Registration Dossier

Administrative data

Description of key information

The key study for the oral route is a 90-day repeated dose oral toxicity study in rats comparable to OECD TG 408 [Hazelton Laboratories (1974)]. The NOAEL was 0.1% (in the diet) or 1000 mg AO/kg diet. Using a food consumption factor of 0.088 kg food/kg bw/day for rats of this strain and age, this translates into a delivered dose of 88 mg AO/kg bw/day. This value represents the highest NOAEL below the lowest LOAEL and was selected for use in the risk assessment to characterize the risk of long term systemic toxicity via the oral and (by route to route extrapolation) dermal and inhalation routes.
With regard to dermal toxicity, repeated dermal treatment of rats (6 hours/day/5 days/week) for 90 days with the substance at dosage levels of 0.27 % AO and 1.33 % AO revealed local signs of irritation but no effects attributable to direct systemic toxicity. A NOAEL regarding systemic effects was therefore not established. Under the conditions of the study the LOEL for local dermal toxicity (irritation) in mice was determined to be 0.27 % AO.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1974-05-08 to 1974-10-30
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study was conducted in methods comparable to OECD guideline 408 " Repeated Dose 90-day Oral Toxicity Study in Rodents" with the following deviations: (1) hematology did not include an evaluate of platelet count or assess a measure of blood clotting; (2) An interim sacrifice of 5 animals/sex/dose was evaluated; however, the total number of animals was not increased accordingly as recommended in the guideline. Pre-GLP.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
See Rationale for Reliability above
Principles of method if other than guideline:
N/A
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Age at study initiation: 35 days old
- Weight at study initiation: Males (120-161 grams), Females (107-138 grams)
- Fasting period before study: N/A
- Housing: Individually in elevated wire mesh cages
- Diet (e.g. ad libitum): Purina Laboratory Rat Chow ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: N/A


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72-78 deg. Fahrenheit
- Humidity (%): 45-50 percent
- Air changes (per hr): N/A
- Photoperiod (hrs dark / hrs light): 14 hrs. dark / 10 hrs. light


IN-LIFE DATES: From: 1974-05-08 To: 1974-08-04
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Test substance UDL-652 was incorporated into basal laboratory diet on a weight-per-weight basis and thoroughly mixed in a twin-shell Patterson-Kelley blender to achieve the desired concentration ot test substance for each dosage level. Doses were 740.7, 3703.7, and 18518.5 ppm UDL-652 in diet. UDL-652 = 26.8% C10-C16 alkyldimethyl N-oxide (DDAO), so diets were 0.02%, 0.1%, and 0.5% DDAO or 200, 1000, and 5000 mg DDAO/kg diet.


DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Purina Laboratory Rat Chow
- Storage temperature of food: Stored in a dark refrigerated area


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:
not specified
Details on analytical verification of doses or concentrations:
Test substance was assumed to be 100% pure for purpose of dosage calculation.
Duration of treatment / exposure:
13-14 weeks
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 0.02, 0.1 and 0.5% AO in diet
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 17.6, 88, 440 mg AO/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
20
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: N/A
- Rationale for animal assignment (if not random): Stratified randomization by body weight was used for animal assignment to account for the difference in body weight, so that a homogenous distribution of weights was obtained between groups.
- Rationale for selecting satellite groups: N/A
- Post-exposure recovery period in satellite groups: N/A
- Section schedule rationale (if not random): N/A
Positive control:
N/A
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations included the following: animals were observed for mortality, morbidity, or other signs of systemic toxicity


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly


BODY WEIGHT: Yes
- Time schedule for examinations: Weekly


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, on a weekly basis
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


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


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): N/A
- Time schedule for examinations: N/A


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: On all animals at pre-treatment, on animals sacrificed at 4 weeks (interim sacrifices), and on all surviving animals at 13 weeks
- Dose groups that were examined: all dose groups were examined


HAEMATOLOGY: Yes
- Time schedule for collection of blood: At 4 and 13 weeks
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 5 rats/sex/dose group
- Parameters examined: hematocrit and hemoglobin determinations, erythrocyte count, total and differential leukocyte counts, mean corpuscular value, mean corpuscular hemoglobin concentration, and mean corpuscular hemoglobin determinations.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At 4 and 13 weeks
- Animals fasted: Yes
- How many animals: 5 rats/sex/dose group
- Parameters examined: fasting blood sugar, blood urea nitrogen, total protein, total albumin, serum sodium, serum potassium, serum chloride, serum calcium, serum glutamic-pyruvic transaminase, serum glutamic-oxaloacetic transaminase, alkaline phosphatase, and bilirubin.


URINALYSIS: Yes
- Time schedule for collection of urine: At 11 and 13 weeks
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters examined: urea nitrogen, pH, specific gravity, 24-hour volume, glucose, protein determinations, and microscopic examination of the sediment.


NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: N/A
- Dose groups that were examined: N/A
- Battery of functions tested: sensory activity / grip strength / motor activity / other: N/A


OTHER: N/A
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. After 4 weeks of treatment, 5 male and 5 female rats from each group (selected at random prior to initiation of treatment) were sacrificed and complete gross necropsies were performed. Following 13 weeks of treatment, an additional 10 male and 10 female animals were sacrificed and complete gross necropsies performed. The remaining 5 male and 5 female animals per group continued on treatment through 14 weeks, at which time they too were sacrificed and necropsied.
HISTOPATHOLOGY: Yes. Tissues examined for histopathology include: brain (with meninges), pituitary, thoracic spinal cord, thyroids, trachea, esophagus, salivary gland, eyes, tongue, lung, heart, liver, kidney, spleen, stomach, pancreas, lymph nodes, small intestine, large intestine, adrenal, urinary bladder, ureters, urethra, testes, ovaries, prostate, uterus, vagina, seminal vesicles, inguinal mammary gland, tibia, psoas muscle, skin, bone marrow, and any unusual lesions or tissue masses. Tissues examined microscopically include all preserved tissues from all control and high level animals sacrificed at 4 and 13 weeks. In addition, liver and kidney sections were examined from 5 male and 5 female animals of the low and intermediate dosage levels at 4 and 13 weeks.
Other examinations:
Organ weights were recorded for each animal for the following tissues: pituitary, heart, liver, adrenal, testes, ovaries, and kidney. Absolute and relative (to body weight) organ weights were determined.
Statistics:
Statistical evaluation was performed for the following criteria: growth rate, food consumption, terminal body weights, organ weights, and organ/body weight ratios. The statistical methods used included the following: analysis of variance, or F-test (5% probability level) and preliminary tests (where applicable) by methods of Rao, Bartlett, Scheffe, and Fisher-Behrens (t-test).
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:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, treatment-related
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
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: Survival was 100% in all groups. A slightly higher frequency of incidental clinical signs (i.e., hunched posture, rough fur coat, stains on fur coat, and soft feces) was observed in the high dose group when compared to the control and the low and intermediate dose groups.


BODY WEIGHT AND WEIGHT GAIN: Analysis of growth rate (body weight gain) data for the high dose group animals revealed a statistically significant suppression of this parameter when compared to the control group and the remaining test groups. The suppressed body weight gains coincided with decreased food consumption, noted as early as week 1, and persisted throughout the study.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Analysis of food consumption data for the high dose group animals revealed a statistically significant suppression of this parameter when compared to the control group and the remaining test groups. Animals in the high dose group displayed a reluctance to consume the diet mixture, due to its palatability, as early as week 1. This sign continued throughout the 14 weeks with growth rates, food consumption, and terminal body weights for these animals being significantly affected.


FOOD EFFICIENCY: There were occasional slight decreases in food efficiency noted among the high dose group animals. However, the finding of statistically decreased food consumption among the high dose group animals accounted for this occasional decrease in food efficiency.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): N/A


OPHTHALMOSCOPIC EXAMINATION: Examination of the 15 male and 15 female animals of the high dose group at week 13 revealed moderate to severe bilateral cataracts in four rats (two males and two females). The etiology of this finding was difficult to determine due to the marked growth retardation displayed by these animals; however, the possibility that ocular changes in these animals were effected by nutritional deficiencies was not substantiated by ophthalmoscopic findings normally associated with the onset of nutritional cataracts. Further, other studies in the testing laboratory wherein test substances produced severe reductions in food consumption and body weight gains for up to 52 weeks failed to reveal an effect of this nature. The relatively high incidence of these findings, when compared to the control group, as well as their appearance in animals of this age suggested a possible effect of administration of C10-C16 alkyldimethyl, N-oxide.


HAEMATOLOGY: Evaluation of the hematology data revealed no distinct test substance-related effects upon test animals at any dosage level.


CLINICAL CHEMISTRY: Blood chemistry data revealed no meaningful alterations in any of the parameters examined.


URINALYSIS: Results of urine analysis revealed no significant alterations in the parameters examined attributable to the treatment program.


NEUROBEHAVIOUR: N/A


ORGAN WEIGHTS: Statistically significant findings in the low and intermediate dose groups include: lower pituitary weights were observed in low dose males at week 4, elevated pituitary weights were observed in intermediate dose group males at week 4, and elevated pituitary/body weight ratios were observed in low dose females at week 13. These findings were considered not biologically relevant due to the lack of a dose-response relationship. Results of analysis of the high dose group animals revealed statistically significant alterations in a number of absolute and relative organ weights for these animals at all intervals. Terminal body weight values for these animals were also significantly lower than corresponding values for male and female control animals. Therefore, the relative lower mean weights for a number of organs probably were a reflection of the growth retardation experienced by animals in this group.


GROSS PATHOLOGY: Gross necropsy findings for the high dose group at 13 and 14 weeks were indicative of suppressed growth (described in the results for "body weight/weight gain") and ocular changes (described in the results for the ophthalmoscopic examination). In addition, animals in the high dose group had darker liver color due to decreased fatty tissue content.


HISTOPATHOLOGY: NON-NEOPLASTIC: There were no tissue alterations attributable to the test substance following 4 weeks of treatment. At the 13-week sacrifice, the only possible test substance-related histomorphologic alterations were observed in the high dose group animals, and consisted of bilateral lenticular cataracts in two males and two females.


HISTOPATHOLOGY: NEOPLASTIC (if applicable): N/A


HISTORICAL CONTROL DATA (if applicable): N/A


OTHER FINDINGS: N/A
Dose descriptor:
NOAEL
Effect level:
0.1 mg/kg diet
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Effect level:
88 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Critical effects observed:
not specified

The following gonadal tissues were examined for all control and high dose group animals at 4 and 13 weeks: testes, ovaries, prostate, uterus, vagina, seminal vesicles, inguinal mammary gland. No test substance-related microscopic or macroscopic changes were seen in any of these tissues at any dose level.

