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Toxicological information

Carcinogenicity

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Description of key information

Two carcinogenicity studies are available; a two year oral feed study in rats and a 2 year dermal study in mice. In the oral study there were no substance related macroscopic changes observed and no neoplastic or non-neoplastic treatment related effects were identified. In the dermal study no treatment related carcinogenic response was noted either dermally or systemically.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1979-02-21 to 1983-04-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted in methods comparable to OECD guideline 451 "Carcinogenicity Studies". GLP. Environmental conditions deviated from those specified in guidelines however, this was not expected to negatively impact the study outcome.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 451 (Carcinogenicity Studies)
Deviations:
yes
Remarks:
environmental conditions deviated from those specified in the guidelines, however, this was not expected to negatively impact the study outcome.
Principles of method if other than guideline:
N/A
GLP compliance:
yes
Species:
rat
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. Michigan
- Age at study initiation: 4 weeks old
- Weight at study initiation: males (162-222g) and females (109-176g)
- Fasting period before study: N/A
- Housing: Individually in stainless steel hanging wire-mesh cages. The rats were transferred into clean cages and the cage racks were repositioned in the animal room once every two weeks.
- Diet: Zeigler NIH-07 powdered diet, ad libitum
- Water: ad libitum
- Acclimation period: 14days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): The usual temperature range was 71-75 degree F with occasional deviant values between 68-79 degree F.
- Humidity (%): The usual relative humidity was 46-70% with occasional deviant values as hight as 92% and as low as 21%.
- Air changes (per hr): The rate of air exchange ranged between 12.8 -13.3 exchanges/hour.
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle was maintained in the animal room.


IN-LIFE DATES: From: 1979-06-04 To: 1981-06 - 04
Route of administration:
oral: feed
Type of inhalation exposure (if applicable):
not specified
Vehicle:
other: Zeigler NIH-07 Rat and Mouse Ration
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was offered in the diet at concentrations of 0.01, 0.1, and 0.2% dodecyl dimethyl amine oxide. For each dietary preparation, the appropriate amount of test substance was added to 4.0 kg of Zeigler NIH-07 Rat and Mouse Ration and mixed in a Hobart Food mixer for 5 minutes. This premix was added to additional Zeigler NIH-07 Rat and Mouse Ration and mixed in a twin shell blender with an intensifier bar for 20 minutes. The intensifier bar was operated throughout the entire mixing period. Fresh batches of control and treated diet were prepared each week, placed in stainless steel containers and stored under refrigeration.


DIET PREPARATION
- Rate of preparation of diet (frequency): once a week
- Mixing appropriate amounts with (Type of food): The test substance was offered in the diet (4.0 kg of Zeigler NIH-07) at concentrations of 0.01, 0.1 and 0.2% dodecyl dimethyl amine oxide.
- Storage temperature of food: Refrigerated

VEHICLE
- Justification for use and choice of vehicle (if other than water): N/A
- Concentration in vehicle: 0.01, 0.1, and 0.2%
- 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:
Test Diet Analysis: Weekly throughout the study, representative 100 gram and 50 gram samples were collected from each control and prepared test diet. The 50 gram samples were shipped frozen to the Sponsor each week for test substance analysis. The 100 gram samples were retained under refrigeration at IRDC.

Test Article Analysis: Throughout the study, a 100 gram sample of the test substance was collected whenever a new container of test substance was opened for use. Additionally, a 100 gram sample was collected when approximately 150 gram of test substance was remaining in the previous container. These samples were shipped frozen to the Sponsor for test substance analysis.
Duration of treatment / exposure:
2 years
Frequency of treatment:
Continuous
Post exposure period:
N/A
Remarks:
Doses / Concentrations:
0.01, 0.1, and 0.2%
Basis:
nominal in diet
No. of animals per sex per dose:
Plain diet control group 1: 30 male/20 female ** to be used solely in the event of early termination resulting from low survival**.
Plain diet control group 2, diet with 0.01% test substance group, 0.1% test substance group, or 0.2% test substance group: 60 males/60 females per group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: N/A
- Rationale for animal assignment (if not random): N/A
- 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: twice daily, seven days a week, for signs of overt toxicity and for mortality.
- Cage side observations checked in table [No.?] were included. N/A

