Registration Dossier

Administrative data

Description of key information

For the whole category of alcohol ethoxysulfates (AES) a oral NOAEL of 300 mg/kg bw/d was established.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
Urinalysis and neurobehaviour were not examined.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
According to Guideline.
Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 25, 75, 225 mg/kg bw
Basis:
nominal in water
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
According to Guideline.
Positive control:
Not necessary
Observations and examinations performed and frequency:
According to Guideline.
Sacrifice and pathology:
According to Guideline.
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):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
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
Dose descriptor:
NOAEL
Effect level:
> 225 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed.
Critical effects observed:
not specified
Conclusions:
For the systemic toxicity after repeated oral application a NOAEL of greater than 225 mg/kg bw/day can be deduced.
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Reason / purpose:
read-across source
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
According to Guideline.
Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 25, 75, 225 mg/kg bw
Basis:
nominal in water
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
According to Guideline.
Positive control:
Not necessary
Observations and examinations performed and frequency:
According to Guideline.
Sacrifice and pathology:
According to Guideline.
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):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
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
Dose descriptor:
NOAEL
Effect level:
> 225 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed.
Critical effects observed:
not specified
Conclusions:
For the systemic toxicity after repeated oral application a NOAEL of greater than 225 mg/kg bw/day can be deduced.
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Reason / purpose:
read-across source
Species:
rat
Strain:
other: CD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River U.K. Ltd., Margate, Kent
- Age at study initiation: 3 weeks
- Weight at study initiation: 40-50 g
- Fasting period before study: No
- Housing: Five rats per cage (suspended polypropylene cages measuring 52 x 35 x 18 cm with stainless steel mesh floors and lids)
- Diet: Complete powdered rodent diet (Spratts Laboratory Animal Diet No. 2), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 3 days, any rat that failed to gain weight at this time was discarded

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 1
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): A pre-mix containing 215,000 ppm test material was prepared each week and, from this, the different dietary concentrations were obtained by direct dilution with further quantities of diet
- Mixing appropriate amounts with (Type of food): Complete powdered rodent diet (Spratts Laboratory Animal Diet No. 2)
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
13 weeks (91 days)
Frequency of treatment:
Continuous through the diet
Remarks:
Doses / Concentrations:
0.05, 0.5 and 5% (w/w) of test material
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0.015, 0.15 and 1.5% active substance
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
10, 100 and 1100 mg/kg bw/day active substance
Basis:
nominal in diet
No. of animals per sex per dose:
20
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Based upon the result of a 14-day preliminary study, which indicated that the test substance incorporated into the diet at 5% w/v would result in a 10% reduction in bodyweight over the treatment period; it was also requested to examine the lower treatment levels of 1/10 and 1/100 the upper level (i.e. 0.5% w/v and 0.05% w/w).
- Rationale for animal assignment (if not random): The mean bodyweights of the male and female subgroups were adjusted by exchange of animals until they all fell within a range of 60-61 g (males) or 59-60 g (females)
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily
- Cage side observations included: Mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily
- All animals were physically examined at appropriate intervals, or otherwise sensible, signs of ill-health or reaction to treatment

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded on the day that treatment commenced and subsequently at weekly intervals throughout the treatment period

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
- The quantity of food eaten by each cage of rats was calculated weekly, by measurements of the amount of food given, and that remaining in the food hoppers

FOOD EFFICIENCY: Yes
- 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 (food conversion ratios calculated on a per cage basis)

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the conclusion of the treatment period
- Anesthetic used for blood collection: No data
- Animals fasted: No data
- Collection method: Blood samples were withdrawn from the retro-orbital sinus using EDTA as anticoagulant
- How many animals: Five male and five female rats from groups 1 and 4 each (on account of findings in the female rats of group 4, the examination of hemoglobin concentration, erythrocyte count and packed cell volume was extended to similar numbers or female rats in groups 2 and 3 at week 13)
- Parameters examined: hemoglobin concentration (Hb) as cyanmethemoglobin, erythrocyte count (RBC), total leukocyte count (WBC), differential leukocyte count (WBC) and packed cell volume (PCV) as microhematocrit
- From the erythrocytic data, the mean cell hemoglobin concentration and mean cell volume were calculated for each sample

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the conclusion of the treatment period
- Animals fasted: No data
- How many animals: Five male and five female rats from groups 1 and 4 each (on account of findings in the rats of group 4, the examination of alkaline phosphatase activity and glutamate-pyruvate transaminase activity was extended to similar numbers or rats in groups 2 and 3 at week 13)
- Parameters examined: alkaline phosphatase activity (SAP) and glutamate-pyruvate transaminase activity (SGPT)

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:
SACRIFICE AND PATHOLOGY
- All rats were killed after 13 weeks of treatment by carbon dioxide inhalation; necropsy followed as soon as possible

GROSS PATHOLOGY: Yes
- A detailed necropsy was performed with opening of the cranial, thoracic and abdominal cavities; the appearance of any abnormal tissues was recorded.

HISTOPATHOLOGY: Yes
- Samples of the following tissues were taken from each rat and preserved in buffered 4% formaldehyde saline and examined microscopically: adrenal glands, brain, duodenum, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (cervical and mesenteric), ovaries, spleen, stomach, testes and urinary bladder
- After dehydration and embedding in paraffin wax, sections of the required tissues were cut at 5 µm thickness and stained with hematoxylin and eosin. Both glandular and non-glandular areas of the stomach, and both auricular and ventricular sections of the heart, were prepared. The brain was sectioned at three levels (cerebellum, cerebral cortex and medulla).
Other examinations:
- Organ weight analysis: adrenal glands, brain, heart, kidneys, liver, ovaries, spleen, testes and thyroid glands
- The ratio of organ to bodyweight was calculated in each case (the ratio of organ weight to brain weight was also determined for the liver, kidney, spleen and heart)
- The following were not examined histopathologically but were taken from ten male and ten female rats per group and stored in fixative for possible future requirement for microscopic evaluation: aorta, colon, eye, esophagus, pituitary, prostate gland, skeletal muscle, skin, spinal cord, trachea, thyroids and uterus.
- A bone marrow smear was taken from each rat, air-dried and fixed in methanol
- The eye was preserved in Davidson’s fluid
Statistics:
The significance of any inter-group differences in organ weights, blood composition, and growth performance was assessed by analysis of variance.
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):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
not specified
Description (incidence and severity):
not applicable
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
- No mortality occurred
- There was on observable difference in the appearance, mood, locomotion, and fecal consistency of treated and control animals, and no animals died during the treatment period.

BODY WEIGHT AND WEIGHT GAIN:
- The bodyweight gains of both sexes in the 5.0% group were reduced from the commencement of treatment, so that their bodyweights were significantly different from the controls when analyzed at week 2 (p < 0.01, analysis of variance); at termination their bodyweights were 84% (males) and 85% (females) of those of the respective controls
-The bodyweight gains of both sexes in the 0.5% group were depressed from week 5 (males) or week 6 (females), although this reduction was statistically significant in the males only
- The growth of either sex was undisturbed in the 0.05% group

FOOD CONSUMPTION AND COMPOUND INTAKE: No information regarding statistical significance provided.
- A small reduction in food consumption occurred in both sexes receiving in the 5.0% group during the first 5 weeks of treatment; total food intakes being 93% (males) and 90% (females) of the respective controls
- The food consumption of females in the 5.0% group remained depressed until week 10, but thereafter was unaffected, whereas the food intake of the males at this treatment level was unaffected from week 6 to week 13
- The food consumption of females in the 0.50% was slightly reduced over the first seven weeks of treatment, but unaffected thereafter; the food intakes of males at this treatment level, and of both sexes at the lowest level, were unaffected throughout the treatment period

FOOD EFFICIENCY: No information on statistical significance provided.
- The food conversion ratios of males in the 5.0% group were consistently greater than those of the controls, indicating inferior food utilization in this group
- The food conversion ratios of females at this treatment level did not demonstrate a clear pattern from week to week, but the overall (week 1-13) value also indicated slightly inferior food utilization
- Rats at the low and intermediate treatment levels showed comparable food conversion ratios to those of the controls.

HAEMATOLOGY
- Females in the 5.0% and 0.5% groups displayed erythrocytic characteristics slightly lower than in controls, statistical significance being achieved only in packed cell volume (5% dose group) and erythrocyte count (0.5% dose group)

CLINICAL CHEMISTRY
- SGPT and SAP levels were significantly elevated in both sexes in the 5.0% group
- The differences in alkaline phosphatase levels in the males were exaggerated by the contribution of one control rat which yielded a very low value (8i.u./L; confirmed by a duplicate analysis). In a statistical analysis omitting this animal, the significance of the difference between the group mean values of the high treatment level and control groups was lower (p< 0.01)
- At the lower treatment levels, occasional animals showed elevations of SGPT or SAP, but neither consistent intergroup variations nor significant deviations from control values were evident.

ORGAN WEIGHTS
- Ratios of organ weights to bodyweight were calculated since bodyweight differences between the control, the 0.5 and 5% dose groups obscured the comparison of organ weights relative to bodyweight
- The liver weights of both sexes in the 5.0% group were significantly elevated.
- The heart weights of males in the 5.0% group were significantly reduced when analyzed in absolute terms and relative to brain weight, as also were spleen and kidney weights relative to brain weight.
- Although there were occasional deviations from the control values in the weights of various organs from males treated at the low and intermediate treatment levels when analyzed relative to bodyweight, there were no consistent changes in these organ weights when analyzed in absolute terms or relative to brain weight.

GROSS PATHOLOGY
- No treatment-related changes were seen in the macroscopic appearance of the tissues examined at necropsy

HISTOPATHOLOGY: NON-NEOPLASTIC
- No histopathological changes or variations from normal were seen that were considered to be related to treatment
Dose descriptor:
NOAEL
Effect level:
0.15 other: % in feed
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Lower body weight gain in males in final eight weeks of the study.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Lower body weight gain in males in final eight weeks of the study.
Dose descriptor:
LOAEL
Effect level:
1.5 other: % in feed
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Lower body weight gain, small reduction in red cell parameters, elevation in serum SGPT and alkaline phosphatase activity, elevations in absolute and relative liver weights.
Dose descriptor:
LOAEL
Effect level:
1 100 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Lower body weight gain, small reduction in red cell parameters, elevation in serum SGPT and alkaline phosphatase activity, elevations in absolute and relative liver weights.
Dose descriptor:
NOEL
Effect level:
0.05 other: % in feed
Based on:
test mat.
Sex:
male/female
Dose descriptor:
LOEL
Effect level:
0.5 other: % in feed
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Lower body weight gain in males in final eight weeks of the study.
Dose descriptor:
LOEL
Effect level:
5 other: % in feed
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Lower body weight gain, small reduction in red cell parameters, elevation in serum SGPT and alkaline phosphatase activity, elevations in absolute and relative liver weights.
Critical effects observed:
not specified

Gonadal tissues were examined for both gross pathology and histopathology and for organ weight analysis and no treatment-related effects were detected.

