Potassium (E,E)-hexa-2,4-dienoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP

Justification for type of information:

Since the sorbate anion in the (eco-) toxicologically relevant moiety and its presence in dissociated or non-dissociated form is entirely determined by pH, extrapolation to potassium sorbate or vice versa is not considered to be restricted in any way.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 1998-09-21

Deviations:

yes

Remarks:

, no sensory reactivity test, no assessment of grip strength, no motor activity assessment and no functional observations were performed in this study. Uterus and ovaries were not weighed upon study termination.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

1988

Deviations:

yes

Remarks:

, no sensory reactivity test, no assessment of grip strength, no motor activity assessment and no functional observations were performed in this study. Uterus and ovaries were not weighed upon study termination.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Hexa-2,4-dienoic acid

EC Number:

203-768-7

EC Name:

Hexa-2,4-dienoic acid

Cas Number:

110-44-1

Molecular formula:

C6H8O2

IUPAC Name:

hexa-2,4-dienoic acid

Details on test material:

- Name of test material: Sorbic acid
- Physical state: White crystalline powder
- Stability: The test substance was considered to be stable for the duration of the study. 

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winckelmann GmbH, Borchen, Germany
- Age at study initiation: 5 to 6 weeks
- Weight at study initiation: Male (mean) = 146.5 g Female (mean) = 133.1 g

Administration / exposure

Route of administration:

oral: feed

Duration of treatment / exposure:

90-92 days, pending on dates for scheduled necropsy.

Frequency of treatment:

continuous

Doses / concentrations

Remarks:

Doses / Concentrations:
25000, 50 000 and 100 000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

Number of animals per group: 20 males and 20 females
Control animals: 20 males and 20 females

Control animals:

yes, plain diet

Details on study design:

Post-exposure period: none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Clinical signs once or twice daily and mortality twice daily
- Cage side observations checked in table [No.?] were included.

BODY WEIGHT: Yes
- Time schedule for examinations: Before treatment and weekly throughout the study

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Before treatment and weekly throughout the study

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations and dose groups that were examined: Examinations were carried out before start of study in all animals from all groups and at study end (week 13) in the control and high dose group  (100 000 ppm).

HAEMATOLOGY: Yes
- Time schedule:  End of study
- How many animals: All animals
- Parameters: Erythrocyte counts, haemoglobin, haematocrit, MCV, MCH, MCHC, reticulocytes, platelet, white blood cell counts, neutrophils, lymphocytes, monocytes, eosinophils, basophils, LU cells, coagulation time

CLINICAL CHEMISTRY: Yes
- Time schedule: End of study
- How many animals: All animals
- Parameters: Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, γ-glutamyltranspeptidase, bilirubin total, cholesterol, triglycerides, glucose, urea, creatinine, sodium, potassium, calcium, chloride, inorganic phosporous, total protein, albumin, globulinalbumin/globulin ratio.

URINALYSIS: No

Sacrifice and pathology:

GROSS PATHOLOGY AND HISTOPATHOLOGY: Yes
- Number of animals: All animals
- Time points: End of study
- Organs: Adrenal glands, aorta (thoracic), brain, caecum, colon, duodenum, epididymides, eyes, femur (with joint), harderian glands, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (submandibular and  mesenteric), oesophagus, optic nerves, ovaries with oviducts, pancreas, pituitary gland, prostate gland, salivary gland (mandibular and sublingual), seminal, vesicles, sciatic, nerve, skeletal muscle (thigh), skin with mammary glands, spinal cord (cervical, mid-thoracic and lumbar), spleen, sternum with marrow, stomach, testes, thymus, thyroid glands (with parathyroids) trachea, urinary bladder, uterus, vagina,  macroscopic abnormalities

ORGAN WEIGHTS: Yes
- Organs: Brain, heart, liver, kidneys, testes, epididymides, thyroid  gland, adrenal glands, spleen, thymus

