Fatty acids, C12-18 and C18-unsatd., 2-sulfoethyl esters, sodium salts

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994-07-04 to 1994-08-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented study, performed to GLP and OECD guidline. Substance certificate of analysis and chemical identity.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

- No functional observational battery (FOB) was performed - There was no assessment of blood clotting potential - The epididymis and thymus were not weighed

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): Jordapon CI
- Physical state: White powder
- Analytical purity: 90%
- Impurities (identity and concentrations): Sodium Isethionate, Free cocoyl fatty acid and cocyl fatty acid sodium salt
- Composition of test material, percentage of components: Sodium Cooyl Isethionate
- Purity test date: Material analysed before the start of thestudy (4thJuly1994) and at end of live phase (1stAugust1994)
- Lot/batch No.: S2052501
- Expiration date of the lot/batch: no information
- Stability under test conditions: The stability and homogenity of Jordapon CI in the diet were investigated under study AH940213. The test diets were sampled after week 1 (1st july 1994) and week 3 (15th july 1994) in order to confirm the achieved concentration of test item in the diet.
- Storage condition of test material: no information
- Other: Test material Jordapon CI analysed in study AC940212, reported in Appendix 9 of study FF940215, confirmed to be Sodium Cocoyl Isethionate.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan UK Ltd
- Age at study initiation: 5-6 weeks old
- Weight at study initiation: 174.7-198.4g (males) and 137.9-157.1g (females)
- Fasting period before study: None, this is a repeat dose toxicity feeding study where test item is dosed in diet.
- Housing: The rats were multiple housed in polycarbonate (Markrolon) cages with stainless steel
mesh bottoms and lids, such that there were five animals of a single sex per cage; 15
cages (five rows x three columns) were accommodated on each holding battery.
The animals were housed in room 12 of the SPF Unit.
- Diet: The animals were given free access to the experimental diets AIN-&^A (MODAIN) at all times ad libitum
- Water: potable tap water (Anglian Water) was supplied to each animal cage by a polycarbonate bottle and a
stainless steel sipper tube, ad libitum.
- Acclimation period: no information


ENVIRONMENTAL CONDITIONS
- Temperature (°C): The room environment was controlled to give conditions within the temperature range 22±3°C.
- Humidity (%): relative humidity 30-70%.
- Air changes (per hr): no information
- Photoperiod (hrs dark / hrs light): a light/dark cycle of 12 hours operated throughout the study


IN-LIFE DATES: From: 04 Jul 1994
To:01 and 02 Aug 1994

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: ESL modified AIN-76A (MODAIN) diet

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): AIN-76A (MODAIN) diet at 0.1, 0.3 and 1.0% (w/w) test item.
- Storage temperature of food: Stored at 0-4oC in chill room, then 10 hours at Ambient temperature before dosing and at +4oC imediately prior to use in SPF unit.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of Jordapon Cl in the diet were investigated under Study
number AH940213.
Jordapon Cl was characterised under Study number AC940212; the material was analysed
before the start of the study and again at the end of the live animal phase to confirm
stability of the test material over the duration of the study.
The test diets were sampled after preparation for week 1 (01 Jul 1994) and week 3 (15 Jul
1994) in order to confirm the achieved concentration of Jordapon Cl in the diets.
Jordapon Cl was extracted from the experimental diets with methanol. The extracts were then diluted with methanol such that the final concentration in the assay would be within the range of standards (4-20j.Lg/ml) prepared in methanol. Aliquots of the standards and samples were then pipetted into test tubes, concentrated to dryness under a stream of nitrogen and the residues redissolved in 2ml of deionised water. Analysis was carried out
by the Methylene Blue Assay in which aqueous Methylene Blue reagent and chloroform were added to the sample and standard tubes and mixed thoroughly. After allowing the organic and aqueous phases to separate the optical density of the organic layer was measured at 650nm on a UVIVis Spectrophotometer. Quantitation was achieved by comparison of absorbance values of the standards and samples, then applying the correction factor for a 93% recovery as determined in Study AH940213.
l

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0% (Group A)
Basis:
other: Control Group

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0.1% (Group B)
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0.3% (Group C)
Basis:
nominal in diet

Doses / concentrations 4

Remarks:

Doses / Concentrations:
1.0% (Group D)
Basis:
nominal in diet

No. of animals per sex per dose:

10 male and 10 female per dose Group (plus 10 males and 10 females for the control group (Group A)).

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: A 14 day palatability study was conducted in rats (FP940214). This was to determine the level of test item to be used in the 28 day feeding study. The maximum level of Jordapon CI to be used in the main study was 1.0% in the diet, based on treatment-related effects on body weight and food intakes at 3% and 5% in the palatability study. Therefore, levels above 1% of the test material showed evidence of reduced palatability.