Additionally, organ weights were performed for each animal for the testes and ovaries; absolute and relative (to body) organ weights were determined. Absolute and relative organ weights for the high dose animals sacrificed at 4, 13, and 14 weeks were significantly altered when compared to control animals. These findings likely reflect the effect of suppressed body weight gain in the high dose group, as a result of the animals' reluctance to consume the diet mixture.

Conclusions:
In conclusion, dietary administration of C10-C16 alkyldimethyl, N-oxide at levels of 0.02, 0.1 and 0.5% in diet produced moderate suppression of food consumption among the high level animals (related to the palatability of the diet mixture), and the possibility of treatment-related cataractogenesis at the high dose level. The NOAEL was deemed to be 0.1% active amine oxide in the diet or 1000 mg/kg DDAO. Using a food consumption factor of 0.088kg food/kg BW/day for rats, this translates into a delivered dose of 88 mg/kg BW/day.
Executive summary:

A 13-week repeated dose toxicity study was conducted where C10-C16 alkyldimethyl, N-oxide was administered by oral administration in the diet daily to three groups of 20 male and 20 female Charles River CD Sprague-Dawley rats at dosage levels of 0.02, 0.1, and 0.5%. A fourth group of 20 male and 20 female rats received the basal diet only and served as the control. Five males and five females from each treatment group (including untreated control group) were sacrificed at 4 weeks, 10 males and 10 females were sacrificed after 13 weeks, and the remaining animals, after review of the previous necropsy findings, were sacrificed at the end of week 14.

The effect of C10-C16 alkyldimethyl, N-oxide on the rats was evaluated according to physical appearance, behavior, growth (weekly), food consumption (weekly), survival, clinical laboratory studies (blood and urine), microscopic eye examinations, organ weights, and gross and microscopic pathology. No adverse effects were observed in rats consuming diets containing 0.02 or 0.1% C10-C16 alkyldimethyl, N-oxide. Male and female rats receiving diets containing 0.5% test substance generally consumed less diet (grams of diet/kg body weight), and a depression in the average body weight gain was observable by the end of the first week. This sign continued throughout the 14 weeks with growth rates, food consumption, and terminal body weights for these animals being significantly affected. The alterations observed in the absolute and relative organ weights (including the testes and ovaries) in the 0.5% treatment group probably were secondary to body weight effects. Clinical laboratory findings for the high dose group animals generally remained within acceptable limits; however, slight electrolyte imbalance and slight elevation of alkaline phosphatase and blood urea nitrogen values were evident in some animals. Subsequent histopathology failed to confirm an indication of alterations in organ morphology and these findings may be related to the decrease in food consumption. Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effects were detected.

Significantly, 2/20 males and 2/20 females in the 0.5% treatment group developed moderate to severe bilateral cataracts. The incidence and early appearance of these cataracts in only the high dose group suggests that these ocular changes may be an effect of test substance treatment. This effect could be a direct effect of C10-C16 alkyldimethyl, N-oxide or an indirect effect caused by the effect of the test substance on food consumption and nutrient absorption or utilization. Based on these results, the NOAEL for the C10 -C16 alkyldimethyl, N-oxide was 0.1% (in the diet) or 1000 mg DDAO/kg diet. Using a food consumption factor of 0.088 kg food/kg bw/day for rats of this strain and age, this translates into a delivered dose of 88 mg AO/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
88 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
There are six reliable (Klimisch score = 2) studies performed using C12-14 AO. In addition, there is a reliable (Klimisch score =1) OECD 422 study for C12-14 AO.

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
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
N/A to: 1978-12-26
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.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
See rationale for reliability above.
Principles of method if other than guideline:
N/A
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
- Age at study initiation: N/A
- Weight at study initiation: female mice: 21 to 31 grams, male mice: 28-31 grams
- Fasting period before study: N/A
- Housing: individually housed in steel hanging wire cages
- Diet: Wayne rodent diet, ad libitum
- Water: ad libitum
- Acclimation period: N/A


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72 - 79
- Humidity (%): 36-61%
- Air changes (per hr): 9.1 and 9 changes/hr
- Photoperiod (hrs dark / hrs light): N/A


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 3cm)
- % coverage: N/A
- Type of wrap if used:N/A
- Time intervals for shavings or clipplings: Clipped prior to initial treatment and weekly

REMOVAL OF TEST SUBSTANCE
- Washing (if done): N/A
- Time after start of exposure: N/A


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 ml
- Concentration (if solution): N/A
- Constant volume or concentration used: N/A
- For solids, paste formed: N/A


VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Amount(s) applied (volume or weight with unit): 0.1 ml
- Concentration (if solution): 0.27% and 1.33%
- Lot/batch no. (if required): N/A
- Purity: N/A


USE OF RESTRAINERS FOR PREVENTING INGESTION: N/A
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
N/A
Duration of treatment / exposure:
91 days
Frequency of treatment:
5 treatments per week
Remarks:
Doses / Concentrations:
0.27 mg DDAO/application
Basis:
other: total amount applied per application
Remarks:
Doses / Concentrations:
1.33 mg DDAO/application
Basis:
other: total amount applied per application
No. of animals per sex per dose:
25/sex/dose
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: N/A
- Rationale for animal assignment: random
- Rationale for selecting satellite groups: N/A
- Post-exposure recovery period in satellite groups: N/A
- Section schedule rationale (if not random): N/A
Positive control:
N/A
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 data
- Time schedule: N/A