Conclusions:
Since the decrease in the erythrocyte count was not dose-dependent, it was concluded that the test substance at dietary levels up to 0.05% test material (corresponding to 100 mg/kg bw/day) elicited no adverse effects.
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Reason / purpose:
read-across source
Species:
rat
Strain:
other: albino
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, Massachusetts.
- Weight at study initiation: 112-159 g (males), 102-132 g (females)
- Housing: individually housed
- Diet: ad libitum
- Water: ad libitum

IN-LIFE DATES: From: 1976-06-29 To: 1976-10-06
Route of administration:
oral: feed
Vehicle:
other: plain diet
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Appropriate amounts of the test material were incorporated into the basal diet on a weight/weight basis and thoroughly mixed.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Purina Laboratory Chow
- Storage temperature of food: 22-26°C


VEHICLE
- Justification for use and choice of vehicle (if other than water): N/A
- Concentration in vehicle: N/A
- Amount of vehicle (if gavage): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each freshly prepared diet were submitted to the sponsor for analysis

Duration of treatment / exposure:
13 weeks
Frequency of treatment:
continuous
Remarks:
Doses / Concentrations:
0.1, 0.5, and 1.0% active
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
60, 300 and 600 mg/kg bw/day active
Basis:
nominal in diet
No. of animals per sex per dose:
20
Control animals:
yes, plain diet
Details on study design:
- Rationale for animal assignment (if not random): by individual, initial body weight so that a homogenous distribution of mean weights and weight ranges was obtained among groups.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- 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: Yes
- 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: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to treatment and at week 13
- Dose groups that were examined: some animals from all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 5 rats/sex/group at week 4 and 10 rats/sex/group at week 13
- How many animals: 5 rats/sex/group at week 4 and 10 rats/sex/group at week 13

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at sacrifice
- Animals fasted: Yes
- How many animals: 5 rats/sex/group at week 4 and 10 rats/sex/group at week 13

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. The following organs of each rat were weighed and the organ/body weight ratios determined: heart, liver, kidneys, testes with epididymides, pituitary, adrenals, and ovaries.

HISTOPATHOLOGY: Yes. Brain with meninges, pituitary, thoracic spinal cord, thyroids, trachea, esophagus, salivary glands, eyes, tongue, lungs, heart, liver, kidneys, spleen, stomach, pancreas, lymph nodes, small and large intestine, adrenals, urinary bladder, ureters, urethra, testes, ovaries, prostate, uterus, vagina, seminal vesicles, inguinal mammary gland, skin, bone, muscle, and any unusual lesions. (5 rats/sex sacrificed at 4 weeks from the control and high-dose groups and 10 rats/sex sacrificed at 13 weeks from the control and high-dose groups. Also, kidney and liver sections from the low- and mid- dose groups from 5 rats/sex, sacrificed at 13 weeks were examined)
Statistics:
The following data from control and treated groups of the same sex were statistically compared: growth rate and total food consumption, clinical laboratory data, and terminal body weights, organ weights, and organ/body weight ratios.
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:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL CHEMISTRY:
Differences of statistical significance noted included the following: lower than control mean glutamic-oxaloacetic transaminase values in all of the treated males at week 4 and all of the treated females at week 13; lower than control mean alkaline phoshatase values in all of the treated females at week 4; higher than control mean glucose values in the low- and high-dose females at week 13; lower than control mean albumin values in the high-dose males at week 4 and the low-dose females at week 13; a higher than control mean sodium values in the high-dose females at week 4; and higher than control mean calcium values in the high-dose females at week 4 and the low-dose females at week 13.

ORGAN WEIGHTS:
The liver weights and ratios of all of the treated groups of both sexes were slightly to moderately higher than respective control data at weeks 4, 13, and 14. These differences were generally (but not always distinctly) dose-related and statistically significant at some of the intervals. The heart weights and ratios of all of the male treated groups were slightly, (not statistically significantly) lower than those of the controls at weeks 4, 13, and 14. Microscopic examination of liver and heart sections did not confirm the presence of any treatment-related hepatic or cardiac alterations.
Dose descriptor:
NOEL
Effect level:
>= 1 other: % in the diet
Based on:
act. ingr.
Sex:
male/female
Dose descriptor:
NOEL
Effect level:
>= 600 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Critical effects observed:
not specified

Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effects were detected.

Conclusions:
No outstanding treatment related toxic effects were noted for the test substance administered up to 1% in the diets. Although there were statistically significant dose-related differences in absolute and relative liver and heart weights noted in test substance-treated groups compared to control animals, no histopathological correlation was found to confirm the presence of any treatment-related alterations. As such, the NOEL for the study was determined to be the highest dose, 1% in the diet.
Executive summary:

This study was designed to evaluate the toxicological effects of the test substance in albino rats (20/sex/group) when administered in the diet for thirteen weeks at levels of 0.0, 0.1, 0.5, and 1.0% active ingredient. Interim sacrifices were performed on five rats/sex/group at four weeks and ten rats/sex/group at thirteen weeks. Five rats/sex/group were maintained for one additional week and sacrificed at Week 14. The criteria evaluated for compound effects were clinical signs, mortality, body weight and food consumption, ophthalmoscopic findings, clinical chemistry and haematology, organ weights, and gross and microscopic pathology. 

No distinct effect attributed to the administration of the test substance was noted in comparisons of the clinical signs, mortality rates, ophthalmoscopic findings or haematology and blood chemistry values of the test groups to the controls.  In addition, no compound related gross or histomorphologic organ or tissue alterations were noted in any of the treated animals. 

When compared to the data of the control group, slightly lower body weight gains were noted in the mid- and high-dose male groups.  These differences were not statistically significant. 

The absolute and relative liver weights of all treated groups of both sexes were slightly to moderately higher than respective control data at Weeks 4, 13 and 14.  These differences were generally (but not always distinctly) dose-related and statistically significant at some of the intervals.  The heart weights and ratios of all of the male treated groups were slightly, (not statistically significantly) lower than those of the controls at Weeks 4, 13 and 14.  However, neither gross pathology, nor microscopic examination of liver and heart sections confirmed the presence of any treatment-related hepatic or cardiac alterations. 

Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effects were detected.

No outstanding treatment related toxic effects were noted when the test substance was administered up to 1% in the diet to albino rats. Although there were statistically significant differences in absolute and relative liver and heart weights noted in test substance-treated groups compared to control animals, and these differences were generally dose-related, no histopathological correlation was found to confirm the presence of any treatment-related alterations. As such the NOEL was determined to be the 1% dose level.

Endpoint:
chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Reason / purpose:
read-across source
Dose descriptor:
NOEL
Effect level:
> 250 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Feeding study
Dose descriptor:
NOEL
Effect level:
> 75 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Drinking water study
Critical effects observed:
not specified
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Reason / purpose:
read-across source
Dose descriptor:
NOAEL
Effect level:
> 250 mg/kg bw/day (nominal)
Sex:
male/female
Critical effects observed:
not specified
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was conducted in methods comparable to OECD guideline 408 "Repeated Dose 90-day Oral Toxicity Study in Rodents". Not GLP.
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other: CD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River U.K. Ltd., Margate, Kent
- Age at study initiation: 3 weeks
- Weight at study initiation: 40-50 g
- Fasting period before study: No
- Housing: Five rats per cage (suspended polypropylene cages measuring 52 x 35 x 18 cm with stainless steel mesh floors and lids)
- Diet: Complete powdered rodent diet (Spratts Laboratory Animal Diet No. 2), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 3 days, any rat that failed to gain weight at this time was discarded

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 1
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): A pre-mix containing 215,000 ppm test material was prepared each week and, from this, the different dietary concentrations were obtained by direct dilution with further quantities of diet
- Mixing appropriate amounts with (Type of food): Complete powdered rodent diet (Spratts Laboratory Animal Diet No. 2)
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
13 weeks (91 days)
Frequency of treatment:
Continuous through the diet
Remarks:
Doses / Concentrations:
0.05, 0.5 and 5% (w/w) of test material
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0.015, 0.15 and 1.5% active substance
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
10, 100 and 1100 mg/kg bw/day active substance
Basis:
nominal in diet
No. of animals per sex per dose:
20
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Based upon the result of a 14-day preliminary study, which indicated that the test substance incorporated into the diet at 5% w/v would result in a 10% reduction in bodyweight over the treatment period; it was also requested to examine the lower treatment levels of 1/10 and 1/100 the upper level (i.e. 0.5% w/v and 0.05% w/w).
- Rationale for animal assignment (if not random): The mean bodyweights of the male and female subgroups were adjusted by exchange of animals until they all fell within a range of 60-61 g (males) or 59-60 g (females)
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily
- Cage side observations included: Mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily
- All animals were physically examined at appropriate intervals, or otherwise sensible, signs of ill-health or reaction to treatment

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded on the day that treatment commenced and subsequently at weekly intervals throughout the treatment period

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
- The quantity of food eaten by each cage of rats was calculated weekly, by measurements of the amount of food given, and that remaining in the food hoppers

FOOD EFFICIENCY: Yes
- 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 (food conversion ratios calculated on a per cage basis)

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the conclusion of the treatment period
- Anesthetic used for blood collection: No data
- Animals fasted: No data
- Collection method: Blood samples were withdrawn from the retro-orbital sinus using EDTA as anticoagulant
- How many animals: Five male and five female rats from groups 1 and 4 each (on account of findings in the female rats of group 4, the examination of hemoglobin concentration, erythrocyte count and packed cell volume was extended to similar numbers or female rats in groups 2 and 3 at week 13)
- Parameters examined: hemoglobin concentration (Hb) as cyanmethemoglobin, erythrocyte count (RBC), total leukocyte count (WBC), differential leukocyte count (WBC) and packed cell volume (PCV) as microhematocrit
- From the erythrocytic data, the mean cell hemoglobin concentration and mean cell volume were calculated for each sample

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the conclusion of the treatment period
- Animals fasted: No data
- How many animals: Five male and five female rats from groups 1 and 4 each (on account of findings in the rats of group 4, the examination of alkaline phosphatase activity and glutamate-pyruvate transaminase activity was extended to similar numbers or rats in groups 2 and 3 at week 13)
- Parameters examined: alkaline phosphatase activity (SAP) and glutamate-pyruvate transaminase activity (SGPT)

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:
SACRIFICE AND PATHOLOGY
- All rats were killed after 13 weeks of treatment by carbon dioxide inhalation; necropsy followed as soon as possible

GROSS PATHOLOGY: Yes
- A detailed necropsy was performed with opening of the cranial, thoracic and abdominal cavities; the appearance of any abnormal tissues was recorded.

HISTOPATHOLOGY: Yes
- Samples of the following tissues were taken from each rat and preserved in buffered 4% formaldehyde saline and examined microscopically: adrenal glands, brain, duodenum, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (cervical and mesenteric), ovaries, spleen, stomach, testes and urinary bladder
- After dehydration and embedding in paraffin wax, sections of the required tissues were cut at 5 µm thickness and stained with hematoxylin and eosin. Both glandular and non-glandular areas of the stomach, and both auricular and ventricular sections of the heart, were prepared. The brain was sectioned at three levels (cerebellum, cerebral cortex and medulla).
Other examinations:
- Organ weight analysis: adrenal glands, brain, heart, kidneys, liver, ovaries, spleen, testes and thyroid glands
- The ratio of organ to bodyweight was calculated in each case (the ratio of organ weight to brain weight was also determined for the liver, kidney, spleen and heart)
- The following were not examined histopathologically but were taken from ten male and ten female rats per group and stored in fixative for possible future requirement for microscopic evaluation: aorta, colon, eye, esophagus, pituitary, prostate gland, skeletal muscle, skin, spinal cord, trachea, thyroids and uterus.
- A bone marrow smear was taken from each rat, air-dried and fixed in methanol
- The eye was preserved in Davidson’s fluid
Statistics:
The significance of any inter-group differences in organ weights, blood composition, and growth performance was assessed by analysis of variance.
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):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
not specified
Description (incidence and severity):
not applicable
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
- No mortality occurred
- There was on observable difference in the appearance, mood, locomotion, and fecal consistency of treated and control animals, and no animals died during the treatment period.