- Rationale for animal assignment (if not random): Five days before the start of the study, each animal was weighed and 80 rats were selected
and randomly allocated to one of the four groups using a stratified randomisation procedure based on body weight.

- Rationale for selecting satellite groups: No satellite groups.

- Post-exposure recovery period in satellite groups: Not applicable.

- Section schedule rationale (if not random): Not applicable.

Positive control:

None.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice a day, but once on Saturday and Sunday.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice a day, but once on Saturday and Sunday.
Such signs, including the time of onset, duration and intensity were recorded on clinical signs sheets at the time of observation and then entered into the Pathtox system at the earliest possible time following the observation.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed at weekly intervals (on Mondays) throughout the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each group determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

For each cage of five animals, food and water intakes were recorded twice-weekly (Mondays and Thursdays) throughout the study and the weekly amounts calculated.

The animals were housed in groups of five per cage and the food and water intakes were measured for each cage, therefore, there were insufficient data points for a statistical analysis to be carried out. Examination of the data did not indicate any treatment-related patterns in the food and water consumption of male rats fed Jordapon Cl. However, there was evidence of a slight reduction in the food and water consumption of female rats fed
Jordapon Cl.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes.

No treatment-related differences in absolute body weights at week 4 were observed. Body weight gains of male rats from all treatment groups were increased significantly during the first week of the study. During the second week of the study body weight gains of female rats fed 0.3 and 1.0% Jordapon Cl were decreased significantly. No further changes were observed.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: For each cage of five animals, food and water intakes were recorded twice-weekly (Mondays and Thursdays) throughout the study and the weekly amounts calculated.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the test period the animals were bled by cardiac puncture under Halothane anaesthesia. The blood was divided between three sample tubes: a plastic tube containing EDTA (potassium salt) for haematological analysis; a glass tube containing lithium heparin for plasma chemistry analysis; and a glass tube containing no anticoagulant for serum protein analysis and electrophoresis.
- Anaesthetic used for blood collection: Yes (Halothane anaesthesia)
- Animals fasted: No
- How many animals: 80
- The following haematological indices were determined using the Technicon HI*(A) haematology analyser:

Red blood cell count
Haemoglobin
Platelets
Total white blood cell count
Differential white cell count (lymphocytes, neutrophils, monocytes, eosinophils, basophils,
large unstained cells)
$ Mean red cell volume (determined from the red cell volume histogram)
$ Mean red cell haemoglobin (haemoglobin/red blood cell count)
$ Haematocrit (red cell count x mean red cell volume)
$ Mean red cell haemoglobin concentration (haemoglobin/haematocrit)

$ Values were calculated

Reticulocytes were determined by manual counting after staining with New Methylene
Blue.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the test period the animals were bled by cardiac puncture under Halothane anaesthesia. The blood was divided between three sample tubes: a plastic tube containing EDTA (potassium salt) for haematological analysis; a glass tube containing lithium heparin for plasma chemistry analysis; and a glass tube containing no anticoagulant for serum protein analysis and electrophoresis.
- Animals fasted: No
- How many animals: 80
- The following parameters were examined:

Plasma chemistry
The following plasma clinical chemistry parameters were measured on the Hitachi 704EC:

Sodium
Potassium
Calcium
Magnesium
Chloride
Inorganic phosphate
Creatinine
Urea
Triglyceride
Glucose
Total cholesterol
Alanine aminow-ansferase (ALT)
Aspartate aminotransferase (AST)
Lactate dehydrogenase
Hydroxy butyrate dehydrogenase (HBD)
Alkaline Phosphatase (ALP)
Pseudocholinesterase
Creatine kinase
5I -Nucleotidase

Serum chemistry

The following serum clinical chemistry parameters were measured on the Hitachi 704EC:

Total protein
Albumin
Albumin:globulin ratio (calculated)
The following parameters were determined by serum electrophoresis on cellulose acetate
plates followed by staining with Ponceau S and quantification of the fractions using the
Profil Ecran scanning densitometer:
Albumin
aI-globulin
a2-globulin
(j-globulin
y-globulin

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

After the blood collection for the Haematology, the animals were killed by carbon dioxide.

GROSS PATHOLOGY: Yes

A detailed necropsy was performed on all animals. All macroscopic observations were recorded as well as an assessment of the level of intra-abdominal fat deposition. The following organs were removed and weighed: adrenal glands, brain, heart, kidneys, liver, spleen and testes. The ratio of organ weight to 100g of body weight was calculated in each case and recorded as relative organ weight.