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

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

FOOD EFFICIENCY: No
- 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: N/A

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: N/A

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: N/A
- Dose groups that were examined: N/A

HAEMATOLOGY: No
- Time schedule for collection of blood: N/A
- Anaesthetic used for blood collection: N/A
- Animals fasted: N/A
- How many animals: N/A
- Parameters checked: N/A

CLINICAL CHEMISTRY: No
- Time schedule for collection of blood: N/A
- Animals fasted: N/A
- How many animals: N/A
- Parameters checked: N/A

URINALYSIS: No
- Time schedule for collection of urine: N/A
- Metabolism cages used for collection of urine: No data
- Animals fasted: N/A
- Parameters checked: N/A

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: N/A
- Dose groups that were examined: N/A
- Battery of functions tested: sensory activity / grip strength / motor activity / other: N/A

OTHER: After termination of the study, 5 females from each group were submitted to the study sponsor for in vitro skin penetration studies. Animals from the 1.33 mg/application group were submitted on day 90, and animals from the 0.27 mg/application group were submitted on day 92.
Sacrifice and pathology:
GROSS PATHOLOGY: After 28 days (20 dermal applications) 10 male and 10 females from each group were sacrificed and necropsied. Gross necropsies were performed.
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 - but only skin was examined histologically.
Other examinations:
N/A
Statistics:
N/A
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
All animals in both dose groups survived until the designated termination at 28 and 91 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 at 28 and 91 days.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): N/A

FOOD EFFICIENCY: N/A

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): N/A

OPHTHALMOSCOPIC EXAMINATION: N/A

HAEMATOLOGY: N/A

CLINICAL CHEMISTRY: N/A

URINALYSIS: N/A

NEUROBEHAVIOUR: N/A

ORGAN WEIGHTS: No data

GROSS PATHOLOGY and HISTOPATHOLOGY NON-NEOPLASTIC:
28 day interim sacrifice:
- 0.27% DDAO dose group:
- At necropsy, one male exhibited small red areas on lobes of the lungs and one female exhibited thickened uterus walls and stomach walls, other than the two animals no other gross pathological signs were observed. No effects were considered to be substance related.
- Minimal dermal irritation was present in six male and four female mice consisting of minimal or slight acanthosis.

- 1.33% DDAO dose group:
- Asmall gray area in the left lobe of one female, which was not considered to be substance related was noted.
- A more pronounced dermal irritation was present with more pronounced acanthosis and the presence of hyperkeratosis than the lower dose group sacrificed at 28 days.

91 day terminal sacrifice:
- 0.27% DDAO dose group:
- At necropsy, four males exhibited individual gross pathologies including: thickening of stomach wall; skin vascular; red areas in all lobes of lungs; and spleen slightly smaller than normal. Three females exhibited the following individual gross pathologies: brachial lymph nodes enlarged numerous small white foci on all lobes of liver, pale liver, and small gray cystic area on cortex of left kidney; left lobe of lung ill-regular in shape and had spongy areas, ovaries enlarged and firm; red cysts around right ovary, spleen slightly enlarged. These effects were considered to be substance related.
- Minimal dermal irritation was present in 11 male and the 10 female mice consisting of minimal or slightly acanthosis.

- 1.33% DDAO dose group:
- At necropsy, several males exhibited thickened dorsal skin. Another male exhibited discoloration of the liver, cystic liver mass and enlarged kidneys. One female exhibited an enlarged cervix and thickened walls in addition to an ovarian cyst. The systemic effects were not considered to be substance related. Only the skin effects at the site of treatment were considered to be test substance related.
- A more pronounced dermal irritation was present with minimal acanthosis in one mouse, slightly acanthosis in 11 mice and moderate acanthosis in 13 mice.

HISTOPATHOLOGY: NEOPLASTIC (if applicable): N/A

HISTORICAL CONTROL DATA (if applicable): N/A

OTHER FINDINGS: N/A
Dose descriptor:
LOEL
Remarks:
localized dermal effects
Effect level:
0.27 other: % DDAO
Sex:
male/female
Basis for effect level:
other: Slight to more pronounced dermal irritation was observed microscopically at both dose levels
Critical effects observed:
not specified

N/A

Conclusions:
Repeated dermal applications of the test substance at a dose of 0.27 mg DDAO/application for 28 and 91 days (5 times per week) resulted minimal to mild acanthosis with the effects being more pronounced with the repeated applications of 1.33 mg DDAO/application, resulting in both acanthosis and hyperkeratosis. No systemic effects were identified; however no histopathology was conducted (except on skin).
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. Treatment groups were 0.27% DDAO (0.27 mg per application) and 1.33% DDAO (1.33 mg per application). An area of 2 X 3 cm of the dorsal area of all animals was clipped and treated with 0.1ml of appropriate treatment 5 days 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 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 processed and 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 substance related lesions with the exception of skin effects at the site of treatment for the 0.27% group after 28 days applications of the test substance. Repeated applications of 1.33% over the 28 days resulted in slight to moderate erythema and scaling in most animals. The histological examinations of the 0.27% dose group after 28 days exhibited dermal irritation consisting of minimal to slight acanthosis. The dermal irritation effects were more pronounced in animals dosed with 1.33% consisting of acathosis and hyperkeratosis.   