BODY WEIGHT AND WEIGHT GAIN:
- The bodyweight gains of both sexes in the 5.0% group were reduced from the commencement of treatment, so that their bodyweights were significantly different from the controls when analyzed at week 2 (p < 0.01, analysis of variance); at termination their bodyweights were 84% (males) and 85% (females) of those of the respective controls
-The bodyweight gains of both sexes in the 0.5% group were depressed from week 5 (males) or week 6 (females), although this reduction was statistically significant in the males only
- The growth of either sex was undisturbed in the 0.05% group

FOOD CONSUMPTION AND COMPOUND INTAKE: No information regarding statistical significance provided.
- A small reduction in food consumption occurred in both sexes receiving in the 5.0% group during the first 5 weeks of treatment; total food intakes being 93% (males) and 90% (females) of the respective controls
- The food consumption of females in the 5.0% group remained depressed until week 10, but thereafter was unaffected, whereas the food intake of the males at this treatment level was unaffected from week 6 to week 13
- The food consumption of females in the 0.50% was slightly reduced over the first seven weeks of treatment, but unaffected thereafter; the food intakes of males at this treatment level, and of both sexes at the lowest level, were unaffected throughout the treatment period

FOOD EFFICIENCY: No information on statistical significance provided.
- The food conversion ratios of males in the 5.0% group were consistently greater than those of the controls, indicating inferior food utilization in this group
- The food conversion ratios of females at this treatment level did not demonstrate a clear pattern from week to week, but the overall (week 1-13) value also indicated slightly inferior food utilization
- Rats at the low and intermediate treatment levels showed comparable food conversion ratios to those of the controls.

HAEMATOLOGY
- Females in the 5.0% and 0.5% groups displayed erythrocytic characteristics slightly lower than in controls, statistical significance being achieved only in packed cell volume (5% dose group) and erythrocyte count (0.5% dose group)

CLINICAL CHEMISTRY
- SGPT and SAP levels were significantly elevated in both sexes in the 5.0% group
- The differences in alkaline phosphatase levels in the males were exaggerated by the contribution of one control rat which yielded a very low value (8i.u./L; confirmed by a duplicate analysis). In a statistical analysis omitting this animal, the significance of the difference between the group mean values of the high treatment level and control groups was lower (p< 0.01)
- At the lower treatment levels, occasional animals showed elevations of SGPT or SAP, but neither consistent intergroup variations nor significant deviations from control values were evident.

ORGAN WEIGHTS
- Ratios of organ weights to bodyweight were calculated since bodyweight differences between the control, the 0.5 and 5% dose groups obscured the comparison of organ weights relative to bodyweight
- The liver weights of both sexes in the 5.0% group were significantly elevated.
- The heart weights of males in the 5.0% group were significantly reduced when analyzed in absolute terms and relative to brain weight, as also were spleen and kidney weights relative to brain weight.
- Although there were occasional deviations from the control values in the weights of various organs from males treated at the low and intermediate treatment levels when analyzed relative to bodyweight, there were no consistent changes in these organ weights when analyzed in absolute terms or relative to brain weight.

GROSS PATHOLOGY
- No treatment-related changes were seen in the macroscopic appearance of the tissues examined at necropsy

HISTOPATHOLOGY: NON-NEOPLASTIC
- No histopathological changes or variations from normal were seen that were considered to be related to treatment
Dose descriptor:
NOAEL
Effect level:
0.15 other: % in feed
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Lower body weight gain in males in final eight weeks of the study.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Lower body weight gain in males in final eight weeks of the study.
Dose descriptor:
LOAEL
Effect level:
1.5 other: % in feed
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Lower body weight gain, small reduction in red cell parameters, elevation in serum SGPT and alkaline phosphatase activity, elevations in absolute and relative liver weights.
Dose descriptor:
LOAEL
Effect level:
1 100 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Lower body weight gain, small reduction in red cell parameters, elevation in serum SGPT and alkaline phosphatase activity, elevations in absolute and relative liver weights.
Dose descriptor:
NOEL
Effect level:
0.05 other: % in feed
Based on:
test mat.
Sex:
male/female
Dose descriptor:
LOEL
Effect level:
0.5 other: % in feed
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Lower body weight gain in males in final eight weeks of the study.
Dose descriptor:
LOEL
Effect level:
5 other: % in feed
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Lower body weight gain, small reduction in red cell parameters, elevation in serum SGPT and alkaline phosphatase activity, elevations in absolute and relative liver weights.
Critical effects observed:
not specified

Gonadal tissues were examined for both gross pathology and histopathology and for organ weight analysis and no treatment-related effects were detected.

Conclusions:
Since the decrease in the erythrocyte count was not dose-dependent, it was concluded that the test substance at dietary levels up to 0.05% test material (corresponding to 100 mg/kg bw/day) elicited no adverse effects.
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
other information
Study period:
1976-06-29 to 1977-02-21
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well conducted and documented guideline study comparable to OECD 408. Deviations include: details on acclimitisation, age, and housing; some recommended organ weights not performed; lacking information on test substance purity/stability/homogeneity.
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
Details on acclimitization, age, and housing; some recommended organ weights not performed; lacking information on test substance purity/stability/homogeneity.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other: albino
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, Massachusetts.
- Weight at study initiation: 112-159 g (males), 102-132 g (females)
- Housing: individually housed
- Diet: ad libitum
- Water: ad libitum

IN-LIFE DATES: From: 1976-06-29 To: 1976-10-06
Route of administration:
oral: feed
Vehicle:
other: plain diet
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Appropriate amounts of the test material were incorporated into the basal diet on a weight/weight basis and thoroughly mixed.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Purina Laboratory Chow
- Storage temperature of food: 22-26°C


VEHICLE
- Justification for use and choice of vehicle (if other than water): N/A
- Concentration in vehicle: N/A
- Amount of vehicle (if gavage): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each freshly prepared diet were submitted to the sponsor for analysis

Duration of treatment / exposure:
13 weeks
Frequency of treatment:
continuous
Remarks:
Doses / Concentrations:
0.1, 0.5, and 1.0% active
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
60, 300 and 600 mg/kg bw/day active
Basis:
nominal in diet
No. of animals per sex per dose:
20
Control animals:
yes, plain diet
Details on study design:
- Rationale for animal assignment (if not random): by individual, initial body weight so that a homogenous distribution of mean weights and weight ranges was obtained among groups.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- 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: Yes
- 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: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to treatment and at week 13
- Dose groups that were examined: some animals from all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 5 rats/sex/group at week 4 and 10 rats/sex/group at week 13
- How many animals: 5 rats/sex/group at week 4 and 10 rats/sex/group at week 13

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at sacrifice
- Animals fasted: Yes
- How many animals: 5 rats/sex/group at week 4 and 10 rats/sex/group at week 13

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. The following organs of each rat were weighed and the organ/body weight ratios determined: heart, liver, kidneys, testes with epididymides, pituitary, adrenals, and ovaries.

HISTOPATHOLOGY: Yes. Brain with meninges, pituitary, thoracic spinal cord, thyroids, trachea, esophagus, salivary glands, eyes, tongue, lungs, heart, liver, kidneys, spleen, stomach, pancreas, lymph nodes, small and large intestine, adrenals, urinary bladder, ureters, urethra, testes, ovaries, prostate, uterus, vagina, seminal vesicles, inguinal mammary gland, skin, bone, muscle, and any unusual lesions. (5 rats/sex sacrificed at 4 weeks from the control and high-dose groups and 10 rats/sex sacrificed at 13 weeks from the control and high-dose groups. Also, kidney and liver sections from the low- and mid- dose groups from 5 rats/sex, sacrificed at 13 weeks were examined)
Statistics:
The following data from control and treated groups of the same sex were statistically compared: growth rate and total food consumption, clinical laboratory data, and terminal body weights, organ weights, and organ/body weight ratios.
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:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL CHEMISTRY:
Differences of statistical significance noted included the following: lower than control mean glutamic-oxaloacetic transaminase values in all of the treated males at week 4 and all of the treated females at week 13; lower than control mean alkaline phoshatase values in all of the treated females at week 4; higher than control mean glucose values in the low- and high-dose females at week 13; lower than control mean albumin values in the high-dose males at week 4 and the low-dose females at week 13; a higher than control mean sodium values in the high-dose females at week 4; and higher than control mean calcium values in the high-dose females at week 4 and the low-dose females at week 13.

ORGAN WEIGHTS:
The liver weights and ratios of all of the treated groups of both sexes were slightly to moderately higher than respective control data at weeks 4, 13, and 14. These differences were generally (but not always distinctly) dose-related and statistically significant at some of the intervals. The heart weights and ratios of all of the male treated groups were slightly, (not statistically significantly) lower than those of the controls at weeks 4, 13, and 14. Microscopic examination of liver and heart sections did not confirm the presence of any treatment-related hepatic or cardiac alterations.
Dose descriptor:
NOEL
Effect level:
>= 1 other: % in the diet
Based on:
act. ingr.
Sex:
male/female
Dose descriptor:
NOEL
Effect level:
>= 600 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Critical effects observed:
not specified

Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effects were detected.

Conclusions:
No outstanding treatment related toxic effects were noted for the test substance administered up to 1% in the diets. Although there were statistically significant dose-related differences in absolute and relative liver and heart weights noted in test substance-treated groups compared to control animals, no histopathological correlation was found to confirm the presence of any treatment-related alterations. As such, the NOEL for the study was determined to be the highest dose, 1% in the diet.
Executive summary:

This study was designed to evaluate the toxicological effects of the test substance in albino rats (20/sex/group) when administered in the diet for thirteen weeks at levels of 0.0, 0.1, 0.5, and 1.0% active ingredient. Interim sacrifices were performed on five rats/sex/group at four weeks and ten rats/sex/group at thirteen weeks. Five rats/sex/group were maintained for one additional week and sacrificed at Week 14. The criteria evaluated for compound effects were clinical signs, mortality, body weight and food consumption, ophthalmoscopic findings, clinical chemistry and haematology, organ weights, and gross and microscopic pathology. 

No distinct effect attributed to the administration of the test substance was noted in comparisons of the clinical signs, mortality rates, ophthalmoscopic findings or haematology and blood chemistry values of the test groups to the controls.  In addition, no compound related gross or histomorphologic organ or tissue alterations were noted in any of the treated animals. 

When compared to the data of the control group, slightly lower body weight gains were noted in the mid- and high-dose male groups.  These differences were not statistically significant. 

The absolute and relative liver weights of all treated groups of both sexes were slightly to moderately higher than respective control data at Weeks 4, 13 and 14.  These differences were generally (but not always distinctly) dose-related and statistically significant at some of the intervals.  The heart weights and ratios of all of the male treated groups were slightly, (not statistically significantly) lower than those of the controls at Weeks 4, 13 and 14.  However, neither gross pathology, nor microscopic examination of liver and heart sections confirmed the presence of any treatment-related hepatic or cardiac alterations. 

Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effects were detected.

No outstanding treatment related toxic effects were noted when the test substance was administered up to 1% in the diet to albino rats. Although there were statistically significant differences in absolute and relative liver and heart weights noted in test substance-treated groups compared to control animals, and these differences were generally dose-related, no histopathological correlation was found to confirm the presence of any treatment-related alterations. As such the NOEL was determined to be the 1% dose level.

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards, well documented and acceptable for assessment.
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
Deviations:
yes
Remarks:
Only limited data are available
Dose descriptor:
NOEL
Effect level:
> 250 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Feeding study
Dose descriptor:
NOEL
Effect level:
> 75 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Drinking water study
Critical effects observed:
not specified
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards, well documented and acceptable for assessment.
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
Only limited data are available
Dose descriptor:
NOAEL
Effect level:
> 250 mg/kg bw/day (nominal)
Sex:
male/female
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The whole data base is conclusive and of high quality.

