Registration Dossier

Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 February 1994 to 09 March 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
animal room humidity was outside protocol limits on one occasion with no impact on the study
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
Commission Directive 92/69/EEC
Deviations:
yes
Remarks:
animal room humidity was outside protocol limits on one occasion with no impact on the study
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Fatty acids, C12-18, α-sulfo, 1-Me esters, sodium salts
EC Number:
288-224-7
EC Name:
Fatty acids, C12-18, α-sulfo, 1-Me esters, sodium salts
Cas Number:
85681-86-3
Molecular formula:
C13H25NaO5S - C19H37NaO5S
IUPAC Name:
Fatty acids, C12-18, α-sulfo, 1-Me esters, sodium salts
Constituent 2
Chemical structure
Reference substance name:
Fatty acids, C12-18, α-sulfo, disodium salts
EC Number:
286-085-7
EC Name:
Fatty acids, C12-18, α-sulfo, disodium salts
Cas Number:
85186-99-8
Molecular formula:
C12H22Na2O5S - C18H34Na2O5S
IUPAC Name:
Fatty acids, C12-18,α-sulfo, disodium salts

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
CD strain
Sex:
male/female
Details on test animals or test system and environmental conditions:
ANIMALS AND ANIMAL HUSBANDRY
- A sufficient number of male and female Sprague-Dawley CD strain rats were obtained from Charles River (UK) Limited, Manston, Kent.
- On receipt the animals were examined for signs of ill-health or injury.
- Animals were acclimatised for seven days during which time their health status was assessed.
- A total of 40 animals (20 males and 20 females) were accepted into the study.
- Animal weights at the start of treatment were 128-161 g (males) and 126-152 g (females).
- The animals were housed in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper.
- Animals were allowed free access to food and water.
- A pelleted diet (Rat and Mouse SQC Expanded Diet No 1; Special Diets Services Limited, Witham, Essex, UK) was used.
- Mains water was supplied from polycarbonate bottles attached to the cage.
- Diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
- Animals were housed in a single air-conditioned room within the barrier-maintained rodent facility.
- Rate of air exchange was at least fifteen air changes per hour.
- Low intensity fluorescent lighting was controlled to give 12 hours continuous light and 12 hours darkness.
- Environmental conditions were continuously monitored by a computerised system and print-outs of the mean temperatures and humidities were included in the study records.
- Room temperature was maintained at 20-21 °C.
- Relative humidity was maintained at 27-55 %. Humidity fell outside protocol limits on one occasion but was not considered to have affected the purpose or integrity of the study.
- Animals were randomly allocated to dose groups using random letter tables and the group mean bodyweights were then determined to ensure similarity between the dose groups. Cage distribution within the holding rack was also randomised.
- The animals were uniquely identified within the study by an ear punching system and colour coded cage labels were used to assist recognition of dose groups.


Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
- Treatment was administered using a stainless steel cannula attached to a disposable plastic syringe.
- Control animals were treated in an identical manner with distilled water.
Vehicle:
other: distilled water
Details on oral exposure:
TEST MATERIAL AND EXPERIMENTAL PREPARATION
- Test material was prepared at appropriate concentrations in distilled water.
- Stability and homogeneity of test item formulations were determined (see Appendix IX, attached).
- Based on demonstrated stability of at least 10 days, formulations were prepared weekly and stored at 4 °C in the dark.

PROCEDURE
- Volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Concentration of test material in the test samples was determined using a colorimetric method (see Appendix IX, attached).
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1; animals 1-5 (males) and 5-10 (females); treatment volume 5 mL/kg
Dose / conc.:
15 mg/kg bw/day (nominal)
Remarks:
Group 2; animals 11-15 (males) and 16-20 (females); treatment volume 5 mL/kg
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
Group 3; animals 21-35 (males) and 26-30 (females); treatment volume 5 mL/kg
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4; animals 31-35 (males) and 36-40 (females); treatment volume 5 mL/kg
No. of animals per sex per dose:
Five males and five females
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CLINICAL SIGNS
- All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing and 1 to 5 hours after dosing during the working week.
- Animals were observed immediately before dosing and one hour after dosing at weekends.
- All observations were recorded.

BODYWEIGHT
- Individual bodyweights were recorded on Day 0 (the day before start of treatment) and on Days 7, 14, 21 and 28.
- Bodyweights were also recorded at necropsy.

FOOD CONSUMPTION
- Food consumption was recorded for each cage group at weekly intervals throughout the study.

WATER CONSUMPTION
- Water intake was measured and recorded daily for each cage group.

LABORATORY INVESTIGATIONS
- Haematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 28).
- Blood samples were obtained from the lateral tail vein.
- Animals were not fasted prior to sampling.