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule: :N/A

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded weekly for the first 14 weeks of study, biweekly for the next 12 weeks and monthly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- 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
- Other: Weekly mean values were calculated for the first 14 weeks of study and at study week 40. Weekly mean values were averaged at 2 week interval (weeks 15-28) and at monthly intervals thereafter. From the average food consumption and body weight values, mean compound consumption and food efficiency values were calculated.

FOOD EFFICIENCY: No data
- 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

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the pretest period and at 3, 6, 12, 16, 19, 22, and 24 months of study.
- Dose groups that were examined: all rats

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 52 and 104
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes, for 23 hours prior to collection at 52 weeks and approximately 16 hours prior to collection at 104 weeks of study.
- How many animals: all rats
- Parameters examined: hemoglobin, hematocrit, erythrocyte count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and total and differential leukocyte count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 52 and 104
- Animals fasted: Yes, for 23 hours prior to collection at 52 weeks and approximately 16 hours prior to collection at 104 weeks of study.
- How many animals: all rats
- Parameters examined: blood urea nitrogen, aspartate amino transferase, (serum glutamic oxaloacetic transaminase - SGOT), alanine aminotransferase (serum glutamic pyruvic transaminase - SGPT), total protein, albumin, and globulin.

URINALYSIS: Yes
- Time schedule for collection of urine: When urinalyses were required, freshly voided urine was collected every 30 minutes during a 6 hour fasting period until the quantity was sufficient to perform all the tests.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, for a 6 hour period
- Parameters examined: color, appearance, microscopic examination of sediment, specific gravity, volume, pH, protein, glucose, occult blood, nitrates, bilirubin, ketones, and urobilnogen.

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: N/A
- Dose groups that were examined: N/A
- Battery of functions tested: N/A

OTHER: N/A
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. At necropsy after a thorough external examination each animal was opened and the contents of the abdominal, thoracic and cranial cavities were examined both in situ and after removal. All macroscopic abnormalities were recorded. The following tissues were trimmed free of fat and connective tissues and weighed: brain, heart, liver, kidney, ovary, and testes.

Representative samples of the following tissues from all animals were collected and placed in neutral buffered formalin for fixation: urinary bladder, skin (dorsal, lumbar, midline) gross lesions, masses/tumors, regional lymph nodes, mandibular lymph nodes, mesenteric lymph nodes, mammary glands (females only), salivary gland (mandibular), stomach (entire), duodenum, jejunum, ileum, colon, cecum, rectum, thyroid/parathyroid, esophagus, seminal vesicle (2), heart, epididymis (2), coatochondral junction- rib, larynx, testis (2), lung (both and mainstem bronchi), pituitary, thigh muscle (right), sciatic nerve (right), ovary (2), uterus, brain (entire), liver (2 sections- one from each major lobe), pancreas, spleen, kidney (2)- cross section of right kidney and longitudinal of left kidney, adrenal (2) prostate(separate from bladder), femur (with marrow), thymus, spinal cord, trachea, eye (2), bone marrow smear, blood smear.

HISTOPATHOLOGY: Yes, Hematoxylin and eosin stained paraffin sections of the following tissues from control group II animals and high dose group animals were prepared and examined microscopically from the 0-12 month deaths and the 12 month interim sacrifice: pituitary, sciatic nerve, adrenal (2), thyroid (2), parathyroid (2), esophagus, trachea, larynx, prostate, uterus, testis (2), ovary (s), seminal vesicle (s), stomach, duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, epididymis (2), brain (3 sections), spinal cord (mid-section), eye (2), skin, muscle (thigh), mammary gland (females), mandibular lymph node, mesenteric lymph node, thymus, pancreas, salivary gland, liver (2), spleen, kidney (2), lungs (2), heart , rib junction, femur, gross lesions, regional lymph nodes of masses or tumors, blood and bone marrow smears.