Statistics:

Statistics: Body weight, body weight gain and food consumption were first transformed using an average time response and linear time response. The average and linear value were then analyzed for statistical significance (p ≤ 0.05) within each sex by a 1-way ANOVA with a two-sided ordinal step-down trend test. Haematological parameters, serum chemistry parameters, urinalysis parameters and organ weights were analyzed either by the step-down trend test, if normally distributed, or by the Jonckheere trend test with corrections for ties.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Statistically significant effects wered observed, but considered as biologically not relevant, see details on results.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Description (incidence and severity):

not applicable

Details on results:

- Clinical signs: No clinical signs of intoxication throughout the study period in any  group.

- Mortality: No unscheduled deaths troughout the study.

- Body weight gain: Body weight development was considered not adversely affected by the administration of the test compound throughout the study in any treated  group. There was a statistically significant negative trend (p < 0.05) of  mean body weight for high dose males (100 000 ppm) on study days 8, 15,  22 and 29 as compared to the control which, however, at each time point was far below 10 %. Hence this finding was considered as not biologically  relevant. In contrast, there was a statistically significant positive trend (p < 0.05) of mean body weight in the high dose females from study day 57 until  the end of the study, and for mid-dose females from study days 71 and 85  until end of study. Accordingly, statistical analysis of mean overall  body weight gains revealed a positive trend for high-dose females on  study days 29, 36 and 50 until the end of the observation period, for  mid-dose females on study day 85 and 91 and even for low-dose females on study day 91.

- Food consumption and compound intake: Mean daily food consumption was slightly reduced in all treated groups over the study period compared to the controls. This finding was considered treatment-related.

- Ophthalmoscopic examination: There were no ophthalmological findings during both the physical and slit lamp examination, which could be related to the dietary administration of  the test compound in any group. Only incidental findings occurred in all  groups including the control at pre-test and at final examination.

- Blood analysis:        
- Haematology: Evaluation of final haematology revealed no findings of toxicological  significance following dietary administration of the test compound in any  treated group as compared to the control. There were marginal statistically significant changes noted for the high dose males, which in  the absence of any corresponding findings in the females, as well as in the absence of any histopathological correlates, were considered as not biologically relevant. Slightly decreased mean corpuscular haemoglobin concentration (MCHC) as well as slightly increased mean reticulocyte counts (both statistically significant) were not  accompanied by changes in RBC, haemoglobin and haematocrit parameters that would be indicative of an anemia. Slightly decreased neutrophils (statistically significant) were accompanied by a non-significant increase in lymphocytes, but there was no dose-response  relationship and there was no clinical and histopathological correlate such as decreased platelets or glomerulopathnephritis, to support  hypersensitivity in this sex. A slight increase in numbers of unidentified lymphocytes, although statistically significant, was very marginal and  without any histopathological correlate in either thymus, spleen or bone  marrow. These findings were considered as incidental.