The gross observations of skin effects of animals treated with 1.33% and sacrificed after 91 days were more severe than the irritation effects observed at 28 days. The gross observations of the skin effects of animals treated with 0.27% were erythema and scaling in most animals compared to no significant irritative effects at 28 days. When histological examinations were conducted, minimal dermal irritation was present in 11 male and the 10 female mice treated with 0.27% and consisted of minimal or slight acanthosis. In mice treated with 1.33%, a more pronounced dermal irritation was present with minimal acathosis in one mouse, slight acanthosis in 11 mice and moderate acanthosis in 13 mice.

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

Repeated dermal applications of 0.1ml of 0.27% DDAO (0.27 mg/application) for five days/week for 28 and 91 days resulted minimal to mild acanthosis. Local effects were more pronounced with the repeated dermal applications of 0.1ml of 1.33% DDAO (1.33 mg/application) for five days/week, resulting in both acanthosis and hyperkeratosis. No systemic effects, based on mortality, clinical signs and gross necropsy observations were identified at either dose level; however, no histopathology was performed except on skin. 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
Two reliable studies available

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
N/A to: 1978-12-26
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.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
See rationale for reliability above.
Principles of method if other than guideline:
N/A
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
- Age at study initiation: N/A
- Weight at study initiation: female mice: 21 to 31 grams, male mice: 28-31 grams
- Fasting period before study: N/A
- Housing: individually housed in steel hanging wire cages
- Diet: Wayne rodent diet, ad libitum
- Water: ad libitum
- Acclimation period: N/A


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72 - 79
- Humidity (%): 36-61%
- Air changes (per hr): 9.1 and 9 changes/hr
- Photoperiod (hrs dark / hrs light): N/A


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 3cm)
- % coverage: N/A
- Type of wrap if used:N/A
- Time intervals for shavings or clipplings: Clipped prior to initial treatment and weekly

REMOVAL OF TEST SUBSTANCE
- Washing (if done): N/A
- Time after start of exposure: N/A


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 ml
- Concentration (if solution): N/A
- Constant volume or concentration used: N/A
- For solids, paste formed: N/A


VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Amount(s) applied (volume or weight with unit): 0.1 ml
- Concentration (if solution): 0.27% and 1.33%
- Lot/batch no. (if required): N/A
- Purity: N/A


USE OF RESTRAINERS FOR PREVENTING INGESTION: N/A
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
N/A
Duration of treatment / exposure:
91 days
Frequency of treatment:
5 treatments per week
Remarks:
Doses / Concentrations:
0.27 mg DDAO/application
Basis:
other: total amount applied per application
Remarks:
Doses / Concentrations:
1.33 mg DDAO/application
Basis:
other: total amount applied per application
No. of animals per sex per dose:
25/sex/dose
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: N/A
- Rationale for animal assignment: random
- Rationale for selecting satellite groups: N/A
- Post-exposure recovery period in satellite groups: N/A
- Section schedule rationale (if not random): N/A
Positive control:
N/A
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 data
- Time schedule: N/A

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

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

FOOD EFFICIENCY: No
- 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: N/A

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: N/A

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: N/A
- Dose groups that were examined: N/A

HAEMATOLOGY: No
- Time schedule for collection of blood: N/A
- Anaesthetic used for blood collection: N/A
- Animals fasted: N/A
- How many animals: N/A
- Parameters checked: N/A

CLINICAL CHEMISTRY: No
- Time schedule for collection of blood: N/A
- Animals fasted: N/A
- How many animals: N/A
- Parameters checked: N/A

URINALYSIS: No
- Time schedule for collection of urine: N/A
- Metabolism cages used for collection of urine: No data
- Animals fasted: N/A
- Parameters checked: N/A

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: N/A
- Dose groups that were examined: N/A
- Battery of functions tested: sensory activity / grip strength / motor activity / other: N/A

OTHER: After termination of the study, 5 females from each group were submitted to the study sponsor for in vitro skin penetration studies. Animals from the 1.33 mg/application group were submitted on day 90, and animals from the 0.27 mg/application group were submitted on day 92.
Sacrifice and pathology:
GROSS PATHOLOGY: After 28 days (20 dermal applications) 10 male and 10 females from each group were sacrificed and necropsied. Gross necropsies were performed.
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 - but only skin was examined histologically.
Other examinations:
N/A
Statistics:
N/A
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
All animals in both dose groups survived until the designated termination at 28 and 91 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 at 28 and 91 days.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): N/A

FOOD EFFICIENCY: N/A

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): N/A

OPHTHALMOSCOPIC EXAMINATION: N/A

HAEMATOLOGY: N/A

CLINICAL CHEMISTRY: N/A

URINALYSIS: N/A

NEUROBEHAVIOUR: N/A

ORGAN WEIGHTS: No data

GROSS PATHOLOGY and HISTOPATHOLOGY NON-NEOPLASTIC:
28 day interim sacrifice:
- 0.27% DDAO dose group:
- At necropsy, one male exhibited small red areas on lobes of the lungs and one female exhibited thickened uterus walls and stomach walls, other than the two animals no other gross pathological signs were observed. No effects were considered to be substance related.
- Minimal dermal irritation was present in six male and four female mice consisting of minimal or slight acanthosis.

- 1.33% DDAO dose group:
- Asmall gray area in the left lobe of one female, which was not considered to be substance related was noted.
- A more pronounced dermal irritation was present with more pronounced acanthosis and the presence of hyperkeratosis than the lower dose group sacrificed at 28 days.

