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Description of key information

ORAL 
In a reasonably 28 days well conducted study, weanling rats exposed, NOAEL of 59 mg/kg/day was determined (Kaiser JA. 1964).
In 90 days repeated dose study: three independent series of feeding experiments were carried out (SERIES I, II, III). Their food intakes (similar for all the groups) and results were compared with those of unmedicated controls in parallel experiments. Results shows that only in SERIE III, with concentrations of from 0.06 to 0.12 per cent of dinitrophenol in the diet, the growth was definitely retarded and the animals attained a final weight some 75 grams lighter than the controls. At necropsy, and histologically, the tissues of the treated rats were indistinguishable from those of the unmedicated controls, there being no lesions which could be ascribed to the action of the drug. With concentrations of from 0.06 to 0.12 per cent of dinitrophenol in the diet, the growth was definitely retarded and the animals attained a final weight some 75 grams lighter than the controls. At necropsy, and histologically, the tissues of the treated rats were indistinguishable from those of the unmedicated controls, there being no lesions which could be ascribed to the action of the drug.
Data obtained from the publication of Mutsuko Koizumi et al., 2001. The study was performed with a dose-finding study (14 days study) and a main study of 28 days. Also an 18 study day was performed on newborn rats but it has not decribed because not considered as a standard 28 days-study.
As an unequivocally toxic level, 80 mg/kg/day is appropriate, based on the clear toxic signs with deaths at 80 mg/kg and the slight effects at 60 mg/kg in the dose-finding study. The NOAEL is presumed to be 20 mg/kg/day from the dose-finding study because adverse effects.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Data have been obtained from pubblication.
Qualifier:
according to
Guideline:
other: Test Guideline of the Japanese Chemical Control Act (Official Name: Law Concerning the Examination and Regulation of Manufacture, etc. of Chemical Substances)
Principles of method if other than guideline:
The study was performed with a dose-finding study (14 days study) and a main study of 28 days.
GLP compliance:
not specified
Remarks:
Rats were examined under GLP
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
not specified
Duration of treatment / exposure:
Dose-ranging study: 14 days
Main study: 28 days
Frequency of treatment:
once a day
Remarks:
Doses / Concentrations:
0
Basis:
other:
Remarks:
Doses / Concentrations:
3
Basis:
actual ingested
Remarks:
Doses / Concentrations:
10
Basis:
actual ingested
Remarks:
Doses / Concentrations:
30
Basis:
actual ingested
Remarks:
Doses / Concentrations:
80
Basis:
actual ingested
No. of animals per sex per dose:
Five to six-week old rats. Referring to the results of preliminary studies including the dose-finding study of 14 days, 5 doses including the control were established.
Control animals:
other: Recovery groups (4NP: 0, 160, 400, 1,000 mg/kg/day, DNP: 0, 30, 80 mg/kg/day) were maintamed for 2 weeks without chemical treatment and fully examined at 11 to 12 weeks of age.
Dose descriptor:
NOAEL
Effect level:
20 other: mg/kg/day
Dose descriptor:
LOAEL
Effect level:
30 other: mg/kg/day
Critical effects observed:
not specified

The number of animals for each sex/dose was 6 for both scheduled sacrifice and recovery. Rats were examined for general

behavior, body weight, food consumption, urinalysis, hematology and blood biochemistry, necropsy finding, organ weights and histopathological finding.

Continuous data were analyzed by Bartletts test (Bartlett, 1937) for homogeneity of distribution (p<0.Olor 0.05). When homogeneity was recognized, Dunnetts test (Dunnett, 1964) or Scheffes test (Scheffe, 1953) (p<0.Olor 0.05) was conducted for group comparison (control vs treatment). If not homogenous, the data were analyzed using the Kruskal-Wallis ranking analysis (Kruskal and Wallis, 1952) (p<0.05) or the mean rank test of Dunnett type (Hollander and Wolfe, 1973) (p<0.01 or 0.05).

Quantitative data for general appearance, functional tests and histopathology were analyzed by Mann-Whitneys U test (Mann and Whitney, 1947) or Fishers exact test (Fisher, 1973) (p<0.005 or 0.025).