HAEMATOLOGY
- Parameters measured for blood collected into tubes containing potassium EDTA anticoagulant were heamatocrit (Hct); haemoglobin (Hb); erythrocyte count (RBC); total leucocyte count (WBC); differential leucocyte count; platelet count (PLT); erythrocyte indices - mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV); mean corpuscular haemoglobin concentration (MCHC).
- Clotting time (CT) was assessed by Hepato Quick time using samples collected into sodium citrate solution (0.11 moL/L).

BLOOD CHEMISTRY
- Parameters measured on plasma from blood collected into tubes containing lithium heparin anticoagulant were blood urea; total protein; albumin; albumin/globulin ratio (by calculation); sodium; potassium; chloride; calcium; inorganic phosphorus; creatinine; alkaline phosphatase (AP); alanine aminotransferase (ALAT); aspartate aminotransferase (ASAT); glucose; total bilirubin.
Sacrifice and pathology:
PATHOLOGY
- On completion of the dosing period, all animals were killed by intravenous administration of sodium pentobarbitone solution (Sagatal, 60 mg/mL; May and Baker Limited, Dagenham, Essex, UK) followed by exsanguination.
- All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS
- Organs dissected free of fat and weighed before fixation were adrenals; brain; gonads; heart; kidneys; liver; pituitary; spleen.