91 day terminal sacrifice:
- 0.27% DDAO dose group:
- At necropsy, four males exhibited individual gross pathologies including: thickening of stomach wall; skin vascular; red areas in all lobes of lungs; and spleen slightly smaller than normal. Three females exhibited the following individual gross pathologies: brachial lymph nodes enlarged numerous small white foci on all lobes of liver, pale liver, and small gray cystic area on cortex of left kidney; left lobe of lung ill-regular in shape and had spongy areas, ovaries enlarged and firm; red cysts around right ovary, spleen slightly enlarged. These effects were considered to be substance related.
- Minimal dermal irritation was present in 11 male and the 10 female mice consisting of minimal or slightly acanthosis.

- 1.33% DDAO dose group:
- At necropsy, several males exhibited thickened dorsal skin. Another male exhibited discoloration of the liver, cystic liver mass and enlarged kidneys. One female exhibited an enlarged cervix and thickened walls in addition to an ovarian cyst. The systemic effects were not considered to be substance related. Only the skin effects at the site of treatment were considered to be test substance related.
- A more pronounced dermal irritation was present with minimal acanthosis in one mouse, slightly acanthosis in 11 mice and moderate acanthosis in 13 mice.

HISTOPATHOLOGY: NEOPLASTIC (if applicable): N/A

HISTORICAL CONTROL DATA (if applicable): N/A

OTHER FINDINGS: N/A
Dose descriptor:
LOEL
Remarks:
localized dermal effects
Effect level:
0.27 other: % DDAO
Sex:
male/female
Basis for effect level:
other: Slight to more pronounced dermal irritation was observed microscopically at both dose levels
Critical effects observed:
not specified

N/A

Conclusions:
Repeated dermal applications of the test substance at a dose of 0.27 mg DDAO/application for 28 and 91 days (5 times per week) resulted minimal to mild acanthosis with the effects being more pronounced with the repeated applications of 1.33 mg DDAO/application, resulting in both acanthosis and hyperkeratosis. No systemic effects were identified; however no histopathology was conducted (except on skin).
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. Treatment groups were 0.27% DDAO (0.27 mg per application) and 1.33% DDAO (1.33 mg per application). An area of 2 X 3 cm of the dorsal area of all animals was clipped and treated with 0.1ml of appropriate treatment 5 days 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 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 processed and 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 substance related lesions with the exception of skin effects at the site of treatment for the 0.27% group after 28 days applications of the test substance. Repeated applications of 1.33% over the 28 days resulted in slight to moderate erythema and scaling in most animals. The histological examinations of the 0.27% dose group after 28 days exhibited dermal irritation consisting of minimal to slight acanthosis. The dermal irritation effects were more pronounced in animals dosed with 1.33% consisting of acathosis and hyperkeratosis.   

The gross observations of skin effects of animals treated with 1.33% and sacrificed after 91 days were more severe than the irritation effects observed at 28 days. The gross observations of the skin effects of animals treated with 0.27% were erythema and scaling in most animals compared to no significant irritative effects at 28 days. When histological examinations were conducted, minimal dermal irritation was present in 11 male and the 10 female mice treated with 0.27% and consisted of minimal or slight acanthosis. In mice treated with 1.33%, a more pronounced dermal irritation was present with minimal acathosis in one mouse, slight acanthosis in 11 mice and moderate acanthosis in 13 mice.

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

Repeated dermal applications of 0.1ml of 0.27% DDAO (0.27 mg/application) for five days/week for 28 and 91 days resulted minimal to mild acanthosis. Local effects were more pronounced with the repeated dermal applications of 0.1ml of 1.33% DDAO (1.33 mg/application) for five days/week, resulting in both acanthosis and hyperkeratosis. No systemic effects, based on mortality, clinical signs and gross necropsy observations were identified at either dose level; however, no histopathology was performed except on skin. 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
0.045 mg/cm²
Study duration:
subchronic
Species:
mouse

Additional information

Repeated dose toxicity oral:

In the key study [Hazelton Laboratories (1974)] using methods comparable to OECD guideline 408 “Repeated Dose 90-day Oral Toxicity Study in Rodents", rats (CD (SD)) received C12 -14 alkyl dimethylamine oxide (AO) via feed at levels of 0, 0.02, 0.1 and 0.5 % for 13 weeks. Twenty animals/sex/group were used. Five males and five females from each treatment group (including untreated control group) were sacrificed at 4 weeks, 10 males and 10 females were sacrificed after 13 weeks, and the remaining animals, after review of the previous necropsy findings, were sacrificed at the end of week 14. The effect of the substance on the rats was evaluated according to physical appearance, behaviour, growth (weekly), food consumption (weekly), survival, clinical laboratory studies (blood and urine), microscopic eye examinations, organ weights, and gross and microscopic pathology. No adverse effects were observed in rats consuming diets containing 0.02 or 0.1% of the substance. Male and female rats receiving diets containing 0.5% test substance generally consumed less diet (grams of diet/kg body weight), and a depression in the average body weight gain was observable by the end of the first week. This sign continued throughout the 14 weeks with growth rates, food consumption, and terminal body weights for these animals being significantly affected. The alterations observed in the absolute and relative organ weights (including the testes and ovaries) in the 0.5% treatment group probably were secondary to body weight effects. Clinical laboratory findings for the high dose group animals generally remained within acceptable limits; however, slight electrolyte imbalance and slight elevation of alkaline phosphatase and blood urea nitrogen values were evident in some animals. Subsequent histopathology failed to confirm an indication of alterations in organ morphology and these findings may be related to the decrease in food consumption. Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effects were detected. Significantly, 2/20 males and 2/20 females in the 0.5% treatment group developed moderate to severe bilateral cataracts. The incidence and early appearance of these cataracts in only the high dose group suggests that these ocular changes may be an effect of test substance treatment. This effect could be a direct effect of the substance or an indirect effect caused by the effect of the test substance on food consumption and nutrient absorption or utilization. Based on these results, the NOAEL for the test substance was 0.1% (in the diet) or 1000 mg AO/kg diet. Using a food consumption factor of 0.088 kg food/kg bw/day for rats of this strain and age, this translates into a delivered dose of 88 mg AO/kg bw/day.