Conclusions:
During the newborn period, the infants may orally intake chemicals not only through their mothers milk but also by mouthing of pacifiers, plastic goods, etc. or eating baby foods containing pollutant chemicals. The present study was therefore conducted to allow comparison of the results for newborn and young rats under the same experimental conditions.
In the overall study, NOAELs based on both the dose-finding study and main study was defined, and compared these values. Estimated appropriate unequivocally toxic levels from both animal studies and compared them in addition were also done. In this study, these were established as doses inducing severe toxic signs, including death or critical histopathological damage.
Many animal studies with oral administration were also conducted, but mostly in the 1930s and 1940s, and no toxicity study in infants has been reported. The present study provides the newest reliable data, with toxicity level and toxic profile in line with those of the old studies.

The unequivocally toxic effect levels were estimated to be 30 and 80 mg/kg/day, and NOAELs to be 10 and 20 mg/kg/day in newborn and young rats, respectively.
In this specific study of 28 days the LOAEL was established at 80 mg/kg/day an NOAEL at 20 mg/kg/day in young rats.
The differences in sensitivity between newborn and young rats range from 2 to 3 times.
Therefore, the toxicity differences between newborn and young rats would be due to variation in metabolic rate including elimination and toxicodynamics. However, there is no supporting information for this speculation in the literature.
Executive summary:

Data have been obtained from the publication of Mutsuko KOIZUMI et al., 2001, Comparative study of toxicity of 4-nitrophenol and 2,4-dinitrophenol in newborn and young rats, The Journal of Toxicological Sciences, Vol.26, No.5, 299 II, 2001. The study was performed with a dose-finding study (14 days study) and a main study of 28 days. Also an 18 study day was performed on newborn rats, but it hasn’t been considered as repeated dose study. A comparison of results is reported, but not details. Experimental protocol is described in scheme 1.

A solution of 85.2 % purity of 2,4-dinitrophenol was tested on Sprague-Dawley SPF rats following the Test Guideline of the Japanese Chemical Control Act (Official Name: Law Concerning the Examination and Regulation of Manufacture, etc. of Chemical Substances) under Good Laboratory Practice conditions.

A dose-finding study was performed for 14 days.

Five to six-week old rats were given the test substances by gastric intubation daily for 28 days and sacrificed after overnight starvation following the last treatment. Referring to the results of preliminary studies

including the above dose-finding study, 5 doses including the control were established for DNP: 0, 3, 10, 30, 80 mg/kg/day. Recovery groups (DNP: 0, 30, 80 mg/kg/day) were maintained for 2 weeks without chemical treatment and fully examined at 11 to 12 weeks of age. The number of animals for each sex/dose was 6 for both scheduled sacrifice and recovery. Rats were examined for general behavior, body weight, food consumption, urinalysis, hematology and blood biochemistry, necropsy finding, organ weights and histopathological finding. Statistical analysis was performed.

Clear toxic signs, such as decrease in locomotor activity, prone position, ptosis, panting, crawling position and salivation, were observed repeatedly during the dosing period at 80 mg/kg in both sexes, and 2males and 6 females died. However, decrease in locomotor activity and salivation in the 30 mg/kg group were mostly observed only after the first dosing. The relative liver weights were increased in both sexes of the 80 mg/kg scheduled-sacrifice group, and this persisted through the recovery period (data not shown). Relative organ weights for brain, kidneys and testes were increased only in 80 mg/kg males. On histopathological examination, mineralization of the corticomedullary junction in kidneys was observed in both sexes at 80 mg/kg in the scheduled-sacrifice and recovery groups, but the change was only statistically significant in males of the scheduled-sacrifice group. On hematological examination increase in Hb and Ht during the treatment, and decrease in RBC, Hb and Ht in the recovery period were observed, limited to 80 mg/kg males (data not shown). Although blood chlorine levels were slightly decreased in 30 and 80 mg/kg males and total bilirubin was slightly increased in females receiving 10 mg/kg and more (data not shown), no changes in histopathology or organ weights were observed at 30 mg/kg or lower.In the dose-finding study, no significant changes were apparent except for a prone position of a few animals given 60 mg/kg during the early dosing period. As anunequivocally toxic level, 80 mg/kg/day is appropriate, based on the clear toxic signs with deaths at 80 mg/kg and the slight effects at 60 mg/kg in the dose-finding study. The NOAEL is presumed to be 20 mg/kg/day from the dose-finding study because adverse effects at 30 mg/kg were mostly observed only after the first dosing in the main study even though the exposure period of the dose-finding study was shorterthan the main study.