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

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was conducted in methods comparable to OECD guideline 411 " Subchronic Dermal Toxicity: 90-day Study". 25 animals per sex per dose, only two dose levels evaluated. Not GLP.
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
25 animals per sex per dose, only two dose levels evaluated.
GLP compliance:
no
Remarks:
However, quality reviews of the study were performed and documented.
Limit test:
no
Species:
mouse
Strain:
other: ICR- Swiss CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Weight at study initiation: female mice: 21 to 31 g, male mice: 28-31 g
- Housing: individually housed in steel hanging wire cages
- Diet: Wayne rodent diet, ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-26
- Humidity (%): 36-61
- Air changes (per hr): 9.1 and 9

IN-LIFE DATES: From: 1977-08-31 To: 1977-11-30
Type of coverage:
not specified
Vehicle:
not specified
Details on exposure:
TEST SITE
- Area of exposure: Dorsal area (2 X 3 cm²)
- Time intervals for shavings or clipplings: clipped

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
91 days
Frequency of treatment:
5 per week
Remarks:
Doses / Concentrations:
2.38 mg/day
Basis:
other: total amount applied/day
Remarks:
Doses / Concentrations:
6.91 mg/day
Basis:
other: total amount applied/day
No. of animals per sex per dose:
25
Control animals:
yes, sham-exposed
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked: general health, mortality, and gross skin irritation effects

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD EFFICIENCY: No

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: After termination of the study, 5 females from each group were submitted to the study sponsor for in vitro skin penetration studies. Group 3, 4, and 6 animals were submitted on Day 90, Group 5 and 7 animals were submitted on Day 91 and Group 2 and 4 animals were submitted on Day 92 after dose initiation.
Sacrifice and pathology:
GROSS PATHOLOGY: After 28 days (21 dermal applications) 10 male and 10 females from each group were sacrificed and necropsied. The remaining animals continued on the treatment until the termination of the study. At study termination 5 animals from each dose group were submitted to the sponsor for skin penetration studies the remaining animals were sacrificed and necropsied. Organs collected and examined: brain, pituitary, thyroid, thymus, large intestine, small intestine, heart, trachea, axillary lymph nodes, stomach, esophagus, uterus, skin from treated area and dorsal untreated area, mesenteric lymph nodes, lungs, liver, spleen, kidneys, adrenals, urinary bladder, ovary, testis, eyes, aorta, pancreas, and carcass.

HISTOPATHOLOGY: Yes - above organs examined histologically.
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
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
- 28 day interim gross skin observations.
2.38 mg/day dose group: No dermal effects with the exception of two animals which exhibited scaling and erythema in the dorsal area. These effects were not significantly different from control animals.
6.91 mg/day dose group: No dermal effects with the exception of two animals which exhibited scaling and erythema in the dorsal area. These effects were not significantly different from control animals.

-91 day gross skin observations.
Animals treated with 2.38 mg/day did not exhibit any gross compound related irritative effects after 91 days of treatment.
The skin of animals treated with 6.91 mg/day exhibited erythema and scaling in most animals compared to no significant effect at 28 days.

BODY WEIGHT AND WEIGHT GAIN
The weekly average body weights of the male and female mice in both dose groups were normal and comparable to the vehicle control group.

GROSS PATHOLOGY
- 28 day interim sacrifice: The organ and tissue lesions identified were few, equally distributed between the dose groups and were not of a consistent type. No effects were considered to be substance related.

- 91 day terminal sacrifice:
2.38 mg/day dose group: The dorsal skin was noted as vascular in two animals. However, the compound was noted as not caused any gross compound related irritative effects. No other treatment related organ or tissue related gross lesions were identified.
6.91 mg/day dose group: The dorsal skin was noted as vascular in two animals. No other treatment related organ or tissue related gross lesions were identified.

HISTOPATHOLOGY: NON-NEOPLASTIC
- 28 day interim sacrifice
Animals treated with 2.38 mg or 6.91 mg of the test substance exhibited comparable histological skin changes as controls at 28 days.

-91 day terminal sacrifice:
Microscopic evaluation revealed a comparable appearance of dermal effects in control mice and mice treated with 2.38 mg of the test substance. Mice treated with 6.91 mg test substance had minimal to slight acanthosis in 12/25 mice.
Dose descriptor:
NOEL
Remarks:
local
Effect level:
2.38 other: mg/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed.
Dose descriptor:
NOEL
Remarks:
local
Effect level:
68 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed.
Dose descriptor:
LOEL
Remarks:
local
Effect level:
6.91 other: mg/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Minimal to slight acanthosis at site of application at study termination.
Dose descriptor:
NOEL
Remarks:
systemic
Effect level:
>= 6.91 other: mg/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed.
Dose descriptor:
NOEL
Remarks:
systemic
Effect level:
>= 195 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed.
Critical effects observed:
not specified

Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effects were detected.

Conclusions:
The test substance did not produce dermal irritation after 91 days of treatment at a dose of 2.38 mg/day. Increasing the dose to 6.91 mg/day over 91 days did induce some irritation effects. No systemic effects were identified at either dose level.
Executive summary:

The objective of the study was to obtain scientific data to determine the histopathological effects of the skin at treatment sites after repeated dermal exposure to the test substance over 91 days with a 28 day interim sacrifice.

Fifty ICR-Swiss CD-1 mice (25M, 25F) per group were assigned to each of the following treatment groups. The dose volume for each group was 0.1mL with the control group being sterile water, a 2.38 mg/day test group, and a 6.91 mg/day test group.

An area of 2 x 3 cm of the dorsal area of all animals was clipped and treated with the appropriate dose five times per week.

All animals were observed daily for signs of general health, mortality and gross skin irritation effects. Gross signs of toxicity and body weights were recorded on a weekly basis throughout the study.

After 28 days (21 dermal applications) 10 males and 10 females from each group were sacrificed and necropsied. The remaining animals continued on the treatment regimen until the termination of the study. At study termination (90-92 days from initiation of the study), 5 females from each group were sent to the sponsor for in-vitro skin penetration studies. The remainder of the animals, were sacrificed and necropsied.

At the 28 and 91 day necropsies, the following tissues were examined and preserved in formalin: brain, pituitary, thyroid, thymus, small and large intestine, heart, trachea, axillary and mesenteric lymph nodes, stomach, esophagus, uterus, skin from treated and dorsal non-treated areas, lungs, liver, spleen, kidneys, adrenals, urinary bladder, ovary, testis, eyes, aorta, pancreas, and carcass. The skin tissues from treated animals and dermal non-treated areas were examined histopathologically.

No mortalities were attributed to treatment and there were no significant differences in body weights in any animals throughout the study. Gross necropsies at interim or terminal sacrifice did not reveal any compounds related lesions with the exception of skin effects at the site of treatment.

At the 28 day interim evaluation, repeated dermal applications of 2.38 mg/day and 6.91 mg/day of the test substance did not result in any gross skin effects with exception of two animals per group, which exhibited scaling and erythema or scales in the dorsal area which were not deemed to be of significance. Histopathologic examinations of skin from animals treated with 2.38 mg/day and 6.91 mg/day of the test substance exhibited comparable skin effects as controls.

Furthermore, animals treated with 2.38 mg/day of the test substance did not exhibit any gross or microscopic compound related irritative effects after 91 days of treatment. However, mice treated with 6.91 mg of the test substance showed minimal or slight acanthosis in 12 of the 25 mice.

Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effects were detected.

The test substance did not produce dermal irritation after 91 days of treatment at a dose of 2.38 mg/day. Increasing the dose to 6.91 mg/day over 91 days did induce some irritation effects. No systemic effects were identified at either dose level.

Endpoint:
sub-chronic toxicity: dermal
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Reason / purpose:
read-across source
Species:
mouse
Strain:
other: ICR- Swiss CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Weight at study initiation: female mice: 21 to 31 g, male mice: 28-31 g
- Housing: individually housed in steel hanging wire cages
- Diet: Wayne rodent diet, ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-26
- Humidity (%): 36-61
- Air changes (per hr): 9.1 and 9

IN-LIFE DATES: From: 1977-08-31 To: 1977-11-30
Type of coverage:
not specified
Vehicle:
not specified
Details on exposure:
TEST SITE
- Area of exposure: Dorsal area (2 X 3 cm²)
- Time intervals for shavings or clipplings: clipped

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
91 days
Frequency of treatment:
5 per week
Remarks:
Doses / Concentrations:
2.38 mg/day
Basis:
other: total amount applied/day
Remarks:
Doses / Concentrations:
6.91 mg/day
Basis:
other: total amount applied/day
No. of animals per sex per dose:
25
Control animals:
yes, sham-exposed
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked: general health, mortality, and gross skin irritation effects

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD EFFICIENCY: No

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: After termination of the study, 5 females from each group were submitted to the study sponsor for in vitro skin penetration studies. Group 3, 4, and 6 animals were submitted on Day 90, Group 5 and 7 animals were submitted on Day 91 and Group 2 and 4 animals were submitted on Day 92 after dose initiation.
Sacrifice and pathology:
GROSS PATHOLOGY: After 28 days (21 dermal applications) 10 male and 10 females from each group were sacrificed and necropsied. The remaining animals continued on the treatment until the termination of the study. At study termination 5 animals from each dose group were submitted to the sponsor for skin penetration studies the remaining animals were sacrificed and necropsied. Organs collected and examined: brain, pituitary, thyroid, thymus, large intestine, small intestine, heart, trachea, axillary lymph nodes, stomach, esophagus, uterus, skin from treated area and dorsal untreated area, mesenteric lymph nodes, lungs, liver, spleen, kidneys, adrenals, urinary bladder, ovary, testis, eyes, aorta, pancreas, and carcass.

HISTOPATHOLOGY: Yes - above organs examined histologically.
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
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
- 28 day interim gross skin observations.
2.38 mg/day dose group: No dermal effects with the exception of two animals which exhibited scaling and erythema in the dorsal area. These effects were not significantly different from control animals.
6.91 mg/day dose group: No dermal effects with the exception of two animals which exhibited scaling and erythema in the dorsal area. These effects were not significantly different from control animals.

-91 day gross skin observations.
Animals treated with 2.38 mg/day did not exhibit any gross compound related irritative effects after 91 days of treatment.
The skin of animals treated with 6.91 mg/day exhibited erythema and scaling in most animals compared to no significant effect at 28 days.

BODY WEIGHT AND WEIGHT GAIN
The weekly average body weights of the male and female mice in both dose groups were normal and comparable to the vehicle control group.

GROSS PATHOLOGY
- 28 day interim sacrifice: The organ and tissue lesions identified were few, equally distributed between the dose groups and were not of a consistent type. No effects were considered to be substance related.

- 91 day terminal sacrifice:
2.38 mg/day dose group: The dorsal skin was noted as vascular in two animals. However, the compound was noted as not caused any gross compound related irritative effects. No other treatment related organ or tissue related gross lesions were identified.
6.91 mg/day dose group: The dorsal skin was noted as vascular in two animals. No other treatment related organ or tissue related gross lesions were identified.

HISTOPATHOLOGY: NON-NEOPLASTIC
- 28 day interim sacrifice
Animals treated with 2.38 mg or 6.91 mg of the test substance exhibited comparable histological skin changes as controls at 28 days.

-91 day terminal sacrifice:
Microscopic evaluation revealed a comparable appearance of dermal effects in control mice and mice treated with 2.38 mg of the test substance. Mice treated with 6.91 mg test substance had minimal to slight acanthosis in 12/25 mice.
Dose descriptor:
NOEL
Remarks:
local
Effect level:
2.38 other: mg/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed.
Dose descriptor:
NOEL
Remarks:
local
Effect level:
68 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed.
Dose descriptor:
LOEL
Remarks:
local
Effect level:
6.91 other: mg/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Minimal to slight acanthosis at site of application at study termination.
Dose descriptor:
NOEL
Remarks:
systemic
Effect level:
>= 6.91 other: mg/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed.
Dose descriptor:
NOEL
Remarks:
systemic
Effect level:
>= 195 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed.
Critical effects observed:
not specified

Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effects were detected.