The following tissues were taken from each rat and preserved in 10% buffered formalin unless otherwise indicated:

Brain
Cervical lymph node
Mesenteric lymph node
Pancreas
Thymus
Ovaries
Fallopian tubes
Vagina *
Uterus
Cervix
Testes *
Epididymides *
Seminal vesicles *
Eyes $
Harderian glands $
Salivary glands *
Adrenal glands
Pituitary
Sciatic nerve
Thyroids
Parathyroids
Liver
Heart
Spleen
Lungs
Larynx
Trachea
Kidneys
Aorta
Bladder
Prostate
Tongue
Caecum
Stomach
Duodenum
Ileum
Jejunum
Colon
Rectum
Oesophagus
Mammary gland (site of)
Skeletal muscle
Skin
Femur and stifle joint
Spinal cord
Sternum
Head

* Tissues were preserved in Bouin's fixative
$ Tissues were fixed in Davidson's fluid

Bone marrow smears were taken and stained with May- Grunwald Giemsa stain.

HISTOPATHOLOGY: Yes

The tissues were processed into paraffin wax, using conventional histological methods, and sections nominally 4Jlm in thickness were prepared and stained with haematoxylin and eosin (H&E) for microscopic examination.

Microscopic examination was performed on the above tissues from all control rats and all rats fed 1.0% Jordapon Cl. Tissues showing macroscopic abnormalities which were designated as lesions at necropsy were examined also. Histopathological findings were entered directly into the KMS V2 pathology software system.

During the examination of the tissues, histopathological changes were recorded as present or absent, or graded according to their morphology, using a numerical grades scale of 0 to 5, in order to assess degrees of severity or activity. This procedure provides a means of ranking degrees of change which may assist in the interpretation of biological differences. The actual scores obtained have no intrinsic value.

Other examinations:

No

Statistics:

Appropriate statistical methods of analysis were chosen using the process map illustrated
in Figure 1 below:

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No deaths; clinical signs observed were ocular discharge, nasal discharge, penile haemorrhage and alopecia. These signs were not treatment related and were considered to form part of the normal background findings seen in this type of study.

Mortality:

mortality observed, treatment-related

Description (incidence):

No deaths; clinical signs observed were ocular discharge, nasal discharge, penile haemorrhage and alopecia. These signs were not treatment related and were considered to form part of the normal background findings seen in this type of study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related differences in absolute body weights at week 4 were observed. Body weight gains of male rats from all treatment groups were increased significantly during the first week of the study. During the second week of the study body weight ga

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The animals were housed in groups of five per cage and the food intakes were measured for each cage, therefore, there were insufficient data points for a statistical analysis to be carried out. Examination of the data did not indicate any treatment-relate

Food efficiency:

not specified

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

effects observed, treatment-related

Description (incidence and severity):

The animals were housed in groups of five per cage and the water intakes were measured for each cage, therefore, there were insufficient data points for a statistical analysis to be carried out. Examination of the data did not indicate any treatment-relat

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Red blood cell indicies/White blood cell indices: No treatment related changes were observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

No treatment related changes were observed in plasma electrolytes, plasma enzyems, and serum electrophoresis. Plasma creatinine was decreased slightly in male rats fed 0.3% and 1.0% test item but increased slightly in female rats fed 0.3% test item. Thes

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The weight of the kidneys relative to terminal body weight was increased slightly in female rats fed 1.0% test item. This minor change was within the normal range expected in this age and strain of rat and was, therefore, considered to be incidental and,

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Male rats fed test item had an increased incidence of grey-green contents in the caecum in contrast to the green contents seen in the Control group. The only other macroscopic finding of note was a pale nodule in the cranial pole of the right kidney of on

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

No microscopic findings considered to be related to the feeding of test item were observed. Female rat DUO, which received 1.0% test item, had a nephroblastoma in the right kidney. A variety of other spontaneous changes was recorded in both control and t

Histopathological findings: neoplastic:

not specified

Details on results:

Chemical analysis

Characterisation and stability of the test substance This was carried out under study number AC940212; the test material was found to be
stable over the study period.

Stability and homogeneity of the experimental diets
The test material was found to be homogeneously dispersed in the diet at concentrations of 0.1, 0.3 and 1.0% (w/w) test item under Study number AH940213. The same concentrations were found to be stable in the diet formulation for 14 days.

Achieved concentration
On both occasions the measured concentrations, for the diets prepared at all three levels, were found to be within ± 10% of the nominal concentration, when the correction factor for a 93% recovery, as determined in Study number AH940213, was applied.

Environmental conditions
Animal room environmental measurements showed that for the duration of the study, temperature and humidity were maintained within the limits specified.

Pre-test health check
Six animals (four male and two female) were found to have total white cell counts in excess of 12.0 x109/1, the preferred maximum value; these animals were rejected on health grounds prior to the start of the study.

