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

Endpoint summary

Description of key information

Data on the main component of the reaction mass:

28-day oral with C16MES: oral NOAEL 865.8 mg/kg bw/d for females and 902.4 mg/kg bw/d for males (dietary)

OECD 422 oral with C16MES: oral NOAEL for systemic effects = 80 mg/kg bw/day

Data on similar substances:

OECD422 oral with C14MES: oral NOAEL = 360 mg/kg bw/day for male rats and 497 mg/kg bw/day for female rats (dietary)

28-day dermal with C12/C14/C16  (1/2/7) MES: dermal NOAEL for local effects is 5% (equivalent to 37 and 55 mg/kg bw/day for male and females, resp.) based on primary skin reactions at high dose. As the systemic effects at the high dose were not considered adverse,  the NOAEL for systemic effects is set at 15%, equivalent to 111 mg/kg bw/day for males and 161 mg/kg bw/day for females.

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

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

The study has been performed according to OECD guidelines. No data was present in the publication on whether the study was performed according to GLP principles.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

not specified

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Males: 56 days including 14 days prior to mating, during the mating and post-mating periods
Females: 41-45 days continueously (from 14 days prior to mating until post-partum Day 4)

Frequency of treatment:

Continueously

Dose / conc.:

0.3 other: % nominal in diet

Dose / conc.:

0.6 other: % nominal in diet

Dose / conc.:

1.2 other: % nominal in diet

No. of animals per sex per dose:

5

Control animals:

yes, plain diet

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no significant differences between groups with respect to body weight or food consumption

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no significant differences between groups with respect to body weight or food consumption

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematological examination revealed decreased fibrinogen levels and increased prothrombin times in females at the 1.2% dosage level as compared to the control values. It has to be noted however that the standard deviations in the controls are very high, making the controls less reliable.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Blood chemistry analysis showed a decrease in the triglyceride levels in both sexes at the 0.6 and 1.2% dosage levels

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A slight increase in absolute and relative weight of testes in males treated at the 1.2% level was observed. At the same dosage level in females, we observed an increase in the absolute and relative weight of liver and the relative weight of kidneys, and a decrease in the absolute and relative weight of adrenals and the absolute weight of thymus, heart and spleen.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross treatment-related changes at necropsy in any treatment group was observed

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Although no gross treatment-related changes at necropsy in any treatment group was observed, histopathological examination revealed hepatocyte enlargement in the centrolobular area of the liver in the 5 treated females, with slight anisonucleosis in 1 and slight focal necrosis in 1, and slight atrophy of the thymus cortex in 2 females. No liver or thymus abnormalities were observed in the 5 corresponding males.

Histopathological findings: neoplastic:

not specified

Dose descriptor:

NOEL

Effect level:

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

Sex:

male

Basis for effect level:

clinical biochemistry

haematology

organ weights and organ / body weight ratios

Dose descriptor:

NOEL

Effect level:

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

Sex:

female

Basis for effect level:

clinical biochemistry

haematology

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Critical effects observed:

not specified

The prolonged prothrombin time and decreased serum fibrinogen level observed in females at the 1.2% dosage level probably reflect decreased synthesis of fibrogen in liver. The finding of lower triglyceride levels at the 0.6 and 1.2% dosage level in both sexes suggest the inhibition of fatty acid absorption in the intestine. This effect was small and furthermore, it should be noted that the controls are not very reliable due to the large standard deviations in the values.

An increase in absolute and relative liver weight accompanied by slight centrilobular hypertrophy in all females treated at the 1.2% dosage level also lead to the conclusion that the liver is the target organ.

The decrease in absolute thymus weight and the focal atrophy in the thymus cortex that was observed in females treated at the 1.2% level was not accompanied by histological abnormalities in other immune system organs. The author considered the atrophy to be stress-induced and not degenerative.

Conclusions:

On the basis of our data, the NOEL of C14MES for the repeated dose toxicity was 0.3%, which corresponds to 175 mg/kg bw/day for male rats and 249 mg/kg bw/day for female rats. The NOAEL of C14MES for the repeated dose toxicity was 0.6%, corresponding to 360 mg/kg bw/d for males and 497 mg/kg bw/d for females.

Reference 2

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

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

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

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

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.

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.

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

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

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

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.

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.

Critical effects observed:

not specified

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.

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.

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2, 2000 - May 29, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study has been performed according to OECD and EC guidelines and according to GLP principles. Although the data in the study report were missing, they were included in the OECD SIDS publication, sponsored by the Ministry of Foreign Affairs of Japan.

