Fatty acids, C12-18 (even numbered)-methyl esters, sulfonated, sodium salts

Endpoint summary

Description of key information

No adverse effects relevant to humans were observed in repeated dose studies via the oral route involving the registered substance and a close analogue. In the key study, acanthosis and hyperkeratosis of the non-glandular gastric epithelium was not considered indicative of serious damage to the health of the animal and a NOAEL of 150 mg/kg bw/day was suggested for the registered substance (OECD 407 and EU Method B.7). The supporting study on an analogue substance suggested a NOAEL of 20 mg/kg bw/day (OECD 422). Further discussion of the proposed NOAEL values is available in the position paper (attached).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

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

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.

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

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

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).

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.

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

Key result

Critical effects observed:

no

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.

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.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crj: CD(SD)IGS

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

olive oil

Duration of treatment / exposure:

- Males: 47 days
- Females: 42-45 days (from 14 days before mating to Day 4 of lactation)

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

5 mg/kg bw/day (nominal)

Dose / conc.:

20 mg/kg bw/day (nominal)

Dose / conc.:

80 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males and 5 females

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS
- General condition was observed once a day.

BODYWEIGHT
- Bodyweights of males were determined on Day 1 (before dosing) and on Days 8, 15, 22, 29, 36, 43 and 49 of treatment.
- Bodyweights of females were determined on Day 1 (before dosing), on Days 1, 8 and 15 of treatment, on Days 0, 7, 14 and 20 of gestation, on Days 0 and 4 of lactation, and on the day of autopsy.

FOOD CONSUMPTION
- Food consumption by males was determined on Days 1, 8, 15, 22, 29, 36, 43 and 48 of treatment.
- Food consumption by females was determined on Days 1, 8 and 15 of treatment, on Days 0, 7, 14 and 20 of gestation, and on Days 0 and 4 of lactation.
- Food consumption of males and females was not determined during the mating period.

URINALYSIS
- Urinalysis was performed for all males on Day 41 or Day 42 of the administration period.

HEAMATOLOGY AND BIOCHEMISTRY
- Investigations were performed at the time of necropsy (after 48 days for males and 5 days after delivery for females).

ORGAN WEIGHTS
- Organ weights from male animals measured at the time of necropsy were brain; heart; liver; kidney; spleen; adrenal; thymus; testis and epididymis.
- Organ weights from female animals measured at the time of necropsy were brain; heart; liver; kidney; spleen; adrenal; thymus.
- Organ weight was determined for 10 males from the control group; 10 males from the 5 mg/kg bw/day group; 9 males from the 20 mg/kg bw/day group; 10 males from the 80 mg/kg bw/day group; 10 males from the 300 mg/kg bw/day group.
- Organ weight was determined for 7 females from the control group; 8 females from the 5 mg/kg bw/day group; 9 females from the 20 mg/kg bw/day group; 10 females from the 80 mg/kg bw/day group; 9 females from the 300 mg/kg bw/day group.

MICROSCOPIC EXAMINATION
- Organs examined in all animals were brain; spinal cord; intestine; liver; kidney; adrenal; spleen; heart; thymus; thyroid; parathyroid; trachea; lung; uterus; ovary; urinary bladder; ischiadic nerve; bone marrow; mesentery lymph node; mandibular lymph node; submandibular gland.
- The sublingual gland from 5 males and 5 females in the 0 and 300 mg/kg bw/day groups was examined.
- The stomach from 5 males and 5 females in the 5, 20 and 80 mg/kg bw/day groups was examined.

Statistics:

STATISTICAL METHODS
- Dunnett's or Scheffe's test for continuous data.
- Chi square test for quantal data.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Transitional softening stools were observed in a few males and females in the 80 and 300 mg/kg bw/day groups.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- No animal deaths related to treatment with test material took place.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no statistically significant changes for males or females.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