- Clinical chemistry: Evaluation of final serum clinical chemistry parameters revealed no  findings of toxicological significance following dietary administration of the test compound in any treated group. Slightly decreased transaminase (AST/ALT) activity for high dose females (statistically significant) indicated metabolic involvement of the test compound in the liver in particular for this sex, but in the absence of any target organ toxicity this finding was of no toxicological relevance. Slightly increased gamma-GT activity for mid- and high-dose  females (statistically significant) was at the upper physiological range for this rat strain and age, indicating a slightly higher liver cell turnover for this sex, but in the absence of a histopathological correlate (necrosis) in the liver, this findings was not considered as toxicologically significant. Slightly increased ALP activity for high dose  males as compared to the control (statistically significant) indicated metabolic involvement of this isoenzyme group (active in liver, bone marrow, kidney, gut) in particular  for this sex, but in the absence of any target organ toxicity this  finding was not considered as toxicologically significant. Slightly to moderately increased triglyceride and cholesterol values for all treated females (statistically significant) indicated involvement of  the lipid metabolism in the liver and correlated well with slightly increased body and liver weights in this sex, but in the absence of a histopathological correlate in the liver, this finding was not considered  as toxicologically significant. The decreased cholesterol for high dose  males was considered as incidental. Slightly decreased blood urea nitrogen and creatinine levels for high dose males (statistically significant) were confined to this sex only and considered as incidental. Slightly increased serum electrolytes (sodium,  potassium, calcium and chloride) for high dose males as well as for low-  and/or mid- and/or high dose females were statistically significant but marginal. However, in the  presence of a dose-response relationship, these findings were regarded as possibly compound related, but in the absence of clinical (blood urea nitrogen,  phosphorus) and/or histopathological correlates (kidney  function/morphology), this finding was not considered as toxicologically  significant. Slightly to moderately increased serum protein  and albumin/globulin ratio for high dose males and all dose females (statistically significant) were considered as an indirect consequence of the involved lipid metabolism (increased availability of transport lipo-proteins) in particular for the  females, but in the absence of any histopathological target organ toxicity these metabolic changes were not considered as toxicologically significant.

- Urinalysis: Not examined

- Sacrifice and pathology:
- Organ weights: The relative brain weights for mid and high dose females showed a  statistically significant slight decrease as compared to the control. The  corresponding absolute brain weights showed no effects. These effects  were considered to be due to slightly higher terminal body weights in these  groups and hence were considered not as a direct effect of the test compound. The absolute and relative liver weights in all treated females showed a statistically significant slight to moderate increase as compared to the control. These effects were considered to have occurred due to compound-induced metabolic activity in the liver for this sex. However, in the  absence of any histopathological correlate in this target organ, these  findings were not considered as toxicologically significant.The absolute and  relative spleen weights in mid- and high dose males showed a  statistically significant slight decrease and in high dose females a  slight increase as compared to the control.  As there was no histopathological correlate in the white and red spleen pulpa which could support this finding, and there were also no corresponding  correlates regarding hypersensitivity in other organs of the reticuloendothelial system, these effects were not considered as toxicologically significant.

- Gross and histopathology: There were no treatment-related macroscopic or microscopic findings. All  of the histopathological findings encountered were considered to have arisen spontaneously.

Effect levels

Dose descriptor:

NOAEL

Effect level:

100 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

food consumption and compound intake

mortality

ophthalmological examination

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

No overt clinical signs of toxicity, no mortalities, no-treatment related effects on food consumption and no ophthalmologic findings were observed during the study. Body weights were initially decreased in males at 100 000 ppm. The deviation from controls was always far below 10 % and returned to normal in study week 5. This finding was considered as toxicologically not relevant. In females, body weights were slightly to moderately increased from 25 000 ppm onwards as compared to the control, indicating sex-specific differences to the males. In both sexes, food intake was constantly lower as compared to the control, indicating weight compensation or even over-compensation through the relatively high caloric substitution via sorbic acid in the diet. The clinical pathology and organ weight data indicate sex-specific metabolic involvement of sorbic acid during the elimination process, which involved the lipid metabolism, transport proteins and liver weights in females, but not in males. In males, the only clinical pathology finding was a slight increase in ALP isoenzyme activity, suggesting a slightly different metabolic pathway for this sex. Therefore it is important to note that none of these metabolic changes identified in clinical pathology and organ weight evaluation in either males or females did result in any histopathologically evident target organ toxicity. These findings due to test compound-induced metabolic changes proved to be toxicologically insignificant in this test system.
Conclusion:
LO(A)EL: Not applicable
NO(A)EL: 100 000 ppm males and females (male: 6800 mg/kg/bw/d and female: 7200 mg/kg/bw/d)

The extrapolation from sorbic acid to potassium sorbate or vice versa is considered not to be restricted in any way, since the determinant of potential toxicity is on the "sorbate" anion.