Reaction mass of sodium 1-methoxy-1-oxohexadecane-2-sulphonate and sodium methyl 2-sulphooctadecanoate

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:

6 April - 3 July 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Hexadecanoic acid, 2-sulfo-,1-methylester, sodium salt.

IUPAC Name:

Hexadecanoic acid, 2-sulfo-,1-methylester, sodium salt.

Test material form:

solid: particulate/powder

Details on test material:

- Physical state: white powder
- Storage condition of test material: ambient temperature in the dark

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Manston Road, Margate, Kent, England
- Age at study initiation: approx. 5 weeks old
- Weight at study initiation: 108 - 144 g (males) and 124 - 147 g (females)
- Fasting period before study: none
- Housing: Animals were housed 5 to a cage on racks, with groups blocked together by sex. Each cage was made of stainless steel with stainless steel grid floors.
- Diet (e.g. ad libitum): ad libitum, except overnight prior to blood sampling
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 9 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 40 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 28 March 2001 To: 4 May 2001

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF FORMULATIONS:
The test substance was used as supplied and administered by admixture in the diet. A pre-mix was prepared each week by grinding the test substance directly into untreated basal diet. Assessment of the homogeneity and stability of the dietary formulations was performed: accuracy of mixing was acceptable and that the concentration of test substance in the diet remained unchanged between preparation and the period of use under the conditions of thestudy.

Control animals received the normal untreated basal diet.
A constant dietary concentration was administered, throughout the treatment period, to each group of treated animals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method involved extraction into methanol after which the extract was measured by reverse phase high performance liquid chromatographic analysis with refractive index detection using external calibration.

The mean concentrations of hexadecanoic acid in test diet formulations analysed during the toxicity study were within 8% of nominal concentrations confirming accurate formulation.
The homogeneity and the stability were confirmed for hexadecanoic acid in rodent diet formulations at nominal concentrations of 700 ppm, 1000 ppm and 10000 ppm. The storage period, 15 days at ambient temperature, represented the maximum time from preparation to completion of feeding.
The analytical procedure was validated with respect to linearity of detector response, precision of injection, specificity of chromatographic analysis, limit of detection, accuracy and precision.

Duration of treatment / exposure:

Treatment continued seven days a week, for a total period of 4 weeks.

Frequency of treatment:

Contineous

Doses / concentrations

Remarks:

Doses / Concentrations:
700, 2000 and 8000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

5

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The high dosage level was selected on the basis of a seven day preliminary dietary toxicity study performed (diet with dosage levels of 0, 2000, 4000 or 8000 ppm). The level of 8000 ppm was chosen because this was approximately equivalent to an achieved intake for both sexes of 1000 mg/kg/day and also represents the limit dose for this design of study. No major toxicity 8000 ppm was not associated toxicity, however slightly lower bodyweight gain for both sexes in comparison with controls, was evident. The low (700 ppm) dose level was selected by the Sponsor on the basis of the lower limit for the sensitivity of the dietary analytical method. The intermediate (2000 ppm) dose level was selected to maintain an approximate 3 to 4 dose increment between these dose levels.

Positive control:

Not applicable.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: yes
- Time schedule: at least once each day during the acclimatisation period. At least twice daily for mortality and any signs of ill health, behavioural changes or reaction to treatment during treatment period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once each week. Any signs of ill health, behavioural change or toxicosis were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: during the acclimatisation period (Day -7), on Day 0 and once each week thereafter, (the last bodyweight being recorded on Day 28 prior to overnight starvation for blood sampling) and on the day of sacrifice.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal 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: Yes.
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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations: Daily monitoring by visual appraisal. No formal measurements were made.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 29
- Anaesthetic used for blood collection: Yes (identity) light isoflurane general anaesthesia
- Animals fasted: Yes
- How many animals: all
- The following estimations were performed using a Bayer-Technicon H1E Haematology Analyser using standard Bayer methodology:
Haematocrit (Hct) L/L
Haemoglobin concentration (Hb) g/dL
Erythrocyte count (RBC) x10E12/L
Mean cell haemoglobin concentration (MCHC) g/dL
Mean cell volume (MCV) fL
Mean cell haemoglobin (MCH) pg
Total leucocyte count (WBC) x10E9/L
Platelet count (Plt) x10E9/L

Differential leucocyte counts:

Neutrophils (Neutrophil) x10E9/L
Lymphocytes (Lymphocyte) x10E9/L
Eosinophils (Eosinophil) x10E9/L
Basophils (Basophil) x10E9/L
Monocytes (Monocyte) x10E9/L
Large unstained cells (LUC) x10E9/L


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day 29
- Animals fasted: Yes
- How many animals: all
- Parameters checked:The following parameters were analysed with a Hitachi 917 Clinical Chemistry Analyser using Hitachi methodology:
Urea (Urea) mmol/L
Creatinine (Creat) µmol/L
Sodium (Na) mmol/L
Potassium (K) mmol/L
Total protein (Total Prot) g/L
Albumin (Alb) g/L
A/G ratio (Calculated from Total protein and Albumin concentrations)
Total Cholesterol- (Chol Total) mmol/L
Glucose (Gluc) - Hexokinase mediated assay mmol/L
Alanine aminotransferase (ALT) Reaction temperature 37ºC U/L
Aspartate aminotransferase (AST) Reaction temperature 37ºC U/L

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at approximately the same time of day, prior to dosing and in Week 4 prior to any laboratory investigations.
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength (FOB)/ motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes: acording to OECD 407

The following organs from each animal were weighed: adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes, thymus. Bilateral organs were weighed together.


HISTOPATHOLOGY: Yes
Some of the tissues subjected to histological processing needed the following specific regions to be examined:
Tissue Regions to be examined
Adrenals : cortex and medulla.
Brain : cerebellum, cerebrum and midbrain.
Femur with joint: longitudinal section including bone marrow.
Heart : including auricular and ventricular regions.
Ileum : Including Peyer’s patch where possible
Kidneys : including cortex, medulla and papilla regions.
Liver : section from all main lobes.
Lungs : section from two major lobes, to include bronchi.
Spinal cord : transverse and longitudinal sections at the cervical, lumbar and thoracic levels.
Stomach : keratinised, glandular and antrum.
Thyroid : includes parathyroid in section, where possible.
Uterus : uterus section separate from cervix section.

For bilateral organs, sections of both the left and right organs were examined.

Statistics:

All statistical analyses were carried out separately for males and females.
The following sequence of statistical tests was used for bodyweight, functional observational battery, haematology, blood chemistry, organ weight and pathology data:
- If 75% of the data (across all groups) were the same value, c, say, then a frequency analysis was applied.
- If Bartlett’s test for variance homogeneity was not significant at the 1% level, then parametric analysis was applied. If the F1 test for monotonicity of dose-response was not significant at the 1% level, Williams’ test for a monotonic trend was applied.If the F1 test was significant, suggesting that the dose-response was not monotone, Dunnett’s test was performed instead.
- If Bartlett’s test was significant at the 1% level, then logarithmic and square-root transformations were tried. If Bartlett’s test was still significant, then non-parametric tests were applied. If the H1 test for monotonicity of dose-response was not significant at the 1% level, Shirley’s test for a monotonic trend was applied. If the H1 test was significant, suggesting that the dose-response was not monotone, Dunn’s test was performed instead.
Where appropriate, (for organ weight data) analysis of covariance was used in place of analysis of variance in the above sequence. For organ weight data, analysis of variance was performed using terminal bodyweight as covariate when the within group relationship between organ weight and bodyweight was significant at the 10% level (Angervall and Carlstrom, 1963), in an attempt to allow for differences in bodyweight which might influence the organ weights.
 Significant differences between control animals and those treated with the test substance were expressed at the 5% or 1% level.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths during the treatment period.
There were no clinical findings observed which were considered to be attributable to treatment
BODY WEIGHT AND WEIGHT GAIN
In the period Days 0 to 8, males receiving 8000 ppm showed a statistically significant lower mean gain, compared with controls. This lower gain was a result of 4/5 individual males at this dosage showing lower gains than the lowest control gain. Thereafter gains improved for these males, such that the mean gains for Days 8 to 28 and Days 0 to 28 for males receiving 8000 ppm were comparable with controls.
Mean gains for males at 700 ppm or 2000 ppm were either similar to or higher than controls, and thus not adversely affected by treatment.
In the period Days 0 to 8, mean gains for all treated females were lower than controls. However, statistical significance was not attained, no dosage-relationship was evident and the majority of individual gains for treated groups were comparable with controls. In addition, in the period Days 8 to 28, mean gains for all treated female groups were comparable with controls.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
In Week 1 both sexes receiving 8000 ppm showed lower group mean food intake, compared with controls. Subsequently during Weeks 2 to 4 of treatment the mean food intakes for these animals were comparable with controls.
Overall there were no adverse effects of treatment on food consumption noted among the remaining treated groups. Although food consumption for males receiving 700 ppm was superior to that of control during the 4 week treatment period, it was not considered to be as adverse effect of treatment and is, therefore, considered to be of no toxicological importance.
The overall calculated group mean intakes of Hexadecanoic acid during the 4 week treatment period followed the expected pattern for a fixed level dietary study, with the achieved intakes in terms of mg/kg/day generally decreasing due to the decline in the ratio of bodyweight increase versus food consumption. The intergroup differences in terms of ‘mg/kg/day’ were similar to those in terms of ‘ppm’ between the treated groups.