Gross lesions were also examined from the low and mid dose groups and animals dying in extremis.

The following tissues were evaluated histopathologically for all animals dying between twelve months and termination and those from terminal sacrifice: testes, eyes, skin, mandibular lymph node, mesenteric lymph node, mammary gland (females only), salivary gland (mandibular), stomach (cardiac, fundic, pyloric), duodenum, jejunum, ileum, colon, cecum, rectum, thyroids, parathyroids, esophagus, seminal vesicle, heart, spinal cord, trachea, costochondral junction (rib), larynx, lungs (with mainstem bronchi), urinary bladder, pituitary, thigh muscle, sciatic nerve, ovaries, uterus, brain (including frontal cortex and basal ganglia, parietal cortex and thalamus, cerebellum and pons), liver, pancreas, spleen, kidneys, adrenals, prostate, femur Including marrow), thymus, and all gross lesions (including tissue masses/tumors and associated regional lymph nodes).
Other examinations:
N/A
Statistics:
All statistical analyses compared each treatment group with control group 2, by sex. Parameters analyzed are the following: body weight, food consumption, efficiency of food utilization, hematological and biochemical parameters, urinalysis parameters and absolute and relative organ weights were compared by analysis of variance (one-way classification), Bartlett’s test for homogeneity of variances and the least significant, and then pair wise comparisons were made using the Mann-Whitney test. All statistical tests were conducted at a 5%, two-sided risk level.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
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 SIGNS AND MORTALITY: N/A

BODY WEIGHT AND WEIGHT GAIN: Mean Body weights were reduced in a dosage related fashion for the treated males when compared with the control group 2 males. The reductions were statisitcally significant at every interval except one for the high dose males and at many of the intervals for the low and mid dose males. By week 104, body weights were only slightly reduced at the low and mid dose levels when compared with the control 2 group. This was likely due to the reduction in control body weights that began at approximately study week 91. In females, statistically significant reductions in mean body weight values were noted at all intervals exceept one at the high dose level. Mean body weights were either slightly increased or similar to the control at the low dose level while only slight decrease were seen at the mid dose level.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): The average food consumption values (weeks 1-104) for the treated groups (male and female) were similar or slightly decreased compared to the control group 2 animals. Several instances of statisitically significant decreases were noted in mean food consumption at the 0.01, 0.1 and 0.2% dose levels in males and at the 0.2% dose level in females, which tended to parallel the decreases in body weight.

FOOD EFFICIENCY: There were no significant changes in food efficiency between the groups.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): From the blind examination of the results, there was a skewed distribution of eye pathology as far as lens lesion were concerned.


OPHTHALMOSCOPIC EXAMINATION:
Two veterinary ophthalmologist reports were included:
1. The results of the individual eye examinations were considered in two steps. First, the individual results were grouped by treatment group and interval but the identity of the actual group was unknown to the ophthalmologist. After an initial opinion had been conveyed, the coding for the groups was broken and a second opinion, with the knowledge of the groups identities, was given.
From the blind examination, it was noted that there was a skewed distribution of eye pathology as far as lens lesions were concerned. While this could have represented a related effect, this was thought to be unlikely because of the following (a) inconsistency of lens pathology observed, (b) inconsistency of bilateral involvement, (c) lens pathology consistent with that observed in all rat studies carried out to 80 weeks, (d) lack of consistent disease progression, and (e) no significant alteration in the normal aging process. The first Ophthalmologist concluded that if the incidence of the lesions was indeed dose related it would represent an unfortunate distribution of a non-spectacular lens pathology. Examination of the noncoded groups did not alter the opinion that there was sufficient evidence to support a contetion of compound related pathology.