HISTOPATHOLOGY
- Tissue samples removed from all animals and preserved in buffered 10 % formalin were adrenals; aorta (thoracic); bone and bone marrow (femur); bone and bone marrow (sternum); brain, caecum; colon; duodenum; eyes; gross lesions; heart; ileum; jejunum; kidneys; liver; lungs; lymph nodes (cervical and mesenteric); muscle (skeletal); oesophagus; ovaries; pancreas; pituitary; prostate; rectum; salivary glands; sciatic nerve; seminal vesicles; skin (hind limb); spleen; stomach; testes; thymus; thyroid/parathyroid; trachea; urinary bladder; uterus.
- All tissues were despatched to Experimental Pathology Services (Willow Court, Netherwood Road, Rotherwas, Hereford, UK) for processing.
- Preserved tissues from all test and control animals (groups 1 to 4) prepared as paraffin blocks, sectioned at nominal thickness of 5 µm and stained with haematoxylin and eosin were adrenals; testes; spleen; stomach; heart; kidneys; liver.
- Macroscopically observed lesions were also processed.
- Microscopic examination was initially performed on control and high dose group tissues (groups 1 and 4) only. The examination was subsequently extended to include sections of stomach from all animals in the remaining dose groups because indications of treatment-related gastric changes were observed.
Statistics:
EVALUATION OF DATA
- Data were processed to give group mean values and standard deviations where appropriate.
- Absolute and relative organ weights, haematological and blood chemical data were analysed by one way analysis of variance incorporating 'F-max' test for homogeneity of variance.
- Data showing heterogeneous variances were analysed using Kruskal Wallis non-parametric analysis of variance and Mann Whitney U-test.
- Probability values were calculated as p < 0.001 (***); p < 0.01 (**); p < 0.05 (*); p ≥ 0.05 (not significant).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Clinical observations are given in Tables 1 to 8 (attached).
- High dose animals of either sex showed short-lived increased salivation either before or immediately after dosing from Day 2 onwards together with associated findings of fur wetting, noisy respiration and red/brown staining of the fur, snout and around the mouth.
- Lethargy, diureses and diarrhoea were also seen on isolated occasions during the first week of treatment with sporadic incidents of more persistent salivation evident throughout the treatment period.
- Two high dose females underwent a noticeable deterioration in condition on Days 4 and 5 of treatment with dehydration, ptosis, abdominal distension, hunched posture, pilo-erection, gasping respiration, decreased respiratory rate and pallor of the extremities seen in addition to the observations common to other animals treated at this dose level. The condition of these two animals rapidly improved and the more severe clinical signs of toxicity were no longer evident by the end of Day 6.
- Intermediate and low dose animals showed no clinically observable signs of toxicity during the study.
- One intermediate dose male had noisy respiration before dosing on Day 18 but this was no longer evident one hour later and was not considered to be indicative of systemic toxicity. One female from the same dose group showed increased salivation before dosing on Day 19 but in isolation this finding was not considered to be toxicologically important.
Mortality:
no mortality observed
Description (incidence):
- No animal deaths took place during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Group mean weekly bodyweights and standard deviations are given in Tables 9 and 10 (attached) and are presented graphically in Figures I and II (attached).
- High dose males showed a slightly lower bodyweight gain than controls over the treatment period.
- There was no adverse effect on bodyweight development at the remaining dose levels.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
- Group mean weekly food consumptions are given in Tables 11 and 12 (attached) and are presented graphically in Figures III and IV.
- There were no convincing effects on dietary intake.
Food efficiency:
no effects observed
Description (incidence and severity):
- Weekly food efficiencies are given in Tables 13 and 14 (attached).
- Animals treated with the test material showed similar weekly food efficiencies to controls during the study.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
- Group mean daily water consumptions are given in Tables 15 and 16 (attached).
- High dose females showed a slightly greater water intake than controls over the treatment period.
- Water consumption of high dose males and animals from the remaining dose groups was unaffected by treatment.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- Group mean values and standard deviations for test and control group animals are given in Tables 17 and 18 (attached).
- No treatment-related changes in measured haematological parameters were observed.
- High dose females showed a sligh but statistically significant increase in clotting time compared with controls whilst males from the same group showed a slight reduction in this parameter.
- All individual values were within the normally expected range for rats of the strain and age used in the study and, in the absence of a consistent effect or any other changes that could be associated with altered clotting potential, the intergroup differences were considered to have arisen fortuitously.
- A statistically significant intergroup difference in neutrophil count was confined to intermediate dose females. The finding was not dose-related and was not considered to be treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
- Group mean values and standard deviations for test and control group animals are given in Tables 19 and 20 (attached).
- There were no treatment-related changes in the blood chemical parameters measured.
- High dose males showed a slight but statistically significant reduction in total plasma protein and plasma alkaline phosphatase concentration compared with controls whilst females from this dose group showed a slight increase in plasma aspartate amino-transferase and bilirubin levels. None of the individual values were outside the normally expected ranges for rats of the strain and age used in the study and the findings were not considered to be toxicologically significant, particularly in the absence of any histopathological evidence of liver damage.
- An increase in plasma albumin was observed in intermediate dose males and an increase in creatinine levels was seen in low dose males. The findings were not dose-related and were not considered to be treatment-related.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- Group mean absolute and relative organ weights are presented in Tables 22 to 25 (attached) together with standard deviations for test and control animals.
- No treatment-related organ weight changes were detected.
- All three female treatment groups showed a statistically significant reduction in kidney weight, both absolute and relative to bodyweight, compared to controls. However, none of the individual values were abnormally low for rats of the strain and age used in the study and, in the absence of any other evidence of renal damage, these intergroup differences were considered to be due to the unusually high control values and of no toxicological significance.
- High dose females also showed a slight but statistically significant reduction in spleen weight, relative to bodyweight, compared to controls. None of the individual values were outside the expected range for rats of the strain and age used in the study and, in the absence of any morphological splenic abnormalities, the intergroup difference was considered to have arisen fortuitously.
- Absolute brain weight was reduced in high dose females but no such intergroup difference existed when brain weight was expressed relative to bodyweight so this finding was not considered to be toxicologically significant.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- Summary incidence of necropsy findings is given in Table 21 (attached).
- Most high dose animals showed thickening of the non-glandular region of the stomach at terminal kill. Three of these animals also had numerous raised white foci on the non-glandular epithelium and/or limiting ridge of the stomach whilst sloughing of the non-glandular region was confined to two females. A small dark focus was also visible in the centre of the non-glandular region of one of these animals whilst the same region of another female contained multiple small light brown adhesions.
- In addition to the gastric abmormalities, the liver of one high dose female had an accentuated lobular pattern at terminal kill whilst two males from this dose group had small and/or pale adrenals. No macroscopic abnormalities were evident at either the intermediate or low dose level.
- Dark patches were detected on the lungs of one high dose female at necropsy and identified histopathologically as areas of haemorrhage, oedeme and pneumonitis. These findings were consistent with accidental introduction of a small amount of an irritant test material into the respiratory tract during dosing and, in isolation, were not considered to be toxicologically important.
- One control male had slight hydronephrosis in both kidneys but this is a common congenital finding in rats of the strain and age used so was clearly unrelated to test material toxicity.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Summary incidence of histopathological findings is given in Table 26 (attached).
- Treatment related gastric changes were observed.
- Epithelial acanthosis, frequently associated with hyperkeratosis, was observed in relation to treatment in the forestomach of rats of either sex dosed at 150 or 1000 mg/kg/day.
- All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed. Since there were no differences in incidence or severity between control and treatment groups, no toxicological significance was attributed to the changes.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
DISCUSSION
- Oral administration of test material by gavage for 28 consecutive days resulted in treatment-related changes at dose levels of 150 and 1000 mg/kg/day.
- The primary effect of treatment at 1000 mg/kg/day was gastric irritation. Macroscopic changes detected in the non-glandular region of the stomach at necropsy were identified histopathologically as epithelial acanthosis and/or hyperkeratosis. Furthermore, the short-lived increased salivation observed clinically at this dose level and the slightly increased water intake demonstrated for the females were also probably associated with the irritant nature of the test material.
- Repeated administration of the test item at 1000 mg/kg/day also resulted in a slight impairment of male bodyweight development over the treatment period. Furthermore, clinical signs of toxicity, indicative of a general deterioration in health, were also evident at this dose level, mostly during the first week of the study. In particular, two of the females showed a much more severe response to treatment than the remaining animals from this dose group on Days 4 and 5 although the aetiology of this response is unclear as sever clinical signs of toxicity were extremely short-lived and were confined to these two animals. The aetiology of the isolated adrenal and liver abnormalities seen at necropsy is equally unclear. Histopathological examination of these tissues failed to show any morphological correlates and, in the absence of any further convincing evidence of an adverse effect on these tissues, the spurious findings are of dubious toxicological significance.
- Effects of treatment at 150 mg/kg/day were limited to morphological gastric changes with epithelial acanthosis and, less frequently, hyperkeratosis detected in the non-glandular region of the stomach. However, there was no further convincing evidence of gastric irritancy at this dose level and, in the absence of any systemic toxicity, the minimal morphological changes are considered not to represent a serious threat to the health of the animals. Futhermore, there is no anatomical equivalent of the non-glandular forestomach in man.
- Animals dosed at 15 mg/kg/day showed no toxicologically significant changes in any of the parameters measured.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
gross pathology
water consumption and compound intake
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
gross pathology
water consumption and compound intake