In a supporting study [Hazelton Laboratories (1980)] using methods comparable to OECD guideline 408 " Repeated Dose 90-day Oral Toxicity Study in Rodents", rats (CD (SD)) received the test substance via feed at levels of 0, 0.1, 0.2 and 0.4% for 13 weeks. The objective of this study was to establish the dose levels of the test substance that would likely to be maximum tolerated levels throughout a two-year chronic feeding study in rats. Microscopic examination of the tissues for all control males and females, all mid dose females, and from 10 rats/sex from the high dose group did not reveal any compound-related alterations. Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effects were detected. Decreased body weights and lenticular lesions were observed at dose levels of 0.2% in the diet (LOAEL) and above when administered to the rat for 91 days. The NOAEL was determined to be 0.1% in the diet or 1000 mg AO/kg diet. Using a food consumption factor of 0.088 kg food/kg bw/day for rats of this strain/age, this translates into a delivered dose of 88 mg AO/kg bw/day.

In a supporting study [Batelle (1980)] which was designed to evaluate ocular toxicity of the test substance, Sprague-Dawley CD rats were administered C12 -14 alkyl dimethylamine oxide (AO) via feed at a level of 0.25% for 10 weeks. Rats were ophthalmoscopically examined prior to the study initiation and at study Weeks 6 and 10. Based on the results the LOEL was determined to be 0.25 % based on lenticular opacities in female animals and decreased body weights and body weight gains in both sexes.

In a supporting study [Procter & Gamble (1978)] using methods comparable to OECD guideline 407 "Repeated Dose 28-day Oral Toxicity Study in Rodents" rats (CD (SD)) received the test substance via feed at a level of 0.35% for 6 weeks. The cataractogenic activity of 0.35% commercial test substance in feed was demonstrated. No NOAEL was identified in this study.

In a supporting study [Gibson (1979)] which was designed to evaluate the cataractogenic potential of the test substance, Sprague-Dawley CD rats were administered C12 -14 alkyl dimethylamine oxide (AO) via feed. Dose levels of 0.12%, 0.35% and 0.5% of a commercial grade material and 0.35%, 0.4% and 0.5% of a laboratory prepared material of the test substance was administered in two phases, one for 16 weeks and the other for 30 weeks. No NOAEL/ LOAEL was identified since all doses affected body weight gain, food consumption, and feed efficiency.

In a supporting study [Hazelton Laboratories (1977)] using methods similar to OECD guideline 408 "Repeated Dose 90-day Oral Toxicity Study in Rodents", except using New Zealand white rabbits, the test substance C12 -14 alkyl dimethylamine oxide (AO) was administered via feed at levels of 0.1, 0.5 and 1.0 % initially for 13 weeks with the low and mid-dose groups exposed for an additional 19 weeks (total 32 weeks) to evaluate the potential for ocular lesions. Treatment related effects attributable to the administration of the high-dose (1% AO in diet) included increased mortality rates, decreased body weight and food consumption, increased incidences of clinical signs (listlessness, hyperpnea, wheezing and thinness), decreased hemogram (hematocrit, haemoglobin, and erythrocyte) values and increased bilirubin and decreased alkaline phosphatase values. Also, dose-related increases in total bilirubin values were noted at Week 13, and statistically significantly higher liver/body weight ratio were noted in the mid-dose males at Week 32. Gross and microscopic pathology examinations revealed findings attributable to the administration of the high-dose level of the test substance at 1% in diet consisting of gross findings of enlarged cervical and mesenteric lymph nodes, the presence of purulent material (including abscesses) in the lungs, and the absence of body fat and compound-related histomorphologic alterations in the lung, spleen, small intestine and mesenteric lymph node characterized by the presence of large foamy appearing macrophages in these tissues. In addition, marked vacuolation of the bronchial epithelium was present in the lung sections from the high-dosed animals. Based on treatment-related effects on mortality, body weight, hematology, clinical chemistry, and gross and histopathology relative to control at high dose and increased liver/BW ratio at mid dose, the NOAEL for the test substance was 0.1% in diet or 1000 mg AO/kg diet. Using a food consumption factor of 0.032 kg diet/kg bw/day for rabbits, this translates to a dose of 31 mg AO/kg bw/day.