General health and survival
There were no decedents during the course of the study. Clinical signs observed during the study were ocular discharge, nasal discharge, penile haemorrhage and alopecia. These signs did not show any treatment-related distribution and were considered to form part of the normal background findings seen in this type of study.

Body weights and body weight gains
No treatment-related differences in absolute body weights at week 4 were observed. Body weight gains of male rats from all treatment groups were increased significantly during the first week of the study. During the second week of the study body weight gains of female rats fed 0.3 and 1.0% test item were decreased significantly. No further changes were observed.

Food and water intakes
The animals were housed in groups of five per cage and the food and water intakes were measured for each cage, therefore, there were insufficient data points for a statistical analysis to be carried out. Examination of the data did not indicate any treatment-related patterns in the food and water consumption of male rats fed test item. However, there was evidence of a slight reduction in the food and water consumption of female rats fed
test item.

Haematology
Red blood cell indices- No treatment-related changes were observed.
White blood cell indices - No treatment-related changes were observed.
Bone marrow smears In the absence of any treatment-related changes in either the red or white blood cell counts it was not considered necessary to examine the bone marrow smears.

Clinical chemistry
Plasma electrolytes - No treatment-related changes were observed.

Plasma enzymes - No treatment-related changes were observed.

Plasma metabolites - Plasma creatinine was decreased slightly in male rats fed 0.3% and 1.0% test item but increased slightly in female rats fed 0.3% test item. These minor changes were within the normal range expected from this age and strain of rat and are, therefore, considered to be incidental and, in the absence of a treatment-related histopathological change, of no toxicological significance.

Serum electrophoresis - No treatment-related changes were observed.

Organ weights
The weight of the kidneys relative to terminal body weight was increased slightly in female rats fed 1.0% test item. This minor change was within the normal range expected in this age and strain of rat and was, therefore, considered to be incidental and, in the absence of a treatment-related histopathological change, of no toxicological significance. No other treatment-related changes were observed.

Macroscopic findings

Male rats fed test item had an increased incidence of grey-green contents in the caecum in contrast to the green contents seen in the '"Control group.
The only other macroscopic finding of note was a pale nodule in the cranial pole of the right kidney of one female rat (D110) fed 1.0% test item.
The quantity of abdominal fat present in rats fed test item was similar to that in control animals.

Microscopic findings
No microscopic findings considered to be related to the feeding of test item were observed.

Female rat DUO, which received 1.0% test item, had a nephroblastoma in the right kidney.

A variety of other spontaneous changes was recorded in both control and treated animals. These findings were within the spectrum of spontaneous lesions commonly encountered in laboratory rats of this age and strain, showed no evidence of a treatment-related distribution in incidence or severity and were considered to be unrelated to the feeding of test item.

Effect levels

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The daily oral (dietary) administration of Jordapol1 Cl to rats (Sprague Dawley strain, bred at Harlan UK Ltd.) did not produce any toxicological changes considered to be related to treatment.
The no-effect level was greater than 1.0% Jordapon Cl in the diet (approximately 1000mg/Kg/Day).

Executive summary:

This study was undertaken to determine whether there was any toxic reaction in rats following oral ingestion of test item for 28 days.

Groups of ten male and ten female rats were fed either 0.1 %,0.3% or 1.0% (w/w) test item in purified diet ad libitum for 28 days. A control group of ten male and ten female rats were fed the purified diet. The animals were observed up to two times per day for signs of ill health or reaction to treatment. Body weights were recorded at weekly intervals throughout the study; food and water intakes were measured twice-weekly and the weekly consumptions calculated. At the end of the treatment period all the rats were killed and subjected to a detailed necropsy, at which stage a number of organs were weighed and a range of tissues was taken for histological examination. Prior to necropsy, blood was removed for clinical pathology determinations.

No animals died or were killed on humane grounds during the study. No clinical signs attributable to treatment were observed. A few minor, but statistically significant changes, were recorded in body weight gains during the first half of the study but no consistent treatment-related changes were observed throughout the study. Food and water consumption were decreased slightly in female rats throughout the study period, but this did not result in any significant changes in the absolute body weights.

The only macroscopic change showing a treatment-related distribution was an alteration in the colour of the caecal contents of male rats fed test item; this was considered to be incidental and of no toxicological significance.

No microscopic findings considered to be related to the feeding of test item were observed.

One female rat which received 1.0% test item had a nephroblastoma in the kidney but this type of tumour occurs occasionally as a spontaneous lesion in young rats of this strain and its presence in a single treated animal is considered to be incidental and unrelated to treatment.

The no-effect level was >1.0% test item in the diet (approximately 1000mg/Kg/Day).