In the overall study (18 days on newborn and 28 days on young rats) the unequivocally toxic effect levels were estimated to be 30 and 80 mg/kg/day, and NOAELs to be 10 and 20 mg/kg/day in newborn and young rats, respectively. The differences in sensitivity between newborn and young rats range from 2 to 3 times. Therefore, the toxicity differences between newborn and young rats would be due to variation in metabolic rate including elimination and toxicodynamics. However, there is no supporting information for this speculation in the literature.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
20 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Clinical studies on humans are available to support animals studies.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
Occupational exposure on humans are available and described on the discussion.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
Occupational exposure on humans are available and described on the discussion.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
Occupational exposure on humans are available and described on the discussion.

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
Occupational exposure on humans are available and described on the discussion.

Additional information

ORAL

In a reasonably well conducted study, weanling rats exposed to 59 mg/kg/day dose of 2,4 -dinitrophenol in the diet for 4 weeks(28 days). No mortality LD0 was observed at dose <= 59 mg/Kg/day, but a NOAEL of 59 mg/kg/day was determined because food consumption and body weight gain in rats exposed to that dose were similar to those of controls (Kaiser JA. 1964).

Data obtained from the publication of Mutsuko Koizumi et al., 2001. The study was performed with a dose-finding study (14 days study) and a main study of 28 days. Also an 18 study day was performed on newborn rats, but it hasn’t been considered as repeated dose study.

A solution of 85.2 % purity of 2,4-dinitrophenol was tested on Sprague-Dawley SPF rats following the Test Guideline of the Japanese Chemical Control Act (Official Name: Law Concerning the Examination and Regulation of Manufacture, etc. of Chemical Substances) under Good Laboratory Practice conditions.A dose-finding study was performed for 14 days.

Five to six-week old rats were given the test substances by gastric intubation daily for 28 days and sacrificedafter overnight starvation following the last treatment. Referring to the results of preliminary studies

including the above dose-finding study, 5 doses including the control were established for DNP: 0, 3, 10, 30, 80 mg/kg/day. Recovery groups (DNP: 0, 30, 80 mg/kg/day) were maintained for 2 weeks without chemical treatment and fully examined at 11 to 12 weeks of age. The number of animals for each sex/dose was 6 for both scheduled sacrifice and recovery. Rats were examined for general behavior, body weight, food consumption, urinalysis, hematology and blood biochemistry, necropsy finding, organ weights and histopathological finding. Statistical analysis was performed.

Clear toxic signs, such as decrease in locomotor activity, prone position, ptosis, panting, crawling position and salivation, were observed repeatedly during the dosing period at 80 mg/kg in both sexes, and 2males and 6 females died. However, decrease in locomotor activity and salivation in the 30 mg/kg group were mostly observed only after the first dosing. The relative liver weights were increased in both sexes of the 80 mg/kg scheduled-sacrifice group, and this persisted through the recovery period (data not shown). Relative organ weights for brain, kidneys and testes were increased only in 80 mg/kg males. On histopathological examination, mineralization of the corticomedullary junction in kidneys was observed in both sexes at 80 mg/kg in the scheduled-sacrifice and recovery groups, but the change was only statistically significant in males of the scheduled-sacrifice group.

On hematological examination increase in Hb and Ht during the treatment, and decrease in RBC, Hb and Ht in the recovery period were observed, limited to 80 mg/kg males (data not shown). Although blood chlorine levels were slightly decreased in 30 and 80 mg/kg males and total bilirubin was slightly increased in females receiving 10 mg/kg and more (data not shown), no changes in histopathology or organ weights were observed at 30 mg/kg or lower.In the dose-finding study, no significant changes were apparent except for a prone position of a few animals given 60 mg/kg during the early dosing period. As an unequivocally toxic level, 80 mg/kg/day is appropriate, based on the clear toxic signs with deaths at 80 mg/kg and the slight effects at 60 mg/kg in the dose-finding study. The NOAEL is presumed to be 20 mg/kg/day from the dose-finding study because adverse effects at 30 mg/kg were mostly observed only after the first dosing in the main study even though the exposure period of the dose-finding study was shorterthan the main study.

In the overall study (18 days on newborn and 28 days on young rats) the unequivocally toxic effect levels were estimated to be 30 and 80 mg/kg/day, and NOAELs to be 10 and 20 mg/kg/day in newborn and young rats, respectively. The differences in sensitivity between newborn and young rats range from 2 to 3 times. Therefore, the toxicity differences between newborn and young rats would be due to variation in metabolic rate including elimination and toxicodynamics. However, there is no supporting information for this speculation in the literature.