Conclusions:
The test substance did not produce dermal irritation after 91 days of treatment at a dose of 2.38 mg/day. Increasing the dose to 6.91 mg/day over 91 days did induce some irritation effects. No systemic effects were identified at either dose level.
Executive summary:

The objective of the study was to obtain scientific data to determine the histopathological effects of the skin at treatment sites after repeated dermal exposure to the test substance over 91 days with a 28 day interim sacrifice.

Fifty ICR-Swiss CD-1 mice (25M, 25F) per group were assigned to each of the following treatment groups. The dose volume for each group was 0.1mL with the control group being sterile water, a 2.38 mg/day test group, and a 6.91 mg/day test group.

An area of 2 x 3 cm of the dorsal area of all animals was clipped and treated with the appropriate dose five times per week.

All animals were observed daily for signs of general health, mortality and gross skin irritation effects. Gross signs of toxicity and body weights were recorded on a weekly basis throughout the study.

After 28 days (21 dermal applications) 10 males and 10 females from each group were sacrificed and necropsied. The remaining animals continued on the treatment regimen until the termination of the study. At study termination (90-92 days from initiation of the study), 5 females from each group were sent to the sponsor for in-vitro skin penetration studies. The remainder of the animals, were sacrificed and necropsied.

At the 28 and 91 day necropsies, the following tissues were examined and preserved in formalin: brain, pituitary, thyroid, thymus, small and large intestine, heart, trachea, axillary and mesenteric lymph nodes, stomach, esophagus, uterus, skin from treated and dorsal non-treated areas, lungs, liver, spleen, kidneys, adrenals, urinary bladder, ovary, testis, eyes, aorta, pancreas, and carcass. The skin tissues from treated animals and dermal non-treated areas were examined histopathologically.

No mortalities were attributed to treatment and there were no significant differences in body weights in any animals throughout the study. Gross necropsies at interim or terminal sacrifice did not reveal any compounds related lesions with the exception of skin effects at the site of treatment.

At the 28 day interim evaluation, repeated dermal applications of 2.38 mg/day and 6.91 mg/day of the test substance did not result in any gross skin effects with exception of two animals per group, which exhibited scaling and erythema or scales in the dorsal area which were not deemed to be of significance. Histopathologic examinations of skin from animals treated with 2.38 mg/day and 6.91 mg/day of the test substance exhibited comparable skin effects as controls.

Furthermore, animals treated with 2.38 mg/day of the test substance did not exhibit any gross or microscopic compound related irritative effects after 91 days of treatment. However, mice treated with 6.91 mg of the test substance showed minimal or slight acanthosis in 12 of the 25 mice.

Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effects were detected.

The test substance did not produce dermal irritation after 91 days of treatment at a dose of 2.38 mg/day. Increasing the dose to 6.91 mg/day over 91 days did induce some irritation effects. No systemic effects were identified at either dose level.

Endpoint:
sub-chronic toxicity: dermal
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Reason / purpose:
read-across source
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 2.2-2.8 kg
- Housing: housed individually in suspended galvanized and stainless steel cages each measuring 0.6 x 0.4 x 0.4 m³ high and fitted with mesh floors and automatic watering.
- Diet: ad libitum to a complete pelleted rabbit diet
- Water: ad libitum to tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18±2
Type of coverage:
open
Vehicle:
water
Details on exposure:
TEST MATERIAL
- Concentration (if solution): 3% aqueous solution of test substance (0.9% active AE3S)
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily, five days a week
Remarks:
Doses / Concentrations:
3% aqueous solution of test substance
Basis:
other: 0.9% active AE3S
No. of animals per sex per dose:
3
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: five days a week for 13 weeks

BODY WEIGHT: Yes
- Time schedule for examinations: 5 times a week for 13 weeks

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

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 data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At necropsy

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy
Sacrifice and pathology:
GROSS PATHOLOGY: Yes: lungs, kidneys, ovaries
HISTOPATHOLOGY: Yes: adrenals, bone marrow, brain, colon, duodenum, heart, jejunum, kidneys, liver, lungs, mesenteric lymph nodes, esophagus, ovaries, pancreas, parathyroid, pituitary, prostate, salivary gland, seminal vesicles, skin: treated and untreated, spleen, stomach, testes, thyroids, tongue, ureters, urethra, urinary bladder, uterus, vaginal wall.
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
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:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
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: Signs of reaction to the test material were apparent only at sites of application: areas of skin treated with the test substance developed a near-continuous low grade erythematous response over the first four weeks of study which persisted until terminal sacrifice without becoming more intense. There were no incidences of edema, scaling, fissuring or necrosis. The application site of control rabbits remained unaffected throughout.
Dose descriptor:
NOEL
Remarks:
systemic
Effect level:
3 other: % aqueous solution
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOEL
Remarks:
systemic
Effect level:
>= 60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Dose descriptor:
LOEL
Remarks:
local
Effect level:
3 other: % aqueous solution
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The only treatment-related response was a low grade erythematous reaction at the site of application.
Dose descriptor:
NOEL
Remarks:
systemic
Effect level:
>= 18 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Critical effects observed:
not specified

Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effected were detected.

Conclusions:
The test substance (30% AE3S) was evaluated on intact; non-occluded skin of New Zealand White rabbits. Under the conditions of this study, a repeated topical application of 3% aqueous suspension of the test substance (equal to 0.9% active AE3S) over 90 days provoked a low grade erythema at the site of treatment without inducing any systemic reaction.
Executive summary:

The objective of the study was to access the sub-chronic percutaneous toxicity of the test substance (30% active AE3S) in the New Zealand White rabbit, to identify any target organs or systems, the nature and time-course of responses and any delayed or irreversible effects resulting from dosages applied without occlusion to the intact skin of the back over 13 weeks.

The animals treated with the test substance developed a low grade erythematous reaction at the site of topical application which persisted at the same intensity for the last nine weeks of the study. Weight-gain, haematology, blood chemistry, liver and kidney weights, macropathology and micropathology displayed no evidence of a systemic reaction to treatment. It was concluded that repeated topical application of 3% aqueous test substance (0.9% active AE3S) provoked only a mild erythema at the site of application without inducing a systemic reaction.

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study was conducted in methods comparable to OECD guideline 411 "Subchronic Dermal Toxicity: 90-day Study". However, only one dose level was tested and there were 3 males and 3 females in each group, incomplete information provided on dosing methodology. Not GLP.
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
only one dose level tested, only 3 animals/sex/group, incomplete information available on application (volume, area of coverage , length of exposure, type of occlusion, washing).
GLP compliance:
no
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 2.2-2.8 kg
- Housing: housed individually in suspended galvanized and stainless steel cages each measuring 0.6 x 0.4 x 0.4 m³ high and fitted with mesh floors and automatic watering.
- Diet: ad libitum to a complete pelleted rabbit diet
- Water: ad libitum to tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18±2
Type of coverage:
open
Vehicle:
water
Details on exposure:
TEST MATERIAL
- Concentration (if solution): 3% aqueous solution of test substance (0.9% active AE3S)
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily, five days a week
Remarks:
Doses / Concentrations:
3% aqueous solution of test substance
Basis:
other: 0.9% active AE3S
No. of animals per sex per dose:
3
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: five days a week for 13 weeks

BODY WEIGHT: Yes
- Time schedule for examinations: 5 times a week for 13 weeks

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

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 data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At necropsy

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy
Sacrifice and pathology:
GROSS PATHOLOGY: Yes: lungs, kidneys, ovaries
HISTOPATHOLOGY: Yes: adrenals, bone marrow, brain, colon, duodenum, heart, jejunum, kidneys, liver, lungs, mesenteric lymph nodes, esophagus, ovaries, pancreas, parathyroid, pituitary, prostate, salivary gland, seminal vesicles, skin: treated and untreated, spleen, stomach, testes, thyroids, tongue, ureters, urethra, urinary bladder, uterus, vaginal wall.
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
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:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
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: Signs of reaction to the test material were apparent only at sites of application: areas of skin treated with the test substance developed a near-continuous low grade erythematous response over the first four weeks of study which persisted until terminal sacrifice without becoming more intense. There were no incidences of edema, scaling, fissuring or necrosis. The application site of control rabbits remained unaffected throughout.
Dose descriptor:
NOEL
Remarks:
systemic
Effect level:
3 other: % aqueous solution
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOEL
Remarks:
systemic
Effect level:
>= 60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Dose descriptor:
LOEL
Remarks:
local
Effect level:
3 other: % aqueous solution
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The only treatment-related response was a low grade erythematous reaction at the site of application.
Dose descriptor:
NOEL
Remarks:
systemic
Effect level:
>= 18 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Critical effects observed:
not specified

Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effected were detected.

Conclusions:
The test substance (30% AE3S) was evaluated on intact; non-occluded skin of New Zealand White rabbits. Under the conditions of this study, a repeated topical application of 3% aqueous suspension of the test substance (equal to 0.9% active AE3S) over 90 days provoked a low grade erythema at the site of treatment without inducing any systemic reaction.
Executive summary:

The objective of the study was to access the sub-chronic percutaneous toxicity of the test substance (30% active AE3S) in the New Zealand White rabbit, to identify any target organs or systems, the nature and time-course of responses and any delayed or irreversible effects resulting from dosages applied without occlusion to the intact skin of the back over 13 weeks.

The animals treated with the test substance developed a low grade erythematous reaction at the site of topical application which persisted at the same intensity for the last nine weeks of the study. Weight-gain, haematology, blood chemistry, liver and kidney weights, macropathology and micropathology displayed no evidence of a systemic reaction to treatment. It was concluded that repeated topical application of 3% aqueous test substance (0.9% active AE3S) provoked only a mild erythema at the site of application without inducing a systemic reaction.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
195 mg/kg bw/day
Study duration:
subchronic
Species:
mouse
Quality of whole database:
The dose levels of the dermal study were chosen to produce irritating effects. Therefore, with regard to the systemic toxicity of the AES the doses are low. In conclusion the NOAELs from the repeated oral toxicity studies were considered for the risk assessment.

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was conducted in methods comparable to OECD guideline 411 " Subchronic Dermal Toxicity: 90-day Study". 25 animals per sex per dose, only two dose levels evaluated. Not GLP.
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
25 animals per sex per dose, only two dose levels evaluated.
GLP compliance:
no
Remarks:
However, quality reviews of the study were performed and documented.
Limit test:
no
Species:
mouse
Strain:
other: ICR- Swiss CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Weight at study initiation: female mice: 21 to 31 g, male mice: 28-31 g
- Housing: individually housed in steel hanging wire cages
- Diet: Wayne rodent diet, ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-26
- Humidity (%): 36-61
- Air changes (per hr): 9.1 and 9

IN-LIFE DATES: From: 1977-08-31 To: 1977-11-30
Type of coverage:
not specified
Vehicle:
not specified
Details on exposure:
TEST SITE
- Area of exposure: Dorsal area (2 X 3 cm²)
- Time intervals for shavings or clipplings: clipped

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
91 days
Frequency of treatment:
5 per week
Remarks:
Doses / Concentrations:
2.38 mg/day
Basis:
other: total amount applied/day
Remarks:
Doses / Concentrations:
6.91 mg/day
Basis:
other: total amount applied/day
No. of animals per sex per dose:
25
Control animals:
yes, sham-exposed
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked: general health, mortality, and gross skin irritation effects

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD EFFICIENCY: No

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: After termination of the study, 5 females from each group were submitted to the study sponsor for in vitro skin penetration studies. Group 3, 4, and 6 animals were submitted on Day 90, Group 5 and 7 animals were submitted on Day 91 and Group 2 and 4 animals were submitted on Day 92 after dose initiation.
Sacrifice and pathology:
GROSS PATHOLOGY: After 28 days (21 dermal applications) 10 male and 10 females from each group were sacrificed and necropsied. The remaining animals continued on the treatment until the termination of the study. At study termination 5 animals from each dose group were submitted to the sponsor for skin penetration studies the remaining animals were sacrificed and necropsied. Organs collected and examined: brain, pituitary, thyroid, thymus, large intestine, small intestine, heart, trachea, axillary lymph nodes, stomach, esophagus, uterus, skin from treated area and dorsal untreated area, mesenteric lymph nodes, lungs, liver, spleen, kidneys, adrenals, urinary bladder, ovary, testis, eyes, aorta, pancreas, and carcass.