In a supporting sub-acute study [Ceccatelli R (2008)] rats (HanRcc: WIST(SPF)) were dosed by oral gavage with the test substance (Lauramine oxide (Ammonyx LO)) at levels of 40, 100 and 250 mg/kg bw/day for at 28 days (males) and for 14 days prior to pairing, through the pairing and gestation period until the F1 generation reached Day 4 post-partum (females). The study was a combined repeated dose toxicity study with reproduction / developmental toxicity screening test. At 250 mg/kg/day, total locomotor activity was statistically significantly reduced in females. At 100 mg/kg/day, lesions in the forestomach were noted at necropsy and histopathology. Based on these results, the overall NOAEL general was established at 40 mg AO/kg/day. The NOEL for reproduction/developmental toxicity was considered to be 100 mg AO/kg/day

In a second supporting sub-acute study [Procter & Gamble (1979)] male rats (Charles River Sprague Dawley derived) were dosed by oral gavage with C12 -14 alkyl dimethylamine oxide (AO) for at 28 days. Each dose was 86 mg of commercial amine oxides (containing approx. 27% amine oxide) in 5 ml of water daily by stomach tube which was calculated to be equivalent to the dose received in a previous study which used dietary administration. When the results of the current study were compared with the results of a previous study in which amine oxides were fed in the diet, it was concluded that amine oxides given by stomach tube were not as toxic as the same amount of amine oxide fed in the diet.

 

Repeated dose toxicity dermal:

There are two repeated dose dermal toxicity studies. In the key study [Hazelton Laboratories (1978)] using methods comparable to OECD guideline 411 " Subchronic Dermal Toxicity Study: 90-day study", ICR Swiss CD-1 mice received daily (6 hours/day/5 days/week) dermal applications of the test substance at dosage levels of 0.27% (0.27 mg per application) or 1.33% (1.33 mg per application) C12 -14 alkyl dimethylamine oxide (AO) for 91 days. After 28 days (20 dermal applications) 10 male and 10 females from each group were sacrificed and necropsied. The remaining animals (15 males and 15 females) continued treatment until the end of the study. 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 substance related lesions with the exception of skin effects at the site of treatment for the 0.27% group after 28 days applications of the test substance. Repeated applications of 1.33% over the 28 days resulted in slight to moderate erythema and scaling in most animals. The histological examinations of the 0.27% dose group after 28 days exhibited dermal irritation consisting of minimal to slight acanthosis. The dermal irritation effects were more pronounced in animals dosed with 1.33% consisting of acanthosis and hyperkeratosis.  As such, a LOEL for localised dermal effects was established of 0.27% test substance for male/female mice.

The second study [Hazelton Laboratories (1976)] was a preliminary sub-acute study conducted to obtain toxicity and mortality data on two sources of the test substance C12 -14 alkyl dimethylamine oxide (AO) for use in setting dose levels in a subsequent chronic study. Initially groups of Charles River ICR Swiss mice received daily (6 hours/day/5 days/week) dermal applications of the test substance at dosage levels of 5, 10, 15 and 20%. However due to severe skin irritation observed at the higher dose levels dosing was terminated. The study was restarted using lower dose levels of 0.5, 1.0 and 2.0% test substance for 4 weeks and a LOEL of 2% and a NOAEL of 1% established. The 2% dose level induced minimal effects in the treatment area of the dorsal skin; however an increase in mortality and some dermal and systemic test substance related effects were seen in some mice at this dose level.

 

Repeated dose inhalation toxicity:

No data are available for the inhalation route. The substance is a solid with very low vapour pressure. It is manufactured and supplied as an aqueous solution therefore there is no possibility of exposure to dust. It is used in some spraying applications, but it is expected that inhalation exposure from these applications will be low and hence the dermal route is more relevant.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The study with the longest duration (90 days) and the highest NOAEL above the lowest LOAEL was chosen (key study).

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
No data are available for the inhalation route. The substance is a solid with very low vapour pressure. It is manufactured and supplied as an aqueous solution therefore there is no possibility of exposure to dust. It is used in some spraying applications, but, based on the risk assessment, it is expected that inhalation exposure from these applications will be low and hence the dermal route is more relevant.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
No data are available for the inhalation route. The substance is a solid with very low vapour pressure. It is manufactured and supplied as an aqueous solution therefore there is no possibility of exposure to dust. It is used in some spraying applications, but, based on the risk assessment, it is expected that inhalation exposure from these applications will be low and hence the dermal route is more relevant.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Study with longest duration

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Study was selected as sufficient information was available in the study to determine the amount dosed.

Justification for classification or non-classification

The NOAEL from the key 90 day repeated dose oral toxicity study is 88 mg AO/kg bw/day [Hazelton Laboratories (1974)]. Adverse effects were seen at the next level of 440 mg AO/kg bw/day. In a second 90 -day study [Hazelton Laboratories (1980)] an NOAEL of 88 mg AO/kg bw/day was also derived. Adverse effects were seen at the next dose level of 176 mg/kg bw/day. According to the Guidance on the Application of Regulation (EC) No 1272/2008 Table 3.9.3, classification in Category 2 is applicable when significant toxic effects observed in a 90 -day repeated dose study conducted in experimental animals are seen to occur within the guidance value range of 10><=100 mg/kg bw/day. The Guidance also states in Section 3.9.2.3.2 that in the case where the NOAEL is below the guidance value then the effective dose level (ED) should be determined in order to establish the need for classification. In the case where the ED is above the guidance value then interpolation between the ED and the NOAEL is required to determine whether the effects expected at or below the guidance value would warrant classification. In the case of this substance, The NOAEL is 88 mg/kg bw/day and the ED is 176 mg/kg bw/day. There is no evidence to suggest that the dose response curve is steep enough that effects would be seen below 100 mg/kg bw/day, hence classification is not warranted.