- No statistically significant changes for males or females.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- No statistically significant changes for males or females.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- An increase in GPT levels and a decrease in triglyceride levels was observed in males from the 300 mg/kg bw/day group.
- No statistically significant differences were observed in females.
- Mean GPT was reported as 41 i.u./L (10 animals; 0 mg/kg bw/day; SD 4 i.u./L); 40 i.u./L (10 animals; 5 mg/kg bw/day; SD 6 i.u./L); 38 i.u./L (9 animals; 20 mg/kg bw/day; SD 3 i.u./L); 44 i.u./L (10 animals; 80 mg/kg bw/day; SD 6 i.u./L); 53 i.u./L (10 animals; 300 mg/kg bw/day; SD 7 i.u./L; statistically significant p < 0.01).
- Mean triglyceride was reported as 63 mg/dL (10 animals; 0 mg/kg bw/day; SD 28 mg/dL); 43 mg/dL (10 animals; 5 mg/kg bw/day; SD 18 mg/dL); 50 mg/dL (9 animals; 20 mg/kg bw/day; SD 22 mg/dL); 49 mg/dL (10 animals; 80 mg/kg bw/day; SD 18 mg/dL); 29 mg/dL (10 animals; 300 mg/kg bw/day; SD 13 mg/dL; statistically significant p < 0.05).

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

- No statistically significant changes.

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):

- No statistically significant changes were observed for males or females.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Thickening of the forestomach mucosa was observed in the 80 mg/kg bw/day group (effect seen in 6 out of 10 males and 10 out of 10 females).
- Thickening of the forestomach mucosa was observed in the 300 mg/kg bw/day group (effect seen in 10 out of 10 males and 9 out of 9 females).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Squamous hyperplasia, erosion and lamina propria and/or submucosa edema and inflammatory infiltration were observed in the forestomach of males and females in the 80 and 300 mg/kg bw/day groups (see table, below).

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Histopathology results  Males            Dose (mg/kg bw/day)  0  5  20  80  300  Number of animals examined  5  5  5  5  5  Forestomach            Hyperplasia, squamous (slight to severe)  0/5  0/5  0/5  4/5  5/5 (p < 0.01)  Erosion (slight to moderate)  0/5  0/5  0/5  0/5  3/5  Edema, lamina propria/submucosa (slight to moderate)  0/5  0/5  0/5  0/5  4/5 (p < 0.05)  Cellular infiltration, inflammatory cell, lamina propria/submucosa (slight to moderate)  0/5  0/5  0/5  0/5  4/5 (p < 0.05)  Females            Dose (mg/kg bw/day)  0  5  20  80  300  Number of animals examined  5  5  5  5  5  Forestomach            Hyperplasia, squamous (slight to moderate)  0/5  0/5  0/5  4/5 (p < 0.05)  5/5 (p < 0.01)  Erosion (moderate)  0/5  0/5  0/5  0/5  1/5  Edema, lamina propria/submucosa (slight to moderate)  0/5  0/5  0/5  2/5  5/5 (p < 0.01)  Cellular infiltration, inflammatory cell, lamina propria/submucosa (slight)  0/5  0/5  0/5  1/5  2/5

 

Conclusions:

The NOAEL for an analogue substance was determined to be 20 mg/kg bw/day when investigated via a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test [OECD 422].

Executive summary:

In a supporting study on an analogue substance, a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test [OECD 422] was used to assess the effect of test material administered to Crj:CD (SD) IGS rats (10 animals/sex/dose) by gavage at 0, 5, 20, 80, or 300 mg/kg bw/day. Males were dosed for 47 days from day 14 before mating and females were dosed for 42-45 days from day 14 before mating to day 4 of lactation throughout the mating and pregnancy period. An increase in the GPT levels and decrease in the triglyceride levels were found in males at 300 mg/kg bw/day. At necropsy, thickening of the forestomach mucosa was observed in six males and nine females at 80 mg/kg bw/day and in 10 males and females at 300 mg/kg bw/day. In histopathological examinations, squamous hyperplasia, erosion, and edema of lamina propria and/or submucosa and inflammatory cell infiltration were observed in the forestomach of both sexes at 80 and 300 mg/kg bw/day. Based on the pathological findings in the forestomach at 80 mg/kg bw/day, the NOAEL for repeated dose toxicity was considered to be 20 mg/kg bw/day in male and female rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

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

The relevance of findings in the forestomach of rodents in short-term toxicity studies is discussed in the attached category justification and position paper (see Section 13).

Additional information

Repeated dose toxicity - oral

In the key study (Wragg & Brooks, 1994) on the registered substance (C12-18 ASME), the substance was administered at dose levels of 15, 150 and 1000 mg/kg bw/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. Haematology and 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 unscheduled 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 change in condition on Days 4 and 5 but they rapidly improved and clinical signs 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; there was no 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 and water intake was similar to controls 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 a s 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 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. 