The calculated group mean intakes of Hexadecanoic acid in mg/kg/day, during the 4 week treatment period were as follows:
700 ppm: 80.7 (males) and 73.1 (females)
2000 ppm: 230.5 (males) and 214.8 (females)
8000 ppm: 902.4 (males) and 865.8 (females)


FOOD EFFICIENCY
The overall (Week 1 to 4) food conversion efficiencies of all treated groups were considered to have been unaffected by treatment with Hexadecanoic acid. However, in Week 1 males receiving 8000 ppm showed less efficient food utilisation compared with controls, as indicated by the lower group value.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Not examined

OPHTHALMOSCOPIC EXAMINATION
Not examined

HAEMATOLOGY
The haematology investigations revealed slightly longer mean prothrombin times (PT) for both sexes receiving 8000 ppm when compared to controls, with the male value attaining statistical significance. There were no other differences from control.


CLINICAL CHEMISTRY
The blood chemistry investigations revealed a statistically significant higher mean alanine amino-transferase (ALT) and albumin levels and related mean A/G ratio among females receiving 8000 ppm, when compared to controls. In addition, higher mean urea, and potassium levels were noted for males receiving 8000 ppm when compared to controls which attained a level of statistical significance.

URINALYSIS
Not examined

NEUROBEHAVIOUR
Assessment of the functional observation battery (FOB) data did not reveal any behavioural changes that were considered indicative of neurotoxicity or to be attributable to treatment.

ORGAN WEIGHTS
Lower mean liver weights were noted among males receiving 2000 and 8000 ppm when compared to control. However this change was not considered to be related to treatment due to the absence of a dosage-relationship, the small magnitude of the difference from control, a lack of a similar difference from control being noted in females and the absence of any treatment-related microscopic changes being observed in the liver.

GROSS PATHOLOGY
The macroscopic examination performed at termination did not reveal any changes that were considered to be attributable to treatment with Hexadecanoic acid.


HISTOPATHOLOGY: NON-NEOPLASTIC
no treatment-related findings

Effect levels

1

Dose descriptor:

NOEL

Effect level:

2 000 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: body weight week 1 lower at 8000 ppm; food consumption week 1 lower at 8000 ppm; haematology: longer PT at 8000 ppm blood chemistry: urea and potasium levels higher at 8000 ppm

2

Dose descriptor:

NOEL

Effect level:

2 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: haematology: longer PT at 8000 ppm Blood chemistry: ALT , Albumine levels and A/G ratio higher at 8000 ppm

3

Key result

Dose descriptor:

NOAEL

Effect level:

865.8 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Effects seen at 8000 ppm were not considered adverse.

4

Key result

Dose descriptor:

NOAEL

Effect level:

902.4 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Effects seen at 8000 ppm were not considered adverse.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

The NOEL of 2000 ppm nominal in the food corresponds to group mean actual intake of 230.5 mg/kg/d for males and 214.8 mg/kg/d for females.

Although at 8000 ppm, effects were seen (longer PT and higher ALT , Albumine levels and A/G ratio), it was decided that these effects were considered minimal and not of any biological importance. Therefore the NOAEL was set to the high dose.

Applicant's summary and conclusion

Conclusions:

In conclusion there was evidence of minor toxicity, manifest as lower initial bodyweight gain, in males at 8000 ppm, thus this level can not be classed as a clear no observed effect level (NOEL). However, there were no effects of treatment noted at 2000 ppm, thus this dosage level can be classed as the no observed effect level (NOEL) on this study. As the effects seen at 8000 ppm were not considered to be adverse, the NOAEL was set at the high dose.