2. A second veterinary ophthalmologist performed eight ocular examinations with a hand held slit lamp and an indirect ophthalmoscope on animals that had been dosed for 0,3,5,12,15,18,21,and 24 months. The Ophthalmologist was unaware of the groups, the compound or any other information that could bias the findings. Although the biological variation was profound the ophthalmologist attempted to categorize his observations into groups. Due to the biologic and anatomic characteristics of the lens, aging was considered. No trends were identified that incriminate test substance related lenticular changes within the confines of this experimental design. As no diferences were discernable between the groups, the ophthalmologist concluded that the changes observed were within the realm of the natural aging or disease common to colones of rats.

HAEMATOLOGY: There were no treatment related changes in any hameatologic parameters. The occasional values which were statistically different from control values were not considered biologically significant as the differences were small and in some instances did not affect higher dose levels.


CLINICAL CHEMISTRY: There were a few mean values where the differences from controls reached statistical significant. In all cases, the differences were slight and in some instances did not affect higher dose levels. They were not considered to be biologically significant.


URINALYSIS: There were no notable features in the urinalysis results.


NEUROBEHAVIOUR: N/A


ORGAN WEIGHTS:
12 month interim sacrifice group:
There was a statistically significant increase (p<0.05) in the mean relative weight of the liver for females at 0.2% dosage level. There was no corresponding absolute or relative weight variation seen in other organs and hence this weight variation was considered to be a reflection of the lower mean body weights seen in this group.
Terminal sacrifice group:
No toxicologically significant changes were observed in mean absolute or relative (to body weight) liver, kidney, gonad, heart or brain weighs after 24 months ingestion of 0.01, 0.1, or 0.2% intake of the test substance. Statistically significant increases in the mean relative kidney, ovary and brain weights for females in the 0.2% dose group and in the mean relative brain weight for males in the 0.2% dose group were considered to be due to significantly lower mean body weights for females and males in these dose groups. A decrease in the mean absolute liver weight of males in the 0.1% dose group, although statistically significant, was considered within the range of normal biologic variability and not considered treatment related.

GROSS PATHOLOGY:
12 month interim sacrifice:
There were no gross morphologic changes observed during post mortem examination of animals dying on study, sacrificed in extremis, or at the 12 month interim sacrifice.
Terminal sacrifice group:
No toxicologically significant changes were observed in male or female rats which died or were sacrificed in extremis during the 12-24 month period, or which were sacrificed at the end of the 12-24 months ingestion of 0.01-0.2% of the test substance in the diet.
HISTOPATHOLOGY: NON-NEOPLASTIC and NEOPLASTIC:
12 month interim sacrifice:
There were no compound related microscopic findings observed in histologic examinations of rats dying on study, sacrificed in extremis, or at the 12 month interim sacrifice. The degenerative, inflammatory and neoplastic histomorphologic changes desribed occurred sporadically among male and female control and test animals. They were therefore considered to be spontaneous in nature and incidental to ingestion of the test substance.
Terminal sacrifice group:
No meaningful alterations were detected in the tissues of test animals which could be attributed to the administration of the test substance. A variety of neoplastic and non-neoplastic alterations were detected in sections of tissues from both control and test animals.

HISTORICAL CONTROL DATA (if applicable): N/A

OTHER FINDINGS: N/A
Relevance of carcinogenic effects / potential:
None
Dose descriptor:
NOEL
Effect level:
0.2 other: % alkyl dimethyl amine oxide in diet
Sex:
male/female
Basis for effect level:
other: No treatment related effects observed in this oral carcinogenicity study.
Remarks on result:
other: Effect type: carcinogenicity (migrated information)

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

Based on the fact that the food consumption paralleled the body weight decreases and that there were no effects seen on food efficiency or utilization, this suggests that the test substance had a negative effect on the palatability of the diet to the test animals.