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

TEST ITEM FORMULTATIONS

- Test material formulations were found to be stable for at least 10 days (see Appendix IX, attached).

- Prepared formulations were found to be ± 9 % of the nominal concentration.

Applicant's summary and conclusion

Conclusions:
Oral administration of the test material to rats by gavage for a period of 28 consecutive days at dose levels up to 1000 mg/kg/day resulted in treatment-related changes at 150 and 1000 mg/kg/day. Acanthosis and hyperkeratosis of the non-glandular gastric epithelium is not indicative of serious damage to the health of the animal and a No Observed Adverse Effect Level (NOAEL) was established at 150 mg/kg/day.
Executive summary:

Test material was administered at dose levels of 15, 150 and 1000 mg/kg/day by gavage to three groups of Sprague-Dawley CD strain rats for 28 consecutive days (five males and five females in each group). A control group of five males and five females was dosed with vehicle alone (distilled water). Clinical signs, bodyweight plus food and water consumption were monitored during the study. Haematologyand blood chemistry were evaluated for all animals at the end of the study. All animals were subjected to gross necropsy and a limited histopathological evaluation of tissues was performed.

No animal deaths took place during the study. With respect to clinical observations, high dose animals of either sex showed short-lived increased salivation from Day 2 onwards together with associated findings of fur wetting, noisy respiration and red/brown staining of the external body surface. Lethargy, diuresis and diarrhoea were also seen on isolated occasions during the first week of treatment whilst sporadic incidents of more persistent increased salivation were evident throughout the treatment period. Two high dose females underwent a noticeable deterioration in condition on Days 4 and 5 but the health of these animals rapidly improved and severe clinical signs of toxicity were no longer evident by the end of Day 6. Intermediate and low dose animals showed no clinically observable signs of toxicity during the study. High dose males showed a slightly lower bodyweight gain than controls over the treatment period but there was no adverse effect on bodyweight development at the remaining dose levels. No convincing effects on dietary intake were noted. High dose females showed a slightly greater water intake than controls over the treatment period but water intake was unaffected by treatment at the remaining dose levels. No treatment-related changes were seen in haematological parameters or blood chemical parameters measured.

Most high dose animals showed thickening of the non-glandular region of the stomach at terminal kill. Several other abnormalities were also observed in this region of the stomach including sloughing, multiple raised white foci, a small dark focus in the centre of the region and multiple light brown adhesions. Multiple raised white foci were also occasionally visible on the limiting ridge of the stomach. In addition to these gastric abnormalities, an accentuated lobular pattern of the liver and dark patches on the lungs were observed for one high dose female at terminal kill. Two high dose males had small and/or pale adrenals. No macroscopic abnormalities were evident at either the intermediate or low dose levels. No treatment-related organ weight changes were detected. Histopathological findings were epithelial acanthosis, frequently with associated hyperkeratosis, in relation to treatment in the non-glandular region of the stomach of intermediate and high dose animals of either sex. No treatment-related microscopic abnormalities were observed at the low dose level.

Oral administration of the test material to rats by gavage for a period of 28 consecutive days at dose levels up to 1000 mg/kg/day resulted in treatment-related changes at 150 and 1000 mg/kg/day. Acanthosis and hyperkeratosis of the non-glandular gastric epithelium is not indicative of serious damage to the health of the animal and a No Observed Adverse Effect Level (NOAEL) was established at 150 mg/kg/day.