HISTOPATHOLOGY: Yes - above organs examined histologically.
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
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
- 28 day interim gross skin observations.
2.38 mg/day dose group: No dermal effects with the exception of two animals which exhibited scaling and erythema in the dorsal area. These effects were not significantly different from control animals.
6.91 mg/day dose group: No dermal effects with the exception of two animals which exhibited scaling and erythema in the dorsal area. These effects were not significantly different from control animals.

-91 day gross skin observations.
Animals treated with 2.38 mg/day did not exhibit any gross compound related irritative effects after 91 days of treatment.
The skin of animals treated with 6.91 mg/day exhibited erythema and scaling in most animals compared to no significant effect at 28 days.

BODY WEIGHT AND WEIGHT GAIN
The weekly average body weights of the male and female mice in both dose groups were normal and comparable to the vehicle control group.

GROSS PATHOLOGY
- 28 day interim sacrifice: The organ and tissue lesions identified were few, equally distributed between the dose groups and were not of a consistent type. No effects were considered to be substance related.

- 91 day terminal sacrifice:
2.38 mg/day dose group: The dorsal skin was noted as vascular in two animals. However, the compound was noted as not caused any gross compound related irritative effects. No other treatment related organ or tissue related gross lesions were identified.
6.91 mg/day dose group: The dorsal skin was noted as vascular in two animals. No other treatment related organ or tissue related gross lesions were identified.

HISTOPATHOLOGY: NON-NEOPLASTIC
- 28 day interim sacrifice
Animals treated with 2.38 mg or 6.91 mg of the test substance exhibited comparable histological skin changes as controls at 28 days.

-91 day terminal sacrifice:
Microscopic evaluation revealed a comparable appearance of dermal effects in control mice and mice treated with 2.38 mg of the test substance. Mice treated with 6.91 mg test substance had minimal to slight acanthosis in 12/25 mice.
Dose descriptor:
NOEL
Remarks:
local
Effect level:
2.38 other: mg/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed.
Dose descriptor:
NOEL
Remarks:
local
Effect level:
68 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed.
Dose descriptor:
LOEL
Remarks:
local
Effect level:
6.91 other: mg/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Minimal to slight acanthosis at site of application at study termination.
Dose descriptor:
NOEL
Remarks:
systemic
Effect level:
>= 6.91 other: mg/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed.
Dose descriptor:
NOEL
Remarks:
systemic
Effect level:
>= 195 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed.
Critical effects observed:
not specified

Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effects were detected.

Conclusions:
The test substance did not produce dermal irritation after 91 days of treatment at a dose of 2.38 mg/day. Increasing the dose to 6.91 mg/day over 91 days did induce some irritation effects. No systemic effects were identified at either dose level.
Executive summary:

The objective of the study was to obtain scientific data to determine the histopathological effects of the skin at treatment sites after repeated dermal exposure to the test substance over 91 days with a 28 day interim sacrifice.

Fifty ICR-Swiss CD-1 mice (25M, 25F) per group were assigned to each of the following treatment groups. The dose volume for each group was 0.1mL with the control group being sterile water, a 2.38 mg/day test group, and a 6.91 mg/day test group.

An area of 2 x 3 cm of the dorsal area of all animals was clipped and treated with the appropriate dose five times per week.

All animals were observed daily for signs of general health, mortality and gross skin irritation effects. Gross signs of toxicity and body weights were recorded on a weekly basis throughout the study.

After 28 days (21 dermal applications) 10 males and 10 females from each group were sacrificed and necropsied. The remaining animals continued on the treatment regimen until the termination of the study. At study termination (90-92 days from initiation of the study), 5 females from each group were sent to the sponsor for in-vitro skin penetration studies. The remainder of the animals, were sacrificed and necropsied.

At the 28 and 91 day necropsies, the following tissues were examined and preserved in formalin: brain, pituitary, thyroid, thymus, small and large intestine, heart, trachea, axillary and mesenteric lymph nodes, stomach, esophagus, uterus, skin from treated and dorsal non-treated areas, lungs, liver, spleen, kidneys, adrenals, urinary bladder, ovary, testis, eyes, aorta, pancreas, and carcass. The skin tissues from treated animals and dermal non-treated areas were examined histopathologically.

No mortalities were attributed to treatment and there were no significant differences in body weights in any animals throughout the study. Gross necropsies at interim or terminal sacrifice did not reveal any compounds related lesions with the exception of skin effects at the site of treatment.

At the 28 day interim evaluation, repeated dermal applications of 2.38 mg/day and 6.91 mg/day of the test substance did not result in any gross skin effects with exception of two animals per group, which exhibited scaling and erythema or scales in the dorsal area which were not deemed to be of significance. Histopathologic examinations of skin from animals treated with 2.38 mg/day and 6.91 mg/day of the test substance exhibited comparable skin effects as controls.

Furthermore, animals treated with 2.38 mg/day of the test substance did not exhibit any gross or microscopic compound related irritative effects after 91 days of treatment. However, mice treated with 6.91 mg of the test substance showed minimal or slight acanthosis in 12 of the 25 mice.

Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effects were detected.

The test substance did not produce dermal irritation after 91 days of treatment at a dose of 2.38 mg/day. Increasing the dose to 6.91 mg/day over 91 days did induce some irritation effects. No systemic effects were identified at either dose level.

Endpoint:
sub-chronic toxicity: dermal
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Reason / purpose:
read-across source
Species:
mouse
Strain:
other: ICR- Swiss CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Weight at study initiation: female mice: 21 to 31 g, male mice: 28-31 g
- Housing: individually housed in steel hanging wire cages
- Diet: Wayne rodent diet, ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-26
- Humidity (%): 36-61
- Air changes (per hr): 9.1 and 9

IN-LIFE DATES: From: 1977-08-31 To: 1977-11-30
Type of coverage:
not specified
Vehicle:
not specified
Details on exposure:
TEST SITE
- Area of exposure: Dorsal area (2 X 3 cm²)
- Time intervals for shavings or clipplings: clipped

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
91 days
Frequency of treatment:
5 per week
Remarks:
Doses / Concentrations:
2.38 mg/day
Basis:
other: total amount applied/day
Remarks:
Doses / Concentrations:
6.91 mg/day
Basis:
other: total amount applied/day
No. of animals per sex per dose:
25
Control animals:
yes, sham-exposed
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked: general health, mortality, and gross skin irritation effects

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD EFFICIENCY: No

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: After termination of the study, 5 females from each group were submitted to the study sponsor for in vitro skin penetration studies. Group 3, 4, and 6 animals were submitted on Day 90, Group 5 and 7 animals were submitted on Day 91 and Group 2 and 4 animals were submitted on Day 92 after dose initiation.
Sacrifice and pathology:
GROSS PATHOLOGY: After 28 days (21 dermal applications) 10 male and 10 females from each group were sacrificed and necropsied. The remaining animals continued on the treatment until the termination of the study. At study termination 5 animals from each dose group were submitted to the sponsor for skin penetration studies the remaining animals were sacrificed and necropsied. Organs collected and examined: brain, pituitary, thyroid, thymus, large intestine, small intestine, heart, trachea, axillary lymph nodes, stomach, esophagus, uterus, skin from treated area and dorsal untreated area, mesenteric lymph nodes, lungs, liver, spleen, kidneys, adrenals, urinary bladder, ovary, testis, eyes, aorta, pancreas, and carcass.

HISTOPATHOLOGY: Yes - above organs examined histologically.
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
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
- 28 day interim gross skin observations.
2.38 mg/day dose group: No dermal effects with the exception of two animals which exhibited scaling and erythema in the dorsal area. These effects were not significantly different from control animals.
6.91 mg/day dose group: No dermal effects with the exception of two animals which exhibited scaling and erythema in the dorsal area. These effects were not significantly different from control animals.

-91 day gross skin observations.
Animals treated with 2.38 mg/day did not exhibit any gross compound related irritative effects after 91 days of treatment.
The skin of animals treated with 6.91 mg/day exhibited erythema and scaling in most animals compared to no significant effect at 28 days.

BODY WEIGHT AND WEIGHT GAIN
The weekly average body weights of the male and female mice in both dose groups were normal and comparable to the vehicle control group.

GROSS PATHOLOGY
- 28 day interim sacrifice: The organ and tissue lesions identified were few, equally distributed between the dose groups and were not of a consistent type. No effects were considered to be substance related.

- 91 day terminal sacrifice:
2.38 mg/day dose group: The dorsal skin was noted as vascular in two animals. However, the compound was noted as not caused any gross compound related irritative effects. No other treatment related organ or tissue related gross lesions were identified.
6.91 mg/day dose group: The dorsal skin was noted as vascular in two animals. No other treatment related organ or tissue related gross lesions were identified.

HISTOPATHOLOGY: NON-NEOPLASTIC
- 28 day interim sacrifice
Animals treated with 2.38 mg or 6.91 mg of the test substance exhibited comparable histological skin changes as controls at 28 days.

-91 day terminal sacrifice:
Microscopic evaluation revealed a comparable appearance of dermal effects in control mice and mice treated with 2.38 mg of the test substance. Mice treated with 6.91 mg test substance had minimal to slight acanthosis in 12/25 mice.
Dose descriptor:
NOEL
Remarks:
local
Effect level:
2.38 other: mg/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed.
Dose descriptor:
NOEL
Remarks:
local
Effect level:
68 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed.
Dose descriptor:
LOEL
Remarks:
local
Effect level:
6.91 other: mg/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Minimal to slight acanthosis at site of application at study termination.
Dose descriptor:
NOEL
Remarks:
systemic
Effect level:
>= 6.91 other: mg/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed.
Dose descriptor:
NOEL
Remarks:
systemic
Effect level:
>= 195 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed.
Critical effects observed:
not specified

Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effects were detected.

Conclusions:
The test substance did not produce dermal irritation after 91 days of treatment at a dose of 2.38 mg/day. Increasing the dose to 6.91 mg/day over 91 days did induce some irritation effects. No systemic effects were identified at either dose level.
Executive summary:

The objective of the study was to obtain scientific data to determine the histopathological effects of the skin at treatment sites after repeated dermal exposure to the test substance over 91 days with a 28 day interim sacrifice.

Fifty ICR-Swiss CD-1 mice (25M, 25F) per group were assigned to each of the following treatment groups. The dose volume for each group was 0.1mL with the control group being sterile water, a 2.38 mg/day test group, and a 6.91 mg/day test group.

An area of 2 x 3 cm of the dorsal area of all animals was clipped and treated with the appropriate dose five times per week.

All animals were observed daily for signs of general health, mortality and gross skin irritation effects. Gross signs of toxicity and body weights were recorded on a weekly basis throughout the study.