The terminal histopathology evaluation was performed by Experimental Pathology Laboratories, Herndon VA

A statistical analyis of the neoplasms noted during the histopathological examinations was performed by the sponsor. All neoplastic lesions with an incidence of 4 or greater in the high dose group were analyzed separately for each sex against control group 2. Additionally, those animals that were sacrifice moribund or as scheduled, were statistically analyzed separately and as one group. No significant differences were found at the 5% risk level.

Conclusions:
The test substance was offered in the diet to Charles River CD rats at concentrations of 0.01, 0.1, and 0.2% dodecyl dimethyl akyl amine. No neoplastic or non-neoplastic treatment related effects were identified.
Executive summary:

The purpose of this study is to evaluate the oral carcinogenicity of alkyl dimethyl amine oxide in rats as this is a common exposure route for this test substance. The test substance was offered in the diet (Zeigler NIH-07) to Charles River CD rats at concentrations of 0.01, 0.1, and 0.2% dodecyl dimethyl amine oxide. Sixty (60) male and 60 female rats were initiated at each dosage level and in a control group. An additional 30 males and 30 females were initiated for a second control group to be used solely in the event of early termination resulting from low survival. The rats were observed twice daily for signs of overt toxicity and for mortality. Detailed observations were recorded weekly. Beginning at approximately 15 weeks of study, a random selection of rats were inspected for dacryoadenitis at weekly intervals.

Individual body weights were recorded weekly for the first 14 weeks of study, biweekly for the next 12 weeks and monthly thereafter. Individual food consumption measurements were recorded weekly. Ophthalmoscopic examinations were conducted for all rats once during the pretest period and at 3, 6, 12, 16, 19, 22 and 24 months of study.

Hematological, biochemical and urinalysis tests were conducted on all rats (predesignated at study initiation) that were sacrificed at 52 weeks of study (10 males and 10 females). Hematological and biochemical tests were conducted for all surviving rats at study termination.

No test substance-related trends in physical appearance and behavior, survival or food (with compound) consumption were established. In treated males, dosage-related reductions were noted in body weights, whereas in females, significant reductions were only seen at the high dose level.

Two veterinary ophthalmologists performed examinations in the study. The first ophthalmologist, Dr. Keller, indicated that there was insufficient evidence to support a contention that there was compound-related pathology. The second ophthalmologist, Dr. Wyman, did not consider any eye lesions to be treatment related.

The few hematological and biochemical values that reached statistically significant differences from the control values were not considered biologically meaningful. There were no notable features in the urinalysis results.

There were no compound-related macroscopic changes observed among animals in this study. Statistically significant increases in the mean relative kidney, ovary and brain weights for high dose females and in the mean relative brain weight for high dose males were observed but these were considered due to the significantly reduced mean body weights in this dosage group. Gonadal tissues were also examined for both gross pathology and histopathology and no treatment-related effects were detected.

Statistical analysis of neoplasm’s and survival times, conducted by the Sponsor, revealed no significant differences at the 5.0% risk level. There was no evidence of treatment related histopathological changes.

Microscopic examination was performed by Experimental Pathology Laboratories, Inc. Herndon Virginia for all animals that died on study during 12 -24 months and animals that were terminally sacrificed.

No neoplastic or non-neoplastic treatment related effects were identified.

The NOAEL for C10 -C16 alkyl dimethyl amine oxide was determined to be 0.2% in diet or 2000 mg AO/kg diet. Using a food consumption factor of 0.045kg diet/kg bw/day for rats, this translates into a dose of 90 mg AO/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
One study available (K=2) performed using a method similar to OECD TG 451

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1979-06-11 to 1982-08-13
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was conducted in methods comparable to OECD guideline 451 "Carcinogenicity study". Not GLP.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 451 (Carcinogenicity Studies)
Deviations:
no
Principles of method if other than guideline:
N/A
GLP compliance:
no
Species:
mouse
Strain:
other: ICR Swiss mice (CD-1)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories from Portage, Michigan mouse colony
- Age at study initiation: N/A
- Weight at study initiation: N/A
- Fasting period before study: N/A
- Housing: housed in individual stainless steel wire mesh cages supported above the droppings.
- Diet: NIH-07 rodent diet (Zeigler Brothers, Inc. Gardners, Pa.); ad libitum
- Water: ad libitium
- Acclimation period: N/A