90 days repeated dose study length of life of growth on white rats was performed. Microscopic appearance of vital organs was also studied. In fact, at time of death, necropsies and histological studies of the tissues were made. The drug 2,4-dinitrophenol was administered in the food to various groups of white rats, beginning shortly after weaning when they weighed about 30 grams, and continuing until death. Three independent series of feeding experiments were carried out (SERIES I, II, III). Their food intakes (similar for all the groups) and results were compared with those of unmedicated controls in parallel experiments. Feed containing different concentrations of 2,4 -dinitrophenol was administered freely to different groups of litter-mate males.

SERIES I (four groups, five rats each group) = 1°group: control; 2° group: 0.0002 %; 3° group: 0.001 %, 4° group: 0.005 % of 2,4 -DNP

SERIES II (five groups, five rats each group) = 1°group: control; 2° group: 0.01 %; 3° group: 0.005 %, 4° group: 0.01 % ; 5° group: 0.02 % of 2,4 -DNP

SERIES III (seven group, six rats each group)= 1°group: control; 2° group: 0.02 %; 3° group: 0.04 %, 4° group: 0.06 % ; 5° group: 0.08 %, 6° group: 0.12 %, 7° group: 0.24 % of 2,4 -DNP.

Results shows that on the I SERIE the rates of growth of the rats and their food intakes were identical with the controls. All of these rats had survived up to the five hundred and first day when the entire series of animals was killed accidentally. In as much as these rats received doses, which in other series were found to be entirely ineffective, it is not necessary to consider these animals further.

In the SERIE II rats weights average was 33 grams at the time the experiment was started. The average weights of these rats were practically identical up to the time when deaths made the averages somewhat irregular. The food consumption, when estimated either per rat or per kilogram of body weight, was not significantly different in the various groups.

SERIE III: the rats receiving concentrations of 0.02 and 0.04 per cent dinitrophenol grew at substantially the same rate as the controls. With concentrations of from 0.06 to 0.12 per cent of dinitrophenol in the diet, the growth was definitely retarded and the animals attained a final weight some 75 grams lighter than the controls. The food intake of the controls tended to be a little higher than of the other rats during the last half of the experiment, but this was not true during the first 400 days, when the growth differences were also present. With the concentration of 0.24 per cent the rats failed to eat or grow, and died promptly.

At necropsy, and histologically, the tissues of the treated rats were indistinguishable from those of the unmedicated controls, there being no lesions which could be ascribed to the action of the drug. Therefore, the administration of dinitrophenol in the maximum doses which would not be acutely fatal throughout almost the entire life-span of white rats, produced only minimal evidences of toxic effects, as indicated by rates of growth, and these only in the highest dosage ranges, far beyond those clinically important (Tainter, 1938).

Despite the well performed study, values ​​ are not well defined in mg/kg dose. These values are subjected to various interpretations because the concentration reported in % of 2,4-DNP coul be compared to weight of total amount of food or animal weight.

A study part of a larger clinical trial of sodium 2,4-DNP involving 170 patients who ingested an average of 4.0 mg/kg/day 2,4-DNP for an average of 88 days defines a LOAEL for 37 obese patients 1.2 mg/kg/day for an average of 14 days (Tainter et al. 1935b).

Data onhuman,a poisoning clinical studies are available.

In a firstclinical observation2,4-dinitrophenol sodium salt caused death at a first dose of c.a 46 mg plus another 46 mg dose 1 week later. It showed toxic effect, considered by the authors to be similar to those seen in heart stroke .

The most significant symptoms were high fever, hyperpnea and chest pain, high rectal temperature and rapid pulse (Tainter and Wood, 1934).

In a secondclinical observation adose of 7 mg of 2,4-dinitrophenol sodium salt has been taken for 5 days by a woman who died after comatose state. She had complained of headache, backache, weakness, dizziness, shortness of breath, and excessive perspiration, high temperature, pulse and respiration rate. Autopsy and histological examination revealed hyperemic and hemorrhagic lungs, degeneration of renal tubules and liver cells, segmentation and fragmentation of cardiac muscles, hemorrhagic spleen, stomach mucosa, spinal cord, pons, and medulla. Slight ganglion cell degeneration was also found in the pons (Poole and Haining, 1934).