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- strain: Crj: CD (SD) IGS
- Source:Charles River Laboratories Japan, Inc
- Age at study initiation: 8 weeks
- Weight at study initiation: 364 to 420 g for males and 223 to 264 g for females.
- Fasting period before study: no
- Housing: in individual stainless steel cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): >= 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Administered suspensions were prepared by mixing the substance with Japanese Pharmacopoeia-grade olive oil to designated concentration doses.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The hexadecanoic acid, 2-sulfo-, 1-methylester, sodium salt suspensions of 0.1 w/v% (for 5 mg/kg group), 0.4 w/v% (for 20 mg/kg group), 1.6 w/v% (for 80 mg/kg group) and 6.0 w/v% (for 300 mg/kg) prepared for the first and last times were analyzed using the HPLC method. The analysis was repeated three times, and the mean values were calculated.

Date of preparation Displayed value (w/v %)
0.1 0.4 1.6 6.0

May 30, 2000 0.10 0.40 1.7 6.2
July 8, 2000 0.09 0.40 1.6 5.8

The results shown below confirm that the suspensions were prepared at the designated concentrations.

Duration of treatment / exposure:

The administration duration ranged from 14 days before start of mating for both males and females, lasting 47 days for males and to 42 days at the shortest to 55 days at longest after birth just before day 4 of lactation for females.

Frequency of treatment:

once a day

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The results of the range finding test indicate that repeated dose toxicity of this substance mainly affects digestion due to local stimulation, especially the stomach. The substance is also considered to influence hepatic and hematologic functions. Based on these results, the doses of 5 mg/kg/day were established as minimum and 300 mg/kg/day as maximum, with 20 and 80 mg/kg/day as intermediate ranges.

Positive control:

Not applicable.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day: mortality, appearance, and behavior, pregnancy, birth, and lactation.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day

BODY WEIGHT: Yes
- Time schedule for examinations: on day 1 (before dosing), 8, 15, 22, 29, 36, 43 and 47 of treatment for males; on day 1, 8 and 15 of treatment and on days 0, 7, 14, and 20 of pregnancy and on days 0 and 4 of lactation and on the day of autopsy for females.

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: Twenty-four hour food consumption per cage was determined from the days of weight measurement through the following days. The last day of food consumption measurement was, however, Day 46 of administration for males and Day 3 of lactation for females. During the mating period, food consumption was not determined for any animals on Day 15 of administration. On Day 22, the weights of male and female rats that had not mated were not measured.

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: No

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: hematology was carried out at the time of necropsy after 48 days for males and at 5 days after deliveray for females.
- Anaesthetic used for blood collection: Yes ether
- Animals fasted: yes
- How many animals: all
- Parameters: red blood cell count, hemoglobin content, hematocrit level, mean red blood cell volume, mean red blood cell hemoglobin content, mean red blood cell hemoglobin concentration, white blood cell and blood platelet counts, reticulocyte count and white blood cell percentage, prothrombin time and activated portion thromboplastin time.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: biochemistry was carried out at the time of necropsy after 48 days for males and at 5 days after deliveray for females.
- Animals fasted: Yes
- How many animals: all
- Parameters: total protein, albumin, A/G ratio, blood sugar, total cholesterol, triglyceride, total bilirubin, urea nitrogen, creatine, GOT, GPT, γ-GTP, ALP, LDH, calcium, and inorganic phosphorous; sodium, potassium, and chlorine.


URINALYSIS: Yes
- Time schedule for collection of urine: for all males, urinalysis was carried out on day 41 or 42 of the administration period.
pH, occult blood, protein, sugar, ketone body, bilirubin, urobilinogen, appearance, specific gravity, sedimentation.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes organ weights measured:
Brain, heart, liver, kidney, spleen, adrenal, thymus, testis and epididymis in males; brain, heart, liver, kidney, spleen, adrenal, thymus in females.
Organ weight was determined in 9 males at 20 mg/kg bw/day, in 10 males at 0 (control), 5, 80, and 300 mg/kg bw/day, in 7 females at 0 (control), 8
females at 5 mg/kg bw/day, in 9 females at 20 and 300 mg/kg bw/day, in 10 females at 80 mg/kg bw/day.

HISTOPATHOLOGY: Yes Microscopic examination:
Brain, spinal cord, stomach, 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, sublingual gland for 5 males and 5 females in 0 and 300 mg/kg bw:/day groups, and stomach for 5 males and 5 females in 5, 20, 80 mg/kg bw/day groups.
The testis, epididymis, prostrate, seminal vesicle, or ovaries were examined in all subjects in the control and 300 mg/kg groups, male and female rats for which no pregnancy resulted, and females indicating the death of the whole embryo after conception. In addition, the uterus and pituitary gland were examined in females for which no pregnancy resulted or indicating the death of the whole embryo after conception. The pituitary gland and thymus were also studied in females whose pups died during delivery and lactation.

