factor responsible for the reduced body weight increase observed in animals administered DHA.
Food efficiency:
no effects observed
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):
A liver enzyme assay showed that alcohol dehydrogenase activity was reduced in the DHA group.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
The amounts of DHA excreted in urine were 2.71 mg, 3.07 mg, 3.02 mg and 2.95 mg in weeks 3, 6, 9 and 12 respectively, corresponding to roughly 15% of the amount administered. Furthermore, a PSP test conducted for the DHA group on the 15th and final week of testing showed that the excretion rate was 27.5% in the administration group versus 51.1% in the control group, indicating that long-term, continuous administration of DHA may result in decreased renal tubular function.
Behaviour (functional findings):
no effects observed
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The relative organ weight (to body weight) of the liver was increased and the kidney decreased.
Gross pathological findings:
no effects observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
A significant decrease in renal tubule function as well as renal atrophy was observed in the DHA group.
Histopathological findings: neoplastic:
not specified
Dose descriptor:
other: Single dose level
Effect level:
ca. 20 mg/kg diet
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
clinical biochemistry
food consumption and compound intake
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Critical effects observed:
yes
Lowest effective dose / conc.:
20 other: mg/day/head
System:
hepatobiliary
Organ:
kidney
liver
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
not specified
Conclusions:
Administration of DHA resulted in transient impaired body weight and food intake performance, decreased hepatic enzyme activity, and compensatory hypertrophy of the liver. Furthermore, a significant decrease in renal tubule function as well as renal atrophy was observed in the DHA group.
Executive summary:

DHA administered in the diet, 20 mg/day/head, to male rats for 15 weeks resulted in transient impaired body weight and food intake performance, decreased hepatic enzyme activity, and compensatory hypertrophy of the liver. Furthermore, a significant decrease in renal tubule function as well as renal atrophy was observed in the DHA group.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
78 mg/kg bw/day
Study duration:
chronic
Species:
rat
System:
other: GI tract, liver
Organ:
liver
stomach

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

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 the liver may be a target, e.g. liver enlargement and enzyme induction. This may be an adaptive response, no overt adverse pathology of the liver stated.

Additional information

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, metabolism) were considered to be in relation to xenobiotic induced changes in metabolism and therefore an adaptive response.