In conclusion: 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. However, acanthosis and hyperkeratosis of the non-glandular gastric epithelium was not considered indicative of serious damage to the health of the animal and a NOAEL of 150 mg/kg bw/day was suggested.

In another study (MHLW, 2002) the test material (hexadecanoic acid, 2-sulfo-, 1-methylester, sodium salt) was dosed by oral gavage to 4 groups of CD rats each consisting of 10 males and 10 females; the treatment groups were: 0 (control), 5, 20, 80 or 300 mg/kg bw/day of the test substance. In life parameters consistent with the OECD 422 study design were recorded. No treatment related effects were seen for the majority of the study parameters investigated. For the males at 300 mg/kg bw/day there was a slight increase in glutamate pyruvate transaminase (GPT) and marginal decrease in triglycerides. At both 80 and 300 mg/kg bw/day, for both sexes, thickening of the forestomach mucosa was seen at necropsy and histopathology revealed squamous hyperplasia, erosion, edema of lamina propria and/or submucosa and inflammatory cell infiltration in the forestomach. Based on these findings a NOAEL for repeat toxicity of 20 mg/kg bw/day was suggested.

An OECD 422 dietary study using tetradecanoic acid, 2-sulfo-, 1-methyl ester, sodium salt is reported in the literature (see sections on Reproductive and Developmental toxicity). This study employed 4 groups of CD rats each consisting of 12 males and 12 females; the treatment groups were: 0 (control), 175, 360 or 740 mg/kg bw/day for males and 0 (control), 249, 497 or 1039 mg/kg bw/day for females of the test substance. In-life parameters consistent with the OECD 422 study design were recorded. No treatment related effects were seen for the majority of the study parameters investigated including no reported effects on the forestomach. At 740 mg/kg bw/day prothrombin time was slightly increased and fibrinogen was marginally decreased for females only; triglycerides were slightly low for both sexes at this dose and at 360 mg/kg bw/day. At the highest dose body weight-relative testis weights were slightly increased as were the liver and kidneys for the females; for the females’ relative adrenal weights were marginally low and for both sexes thymus weights tended to be low. The effects noted on the blood parameters, liver and thymus weights were not considered of toxicological significance (lacking a dose response relationship, due to hepatic metabolic activation or with no histopathological correlates respectively). The no observed effect level (NOEL) was suggested as 175 mg/kg bw/day for males and 249 mg/kg bw/day for females. However, a NOAEL for general toxicity of at least 360 or 497 mg/kg bw/day for males or females should be considered given the authors comments on the lack of toxicological significance of the findings at the mid-dose.

From the available short-term toxicity database with C12-18 ASME and analogues, the key OECD 407 study on C12-18 ASME had a NOAEL of 150 mg/kg bw/day based on forestomach pathology (epithelial acanthosis/ hyperkeratosis). The other findings in this study were unremarkable and consisted of a small number of early clinical observations, a slightly lower bodyweight gain and slightly greater water intake only at the highest dosage of 1000 mg/kg bw/day. As described earlier, the findings associated with the rat forestomach, particularly when oral gavage dosing is used, are of questionable toxicological significance when considering such findings in terms of relevance to humans. Indeed, in the dietary OECD 422 study on the C14 analogue no such findings related to the rat forestomach were evident. The findings on the forestomach from the oral gavage dosing study (OECD 422) should therefore be treated with caution and take into account the real possibility that local effects (e.g. irritationviathe test substance and/or mechanical irritation caused by oral gavage dosing) were most probably the key factors in the pathology seen. As discussed in the position paper attached in Section 13, such conclusions have been arrived at by regulatory agencies for other substances where local irritant effects on the rat forestomach were evident. Taking this into consideration, the effects on the rat forestomach, evident in the oral gavage OECD 407 study on C12-18 ASME, are most likely rat specific local effects and therefore not relevant for use in human risk assessments. As such a NOAEL in this study of ≥1000 mg/kg bw/day would be appropriate. 

Justification for classification or non-classification

No adverse effects relevant to humans have been reported and, on this basis, classification for repeated dose toxicity is not required.