Statistics:

Bartlett’s test was used to test homogeneity of variances in various parametric samples. When variances were equal across samples of data, the one-way analysis of variance (ANOVA) was performed.
If significant differences were found, Dunnett’s or Scheffe’s test (when the number of specimens differed between groups) was applied to compare each study group with the control group. When variances were not equal across samples of data or non-parametric data such as the number of implantations, birth rate, delivery rate, newborn survival rate, and qualitative data of urine test were used, the Kruskal-Wallis rank test was performed. If significant difference was found, Dunnett’s or Scheffe's test (when the number of specimens differed between groups) was applied to compare each study group with the control group. Regarding categorical data, a chi-square test was used to test mating, conception, delivery rate, and offspring sex ratio, while the Fisher exact probability test was used to examine the incidence of abnormal cases in the observation of general conditions and pathological examination.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There was no mortality related to the test substance treatment.
Transitional softening stools in a few males and females were observed in the 80 and 300 mg/kg bw/day groups.

BODY WEIGHT AND WEIGHT GAIN
No statistically significant changes for males and females.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No statistically significant changes for males and females.

HAEMATOLOGY
No statistically significant changes for males and females.

CLINICAL CHEMISTRY
Blood chemistry: Males: An increase in GPT levels and a decrease in triglyceride levels in 300 mg/kg bw/day groups. No changes seen in the females.

URINALYSIS
No statistically significant changes.

ORGAN WEIGHTS
No statistically significant changes for males and females.

GROSS PATHOLOGY
Thickening of the forestomach mucosa was observed in 6 of 10 males and 10 of 10 females at 80 mg/kg bw/day and 10 of 10 males and 9 of 9 females at 300 mg/kg bw/day.

HISTOPATHOLOGY: NON-NEOPLASTIC
Squamous hyperplasia and hyperkeratosis in the 80 and 300 mg/kg bw/d groups, erosion, and lamina propria and/or submucosa edema and inflammatory cell infiltration ( mainly neutrophils) were observed in the forestomach in both sexes of 80 and 300 mg/kg bw/day groups.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: blood chemistry

Critical effects observed:

not specified

The NOAEL for local effects was determined to be 20 mg/kg bw/d based on effects on forstomach mucosa. This is however not of relevance in the hazard assessment.

Conclusions:

The NOAEL for systemic effects was determined to be 80 mg/kg bw/day based on higher GPT and lower triglyceride levels in the 300 mg/kg/bw group, indicating the liver as target organ.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

80 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:

adverse effect observed

Dose descriptor:

NOAEL

111 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral:

In a 28 -day oral repeated dose toxicity study, performed according to OECD 407 test guidelines, rats in groups of 5/sex/dose were exposed to 0, 700, 2000 and 8000 ppm of C16MES via the diet. These doses were based on a 7 -day range finding study. The following parameters were evaluated: mortality, clinical signs, FOB, body weight, food consumption, haematology, biochemical parameters, macroscopy, organ weights and microscopy. No toxicologically significant changes were noted in any of the parameters investigated in this study. Therefore, a NOAEL of >=865.8 mg/kg bw/day (equivalent to 8000 ppm) was derived.

In an oral reproduction/developmental screening study, performed according to OECD 422 test guidelines, rats were exposed to 0, 5, 20, 80 and 300 mg/kg bw/d C16MES by gavage. No toxicologically significant changes were noted in any of the following parental parameters investigated, i.e. clinical appearance, body weight, food consumption, macroscopic examination, organ weights and microscopic examination. Some significant biochemical effects were observed at the highest dose tested. Therefore, the parental NOAEL was determined to be 80 mg/kg bw/d.

In a supporting oral reproduction/developmental screening study, performed according to OECD 422 test guidelines, rats were exposed to C14MES by gavage, showing effects at the highest dose tested with a NOAEL of 360 mg/kg bw/day.

Dermal:

In a 28 -day dermal repeated dose toxicity study, performed according to OECD 410 test guidelines, rats in groups of 5/sex/dose were exposed to 0, 1.5, 5 and 15 w/v% of a mixture of C12/C14/C16 (1/2/7) MES via dermal application. These doses were based on a 5 -day range finding study. The following parameters were evaluated: mortality, clinical signs, body weight, food consumption, haematology, biochemical parameters, macroscopy, organ weights and microscopy. No toxicologically significant changes systemically were noted in any of the parameters investigated in this study. Therefore, a systemic NOAEL of >=15%, equivalent to 111 mg/kg bw/day, was derived. At the application site mild irritation occurred, accompanied by histopathological changes, e.g. mild parakeratosis and thickened epidermis in high dose males and females. Therefore, the local NOAEL is set at 5%, equivalent to 37 mg/kg bw/day.

Although no data is available on C18MES, it is judged that the conclusive information on the main component C16MES (>45% - <85%) can be used to describe the potential effects of the reaction mass concerning the repeated dose toxicity.

Justification for classification or non-classification

Based on the available data, the reaction mass does not have to be classified for repeated dose toxicity according to CLP Regulation 1272/2008.