After 28 days (21 dermal applications) 10 males and 10 females from each group were sacrificed and necropsied. The remaining animals continued on the treatment regimen until the termination of the study. At study termination (90-92 days from initiation of the study), 5 females from each group were sent to the sponsor for in-vitro skin penetration studies. The remainder of the animals, were sacrificed and necropsied.

At the 28 and 91 day necropsies, the following tissues were examined and preserved in formalin: brain, pituitary, thyroid, thymus, small and large intestine, heart, trachea, axillary and mesenteric lymph nodes, stomach, esophagus, uterus, skin from treated and dorsal non-treated areas, lungs, liver, spleen, kidneys, adrenals, urinary bladder, ovary, testis, eyes, aorta, pancreas, and carcass. The skin tissues from treated animals and dermal non-treated areas were examined histopathologically.

No mortalities were attributed to treatment and there were no significant differences in body weights in any animals throughout the study. Gross necropsies at interim or terminal sacrifice did not reveal any compounds related lesions with the exception of skin effects at the site of treatment.

At the 28 day interim evaluation, repeated dermal applications of 2.38 mg/day and 6.91 mg/day of the test substance did not result in any gross skin effects with exception of two animals per group, which exhibited scaling and erythema or scales in the dorsal area which were not deemed to be of significance. Histopathologic examinations of skin from animals treated with 2.38 mg/day and 6.91 mg/day of the test substance exhibited comparable skin effects as controls.

Furthermore, animals treated with 2.38 mg/day of the test substance did not exhibit any gross or microscopic compound related irritative effects after 91 days of treatment. However, mice treated with 6.91 mg of the test substance showed minimal or slight acanthosis in 12 of the 25 mice.

Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effects were detected.

The test substance did not produce dermal irritation after 91 days of treatment at a dose of 2.38 mg/day. Increasing the dose to 6.91 mg/day over 91 days did induce some irritation effects. No systemic effects were identified at either dose level.

Endpoint:
sub-chronic toxicity: dermal
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Reason / purpose:
read-across source
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 2.2-2.8 kg
- Housing: housed individually in suspended galvanized and stainless steel cages each measuring 0.6 x 0.4 x 0.4 m³ high and fitted with mesh floors and automatic watering.
- Diet: ad libitum to a complete pelleted rabbit diet
- Water: ad libitum to tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18±2
Type of coverage:
open
Vehicle:
water
Details on exposure:
TEST MATERIAL
- Concentration (if solution): 3% aqueous solution of test substance (0.9% active AE3S)
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily, five days a week
Remarks:
Doses / Concentrations:
3% aqueous solution of test substance
Basis:
other: 0.9% active AE3S
No. of animals per sex per dose:
3
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: five days a week for 13 weeks

BODY WEIGHT: Yes
- Time schedule for examinations: 5 times a week for 13 weeks

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

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 data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At necropsy

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy
Sacrifice and pathology:
GROSS PATHOLOGY: Yes: lungs, kidneys, ovaries
HISTOPATHOLOGY: Yes: adrenals, bone marrow, brain, colon, duodenum, heart, jejunum, kidneys, liver, lungs, mesenteric lymph nodes, esophagus, ovaries, pancreas, parathyroid, pituitary, prostate, salivary gland, seminal vesicles, skin: treated and untreated, spleen, stomach, testes, thyroids, tongue, ureters, urethra, urinary bladder, uterus, vaginal wall.
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
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:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
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: Signs of reaction to the test material were apparent only at sites of application: areas of skin treated with the test substance developed a near-continuous low grade erythematous response over the first four weeks of study which persisted until terminal sacrifice without becoming more intense. There were no incidences of edema, scaling, fissuring or necrosis. The application site of control rabbits remained unaffected throughout.
Dose descriptor:
NOEL
Remarks:
systemic
Effect level:
3 other: % aqueous solution
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOEL
Remarks:
systemic
Effect level:
>= 60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Dose descriptor:
LOEL
Remarks:
local
Effect level:
3 other: % aqueous solution
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The only treatment-related response was a low grade erythematous reaction at the site of application.
Dose descriptor:
NOEL
Remarks:
systemic
Effect level:
>= 18 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Critical effects observed:
not specified

Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effected were detected.

Conclusions:
The test substance (30% AE3S) was evaluated on intact; non-occluded skin of New Zealand White rabbits. Under the conditions of this study, a repeated topical application of 3% aqueous suspension of the test substance (equal to 0.9% active AE3S) over 90 days provoked a low grade erythema at the site of treatment without inducing any systemic reaction.
Executive summary:

The objective of the study was to access the sub-chronic percutaneous toxicity of the test substance (30% active AE3S) in the New Zealand White rabbit, to identify any target organs or systems, the nature and time-course of responses and any delayed or irreversible effects resulting from dosages applied without occlusion to the intact skin of the back over 13 weeks.

The animals treated with the test substance developed a low grade erythematous reaction at the site of topical application which persisted at the same intensity for the last nine weeks of the study. Weight-gain, haematology, blood chemistry, liver and kidney weights, macropathology and micropathology displayed no evidence of a systemic reaction to treatment. It was concluded that repeated topical application of 3% aqueous test substance (0.9% active AE3S) provoked only a mild erythema at the site of application without inducing a systemic reaction.

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study was conducted in methods comparable to OECD guideline 411 "Subchronic Dermal Toxicity: 90-day Study". However, only one dose level was tested and there were 3 males and 3 females in each group, incomplete information provided on dosing methodology. Not GLP.
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
only one dose level tested, only 3 animals/sex/group, incomplete information available on application (volume, area of coverage , length of exposure, type of occlusion, washing).
GLP compliance:
no
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 2.2-2.8 kg
- Housing: housed individually in suspended galvanized and stainless steel cages each measuring 0.6 x 0.4 x 0.4 m³ high and fitted with mesh floors and automatic watering.
- Diet: ad libitum to a complete pelleted rabbit diet
- Water: ad libitum to tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18±2
Type of coverage:
open
Vehicle:
water
Details on exposure:
TEST MATERIAL
- Concentration (if solution): 3% aqueous solution of test substance (0.9% active AE3S)
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily, five days a week
Remarks:
Doses / Concentrations:
3% aqueous solution of test substance
Basis:
other: 0.9% active AE3S
No. of animals per sex per dose:
3
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: five days a week for 13 weeks

BODY WEIGHT: Yes
- Time schedule for examinations: 5 times a week for 13 weeks

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

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 data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At necropsy

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy
Sacrifice and pathology:
GROSS PATHOLOGY: Yes: lungs, kidneys, ovaries
HISTOPATHOLOGY: Yes: adrenals, bone marrow, brain, colon, duodenum, heart, jejunum, kidneys, liver, lungs, mesenteric lymph nodes, esophagus, ovaries, pancreas, parathyroid, pituitary, prostate, salivary gland, seminal vesicles, skin: treated and untreated, spleen, stomach, testes, thyroids, tongue, ureters, urethra, urinary bladder, uterus, vaginal wall.
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
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:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
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: Signs of reaction to the test material were apparent only at sites of application: areas of skin treated with the test substance developed a near-continuous low grade erythematous response over the first four weeks of study which persisted until terminal sacrifice without becoming more intense. There were no incidences of edema, scaling, fissuring or necrosis. The application site of control rabbits remained unaffected throughout.
Dose descriptor:
NOEL
Remarks:
systemic
Effect level:
3 other: % aqueous solution
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOEL
Remarks:
systemic
Effect level:
>= 60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Dose descriptor:
LOEL
Remarks:
local
Effect level:
3 other: % aqueous solution
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The only treatment-related response was a low grade erythematous reaction at the site of application.
Dose descriptor:
NOEL
Remarks:
systemic
Effect level:
>= 18 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Critical effects observed:
not specified

Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effected were detected.

Conclusions:
The test substance (30% AE3S) was evaluated on intact; non-occluded skin of New Zealand White rabbits. Under the conditions of this study, a repeated topical application of 3% aqueous suspension of the test substance (equal to 0.9% active AE3S) over 90 days provoked a low grade erythema at the site of treatment without inducing any systemic reaction.
Executive summary:

The objective of the study was to access the sub-chronic percutaneous toxicity of the test substance (30% active AE3S) in the New Zealand White rabbit, to identify any target organs or systems, the nature and time-course of responses and any delayed or irreversible effects resulting from dosages applied without occlusion to the intact skin of the back over 13 weeks.

The animals treated with the test substance developed a low grade erythematous reaction at the site of topical application which persisted at the same intensity for the last nine weeks of the study. Weight-gain, haematology, blood chemistry, liver and kidney weights, macropathology and micropathology displayed no evidence of a systemic reaction to treatment. It was concluded that repeated topical application of 3% aqueous test substance (0.9% active AE3S) provoked only a mild erythema at the site of application without inducing a systemic reaction.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
397 µg/cm²
Study duration:
subchronic
Species:
rat
Quality of whole database:
The whole data base is conclusive and of high quality.

Additional information

Data on repeated dose toxicity are available for AES (C12-14; 2EO) Na (CAS 68891-38-3), AES (C10-16; 3 EO) Na (CAS 68585-34-2), and lauryl(3EO)ethoxysulphate. These data are also used to cover this endpoint for the other AES within the AES-category, i.e. AES (C9-11) Na (CAS 160901-27-9), AES (C8-10) NH4 (CAS 68891-29-2), AES (C12-14) NH4 (CAS 125301-88-4), AES (C12-14) MIPA (EC 932-185-7), AES (C12-14) Mg (CAS 160104-51-8), AES (C10-16) Mg (CAS 67762-21-4), AES (C8-10) Na, AES (C9-11) Na (CAS 160901-28-0), AES (C10-12) Na (CAS 68610-66-2), AES (C12-13) Na (CAS161074-79-9), AES (C12-14) Na (CAS 68891-38-3), AES (C12-18) Na (CAS 68081-91-4).

The AES reported within the category show similar structural, physico-chemical, environmental and toxicological properties. The approach of grouping different AES for the evaluation of their effects on human health and the environment was also made by the Danish EPA (2001) and HERA (2003), supporting the read-across approach between structurally related AES.

In a subchronic oral gavage study according to OECD Guideline 408, AES (C12-14) Na (CAS 68891-38-3, no data on grade of ethoxylation) was tested for systemic toxicity at doses of 0, 25, 75, 225 mg/kg bw/day on Wistar rats (BASF, 1994a). No clinical signs of toxicity, no mortalities and no effects on any other investigated parameter (body weight, body weight gain, food consumption, water consumption, ophthalmoscopic examinations, haematology, clinical chemistry, organ weights, gross pathology and histopathology) were seen at the highest dose level. Apart from the missing systemic toxicity, local treatment-related concentration-dependent irritation to different degrees in the forestomach was seen in all test groups. Thus, a NOEL-value was not determined. Since there is no human equivalent to the rat forestomach, these effects are not considered to be relevant to human health assessment. Thus, a no adverse effect level (NOAEL) of greater than 225 mg/kg could be established.