In aclinical psychiatric studya dose of 2.66 mg/kg/day of 2,4-dinitrophenol has been taken for 14 days by a woman who died. Before death her pulse, respiration rate and temperature increased and she she became comatose. Because autopsy was delayed for 4 days, no conclusions regarding histopathological lesions could be made (Masserman and Goldsmith, 1934).

In aclinical observation2,4-dinitrophenol caused toxic effect as body temperature increasing, changing in cardiac rate and coma. The Lowest published lethal dose LDLo on human is 36 mg/kg.

A patient who took 2.3 mg/kg/day 2,4-DNP for 14 days and had severe dermatological reactions: severe pruritus, edema, maculopapular eruptions covered the entire body, with the exception of the face and scalp (LOAEL for dermatological effects). She developed pain in her fingers and all large joints after taking the dose (LOAEL of 2.3 mg/kg for neuritis). (Anderson et al. 1933).

A young obese woman died taking a dose of 1.03 mg/Kg/day for 46 days by capsules.Elevated respiratory rates, dyspnea, cyanosis,vascular congestion in the lungs and have been reported.Elevated pulse rate, tachycardia, hyigh body temperature and excessive perspiration were also noted.At the same dose, necrosis and ulceration of small intestine, severe neutropenia, agranulocytosis, servere fatty changes, hemorrhagic nephritis, extensive vascularization of spleen and pituary, goiter an thyroid were serious symptoms revealed at 1.03 mg/kg/day (LOAEL) (Goldman and haber, 1936).

Cataracts developed in a small percentage of patients who took 2,4-DNP or sodium 2,4-DNP as a weight reduction aid for acute, intermediate, and chronic durations (Haber, 1942).Cataracts developed also in patients who were at an age when senile cataracts do not occur.at dose of 1.86 mg/kg/day 2,4-DNP (LOAEL),developing blurred vision which was attributed to bilateral cataracts.

INHALATION/DERMAL ON HUMANS

Occupational exposuresindicate that inhalation ad dermal routes are the major way of exposure in workplaces. Illness and death are associated to workers exposure.Von Oettinger in 1949 stated that there had been 27 reported cases of fatal occupational dinitrophenol poisoning in the United States between 1914 and 1916.Deaths by poisoning occurred after exposure to airborne vapour and dust and direct dermal contact in a munitions industry (Perkins, 1919). Toxic effects included: respiratory, gastrointestinal effect with losing of body weight and metabolic effects.

Is to be specified that fatal cases occurred particularly among alcoholics and workers with renal or hepatic disease

Anotheroccupation al observationof two workers in a U.S manufacture exposed to mists and dust for a few months, with significant dermal exposure and even oral absorption, indicates signs of metabolic toxicity (fever, profuse sweating, restlessness) until death. Workroom air levels, determined after the deaths occurred, were “normally” at least 40 mg/m^3, but this value may underestimate breathing zone levels. The warmer weather during the second period of could have contributed on increasing dermal absorption and exacerbated the effects.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Of the three studies on repeated toxicity available (28 and 90 days), the 28 days of Koizumi is the most appropriate due to the exposure on young rats, more sensible than the adults. The 90-day study ( Tainter, 1938) indicates toxicity values ​ not defined in mg/kg and subject to various interpretations.

Justification for classification or non-classification

In general, toxicity effects are consequences of the uncoupling of oxidative phosphorylation.

2,4-DNP is an uncoupler of oxidative phosphorylation. In humans or animals exposed to 2,4-DNP, the energy produced from the Krebs cycle is not stored in adenosine triphosphate (ATP), but is released as heat. This short-circuiting of metabolism results in the characteristic clinical signs of increased basal metabolic rate, oxygen consumption, perspiration, and body temperature. Elevated environmental temperatures may compromise the body’s ability to dissipate the heat. Damages to several organs could be caused by uncoupling of oxidative phosphorylation. Gastrointestinal, body weight, metabolic, neurologic, Respiratory, cardiovascular, hepatic, musculoskeletal hematological and neurological, muskoloskeletal, renal, endocrine, dermal ocular and other systemic effect can be generate from the assumption of this drug (further details on these effects are described in section 7.9- specific investigations).

2,4- dinitrophenol produce significant toxicity in humans in animals and humans. It can be classified according to Regulation 1272/2008 (CLP) as STOT RE 1. H373: may causes damage to organs affected, through prolonged exposure. This classification according to the European Commission decision for the inclusion in annex I of the Directive 67/548/EEC.