In another subchronic feeding study four groups of 20 rats per sex were fed 0, 0.05, 0.5 and 5.0% (w/w) of the AES (C10-16; 3 EO) Na (CAS 68585-34-2) over a period of 91 days (P&G, 1977a). The test material contained 30% active ingredient, converting the doses to 0, 10, 100 and 1100 mg/kg bw/day active substance. There were no observable differences in the appearance, mood, locomotion, and fecal consistency of treated and control animals and no animals died during the treatment period. No statistically significant changes in food consumption occurred during the study. The body weight gains of both sexes in the 5.0% group were reduced from the commencement of treatment, so that their body weights were significantly different from the controls when analyzed at Week 2, at termination their body weights were 84% (males) and 85% (females) of those of the respective controls. The body weight gains of both sexes in the 0.5% group were lower from Week 5 (males) or 6 (females), although this reduction was statistically significant in the males only. The food conversion ratio of males in the 5.0% group was consistently reduced, indicating inferior food utilization; however, it is not specified if this finding was statistically significant. Females in the 5.0% group displayed erythrocytic characteristics slightly lower than in the controls, with statistically significant effects on packed cell volume and erythrocyte count. Serum glutamate pyruvate transaminase and alkaline phosphatase levels were significantly elevated in both sexes in the 5.0% group. The liver weights of both sexes in the 5.0% group were significantly elevated. The heart weights of both sexes in the 5.0% group were significantly reduced when analyzed in absolute terms and relative to brain weight. No treatment-related changes were seen in the macroscopic appearance of the tissues examined at necropsy. No histopathological changes or variations that were considered treatment-related were observed in any of the tissues and organs examined. Several effects were seen in the lungs, liver and kidneys; however, they were not statistically significant and were considered not to be of toxicological significance. There were no treatment-related effects on the organ weights (both absolute and relative), or macroscopic or microscopic pathology of the gonadal tissues examined. Since the decrease in the erythrocyte count was not dose-dependent, it was concluded that the test substance at dietary levels up to 0.05% test material (corresponding to 100 mg/kg bw/day) elicited no adverse effects.

A third subchronic study was designed to evaluate the toxicol ogical effects of AES (C10-16; 3 EO) Na (CAS 68585-34-2) in albino rats (20/sex/group) when administered in the diet for thirteen weeks at levels of 0.0, 0.1, 0.5, and 1.0% active ingredient (corresponding to 0, 60, 300 and 600 mg/kg bw/day) (P&G, 1977b). Interim sacrifices were performed on five rats/sex/group at four weeks and ten rats/sex/group at thirteen weeks. Five rats/sex/group were maintained on study for one additional week and sacrificed at Week 14. The criteria evaluated for compound effects were clinical signs, mortality, body weight and food consumption, ophthalmoscopic findings, clinical chemistry and haematology, organ weights, and gross and microscopic pathology. No distinct effect attributed to the administration of the test substance were noted in comparisons of the clinical signs, mortality rates, ophthalmoscopic findings or haematology and blood chemistry values of the test groups to the controls. In addition, no compound related gross or histomorphologic organ or tissue alterations were noted in any of the treated animals. When compared to the data of the control group, slightly lower body weight gains were noted in the mid- and high-dose male groups. These differences were not statistically significant. The absolute and relative liver weights of all treated groups of both sexes were slightly, to moderately higher than respective control data at Weeks 4, 13 and 14. These differences were generally (but not always distinctly) dose-related and statistically significant at some of the intervals. The heart weights and ratios of all of the male treated groups were slightly (not statistically significantly) lower than those of the controls at Weeks 4, 13 and 14. However, neither gross pathology, nor microscopic examination of liver and heart sections confirmed the presence of any treatment-related hepatic or cardiac alterations. Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effects were detected. No outstanding treatment related toxic effects were noted when the test substance was administrated up to 600 mg/kg bw/day in the diet to albino rats. Although there were statistically significant differences in absolute and relative liver and heart weights noted in test substance-treated groups compared to control animals, and these differences were generally dose-related, no histopathological correlation was found to confirm the presence of any treatment-related alterations. As such the NOEL was determined to be 600 mg/kg bw/day.

Synthetic AES (C12-15; 3 EO) Na and natural AES (C12; 3 EO) Na (sodium lauryl(3EO)ethoxysulphate) were tested in a 90-day rat diet study at dose levels of 0, 40, 200, 1000 and 5000 ppm active material, corresponding to 0, 2, 10, 50 and 250 mg/kg bw/day (Walker, 1967). Health, behaviour, body weight, food intake, haematological and urinary parameters remained within normal limits at all doses. In both studies organ weight and blood chemistry effects observed were unaccompanied by any pathological changes. Based on the available information and taking into account that the study was conducted prior to the development of GLP and OECD guidelines, a NOAEL could be established at the dose level of greater than 250 mg/kg bw/day.

 

Further information on subchronic toxicity can be deduced from a two-generation reproduction study with AES (C12-14, no data on grade of ethoxylation) Na (CAS 68891-38-3) (BASF, 1999). Sprague Dawley rats were dosed via the drinking water at the concentrations 0, 0.03, 0.1 and 0.3%, which corresponded to daily doses of ca. 0, 30, 100 and 300 mg/kg bw. There were some changes indicative of parental toxicity in the group treated with 0.3% of the test substance. Slight but significantly reduced straight line velocity (VSL) of the sperm was without any significant effects on averaged path velocity (VAP) or total motility. Moreover, in the available subchronic and chronic toxicity studies on various AES the primary sex organs of the males and females did not show evidence for treatment-related adverse effects. The observed reduced triglyceride levels (female) and increased percentage neutrophil counts (males) were slight and within the range of the historical control data. The male F0 generation showed a small but significant reduction in body weight-liver weight ratios, but the corresponding brain related liver weights and the absolute liver weights developed not in a dose dependant way. For the F1 generation where similar results were reported, no dose-response relationship was detected either. No influence on liver weight development was seen in the F2 generation. None of the groups revealed any histopathological or clinical-chemical findings, which could be attributed to hepatotoxicity. This led to the conclusion that this untypical liver weight reduction was of no toxicological relevance, additionally underlined by the absence of such effects in the studies for subchronic toxicity mentioned above. There was evidence of toxicity on pup development at this dose level that was characterised by an increase in the time taken for sexual development of the male (not significant) and female (significant) offspring. This was investigated in more detail in the developmental toxicity studies up to 1500 mg/kg bw and no effects were noted there. Considering all these facts the subchronic NOAEL for systemic toxicity can be set to greater than 300 mg/kg bw.

No unusual findings regarding systemic toxicity were noted in a 2-year chronic feeding study in rats in which AES (C12; 3EO) (lauryl(3EO)ethoxysulphate) was given at 0, 0.1 or 0.5% (corresponding to 0, 50, 250 mg/kg bw/day) in the diet (Little, 1991). The results of this study suggest that the NOEL for AES (C12; 3EO) in this 2-year chronic feeding study in rats was greater than 250 mg/kg bw. In another 2-year study (Little, 1991), rats were administered AES (C12; 3 EO) (lauryl(3EO)ethoxysulphate) in the drinking water at a concentration of 0.1% (equals a dose of 75 mg/kg bw/day). The only unusual finding was slight, but consistently higher water consumption by all rats receiving the test compound in their drinking water and a significant difference in the empty caecum to body weight ratio of females. Absolute organ weights were all comparable to controls and no consistent gross or histopathology was found. A NOEL greater than 75 mg/kg bw can be estimated on the basis of the available information.

 

There are also two repeated dose toxicity studies with dermal application available.

The objective of the first study (P&G, 1978b) was to determine the histopathological effects on the skin at treatment sites after repeated dermal exposure to AES (C10-16; 3 EO) Na (CAS 68585-34-2) over 91 days with a 28 day interim sacrifice. 25 ICR-Swiss CD-1 mice per sex and group were assigned to each of the following treatment groups. The dose volume for each group was 0.1 mL with the control group being sterile water, a 2.38 mg/day test group, and a 6.91 mg/day test group. An area of 2 x 3 cm of the dorsal area of all animals was clipped and treated with the appropriate dose five times per week. All animals were observed daily for signs of general health, mortality and gross skin irritation effects. Gross signs of toxicity and body weights were recorded on a weekly basis throughout the study. After 28 days (21 dermal applications) ten animals per sex from each group were sacrificed and necropsied. The remaining animals continued on the treatment regimen until the termination of the study. At study termination (90-92 days from initiation of the study), five females from each group were sent to the sponsor for in-vitro skin penetration studies. The remainder of the animals were sacrificed and necropsied. No mortalities were attributed to treatment and there were no significant differences in body weights in any animals throughout the study. Gross necropsies at interim or terminal sacrifice did not reveal any compound related lesions with the exception of skin effects at the site of treatment. At the 28 day interim evaluation, repeated dermal applications in both test groups did not result in any gross skin effects with exception of two animals per group, which exhibited scaling and erythema or scales in the dorsal area which were not deemed to be of significance. Histopathological examinations of the skin exhibited comparable skin effects as controls. Furthermore, animals treated with 2.38 mg/day of the test substance did not exhibit any gross or microscopic compound related irritative effects after 91 days of treatment. However, mice treated with 6.91 mg/day of the test substance showed minimal or slight acanthosis in 12 of the 25 mice. Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effects were detected. The test substance did not produce dermal irritation after 91 days of treatment at a dose of 2.38 mg/day. When calculating the concentrations based on mg/cm2 the NOAEL for local effects was 0.4 mg/cm2. Increasing the dose to 6.91 mg/day (corresponds to 1.15 mg/cm2) over 91 days did induce some irritation effects. No systemic effects were identified at either dose level. Thus, the systemic NOEL was the highest dose of 195 mg/kg bw/d.

 

In the second subchronic percutaneous toxicity study (P&G, 1976) with AES (C10-16; 3 EO) Na (CAS 68585-34-2), three New Zealand White rabbits per sex were exposed to 0.9% active ingredient (corresponding to 18 mg/kg bw/day) to the intact non-occluded skin of the back over 13 weeks. The animals treated with the test substance developed a low grade erythematous reaction at the site of topical application which persisted at the same intensity for the last nine weeks of the study. Weight-gain, haematology, blood chemistry, liver and kidney weights, macropathology and micropathology displayed no evidence of a systemic reaction to treatment. It was concluded that repeated topical application provoked only a mild erythema at the site of application without inducing a systemic reaction.

 

Since the dose levels from the repeated dermal toxicity studies were very low and did not produce any systemic signs, only the NOAELs from the repeated oral toxicity studies were considered for the risk assessment.The respective NOAELs and LOAELs are listed in Table 1. 

 

Table 1: Dietary NOAELs and LOAELs (a.i.) for repeated oral toxicity studies of AES

Substance

Duration

(weeks)

NOAEL/NOEL

(mg/kg bw/day)

LOAEL

(mg/kg bw/day)

Reference

AES(C12-14)Na

13

225

> 225

BASF (1994a)

AES(C12-15;3EO)Na

AES(C12;3EO)Na

13

250

> 250

Walker (1967)

AES(C10-16;3EO)Na

13

100

1100

P&G (1977a)

AES(C10-16;3EO)Na

13

600

> 600

P&G (1977b)

AES(C12-14)Na

13

300

> 300

BASF (1999)

AES(C12;3EO)

104

250

> 250

Little (1991)

No LOAELs could be detected in the most studies, except for one study were a LOAEL was established at 1100 mg/kg bw/d (P&G, 1977a). However the dose tested exceeded the next lower dose within this study by 11 times. In addition the LOAEL is almost twice of the highest reported NOAEL. With exception of this study all NOAELs represented the highest dose level. Therefore an average of all NOAEL was chosen as basis for the risk assessment.

The available oral toxicity studies provide a coherent picture on the subchronic and chronic oral toxicity of AES. Based on the described effects, the NOAEL of 300 mg/kg bw/day (BASF, 1999) representing an average of all NOAELs, was chosen for the risk assessment.

 

References:

Danish EPA - Environmental and Health Assessment of Substances in Household Detergents and Cosmetic Detergent Products (2001). Environmental Project No. 615, pp. 24-28

HERA (2003). Human & Environmental Risk Assessment on ingredients of European household cleaning products Alcohol Ethoxysulphates, Human Health Risk Assessment Draft, 2003. http: //www. heraproject. com.


Justification for classification or non-classification

The available data on repeated dose toxicity do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.