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72-78 F
- Humidity (%): 40-65%
- Air changes (per hr): 10-20
- Photoperiod (hrs dark / hrs light): 12 hr dark/12 hr light


IN-LIFE DATES: From: 1979-06-11 To: N/A
Route of administration:
dermal
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: The application site was an area approximately 2 x 3 cm, centered on the dorsal midline skin.
- % coverage: N/A
- Type of wrap if used: Dermal doses were applied with an automatic pipette with a disposable application tip, discarded after treatments.
- Time intervals for shavings or clipplings: prior to the first dermal treament and once weekly thereafter

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: yes
- For solids, paste formed: no
- Other: A bottle of the stock solution of the test substance was used for four weekly preparations of the use dilution solutions. The 0.06% use dilution was prepared by mixing 0.46 ml of the test substance with distilled water to a total volume of 250 ml. The 0.13% use dilution was prepared by mixing 1.2 ml of the test substance stock solution with distilled water to a total volume of 250ml. The 0.25% use dilution was prepared by mixing 2.4 ml of test substance with distilled water to a total volume of 250 ml. Dosing solutions of the test material were prepared once weekly, usually on Tuesday. A volume of 0.1 ml was expelled on the dorsal midline skin approximately 3 cm posterior to the nape of the neck.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Through experimental day 186 deionized water was used to make the use dilutions and after that date distilled water was used.
- Amount(s) applied (volume or weight with unit): 0.1 ml
- Concentration (if solution): 0.05%, 0.13%, and 0.26% active in water vehicle
- Lot/batch no. (if required): N/A
- Purity: N/A


USE OF RESTRAINERS FOR PREVENTING INGESTION: N/A
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to initiation of the study, homeogeneity and stability studies were performed on use dilution preparations. The stability was done on samples after storage for 10 days in the refrigerator. These stability determinations were repeated on these use dilutions three months later. All values were within the specified acceptable limits.
Duration of treatment / exposure:
2 years
Frequency of treatment:
3 times/week (Monday, Wednesday, and Friday)
Post exposure period:
N/A
Remarks:
Doses / Concentrations:
0.05%, 0.13%, 0.26% dodecyl dimethyl amine oxide
Basis:
nominal conc.
No. of animals per sex per dose:
74 animals per sex/per dose
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale:
Group 1 (0.1 ml distilled water)
Group 2 (0.1 ml of 0.05% test substance)
Group 3 (0.1 ml of 0.13% test substance)
Group 4 (0.1 ml of 0.26% test substance)
Group 5 (0.1 ml of distilled water)
- Rationale for animal assignment (if not random): A computer program matched animals by sex and quarantine cage number with random numbers, and assigned the individual animals to the experimental and control groups. Using the computer printout of random animal assignments, 690 normal healthy mice, 345 males and 345 females, were assigned to control and expeirmental groups.

Mice in control group 5 were selected for terminal sacrifice as determined by survival rate of test Groups 2, 3, and 4, using a table of random numbers and randomization cards. Subgroups of 15 male and 15 female mice in Group 5 were designated by random selection to be sacrificed if the survival rate of an experimental group reached 20% prior to 104 weeks. The mice in group 5 were continugency controls designated to be sacrificed in the event a 20% surivival rate prevailed in a test group prior to the scheduled termination of the study. Their utilization for this purpose was not required since all experimental groups had a survival rate greater than 20% at 104 weeks.
- 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: Mortality checks of the animals were made twice daily-morning and afternoon.
- Cage side observations checked in table [No.?] were included.


DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule: N/A


DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: daily


BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded weekly during the first 13 weeks of the study and monthly thereafter.


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 / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / 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


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


OPHTHALMOSCOPIC EXAMINATION: No data
- Time schedule for examinations:
- Dose groups that were examined:


HAEMATOLOGY: No data
- 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 in table [No.?] were examined. N/A


CLINICAL CHEMISTRY: N/A
- Time schedule for collection of blood: N/A
- Animals fasted: N/A
- How many animals: N/A
- Parameters checked in table [No.?] were examined. N/A


URINALYSIS: N/A
- Time schedule for collection of urine: N/A
- Metabolism cages used for collection of urine: N/A
- Animals fasted: N/A
- Parameters checked in table [No.?] were examined. N/A


NEUROBEHAVIOURAL EXAMINATION: N/A
- 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: Surviving animals were sacrificed on experimental days 730-738.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; The mice were observed for gross signs at each treatment period and written observations were made at least once weekly. Necropies were performed on dead mice, those sacrificed in a moribund condition and at termination of the study. At necropsy the following tissues were fixed in 10% neutral buffered formalin: skin from treatment area, gross lesions-including tissue masses/tumors and associated regional lymph nodes, mandibular lymph node, ileo-cecocolic lymph nodes, mammary gland (females), salivary gland (mandibular gland), thyroids, and parathyroids, esophagus, seminal vesicles, testes, ovaries, uterus, brain, liver, stomach-to include cardiac, fundic and pyloric regions, small intestine-to include the duodenum, jejunum and ileum, colon, cecum, rectum, kidneys, adrenal, urinary bladder, prostate, femur including marrow, thymus, trachea, lungs (both including mainstem bronchi, spleen, pancreas, heart, pituitary, eyes, sciactic nerve, thigh muscle, costochondral junction, gallbladder, spinal cord, larynx, blood smear, carcass, tissues from male mice which exhibit urogenital lesions, urethra, ureter, pulbourethral glands with bulbocavernosus muscles, preputial glands
The following tissues from each sacrificed animal were trimmed to exclude fat or other extraneous tissue and weighed: Liver, brain, heart, kidneys, and gonads (in pairs, ovaries trimmed of fat; testes include epididymis, through experimetal day 386-thereafter epididymis excluded).
HISTOPATHOLOGY: Yes;
Histopathology was performed on the tissues listed under gross pathology from Group 1 and Group 4 males and selected tissues from Group 1 and 4 males which died or were sacrificed during the first year. After completion of the study, the above tissues from all Group 1 and Group 4 mice were sectioned and examined. In addition, all tumors and selected tissues from Groups 2 and 3 were also examined.
Other examinations:
Beginning in the sixth month of the study (December, 1979), monthly examinations of the male mice were initiated. The incidence of urine stains, distended bladder, irritated penis, and irritated scrotum were recorded. The size of the preputial gland was estimated following palpation.
Statistics:
Statistical evalaution of these data from animals killed at termination of the study was performed by one-way analysis of variance (ANOVA). Treated groups were compared against the controll by least significant difference test (LSD) provided there was homogeneity of variances by Bartlett's test. IN the case of heterogeneity of variances group comparisons were performed by (Cochran's modified t-test) and WIlcoxon's (Mann-Whitney's) non-parametric two samle rank sum test. Whenever permitted, the nonparametric test was preferred for conclusion regarding group comparisons in the event of heterogeneity of heterogeneity of variances. A linear regression on dose levels was perfomed which included F-tests for regression and lack of fit. Group comparisons were performed at two-tailed five percent level of significance.
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):
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:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
The survival rates were comparable among the control and experimental groups in the normal range for this strain. The average number of experimental days for the males varied from 587 to 627 and for the females, 542 to 644. The mice which survived the experimental period of 726 days received a maximum of 304 dermal treatments and a total volume of 30.4 ml of distilled water.

Gross observationsin th treatment groups included distended bladder, urine stains, unkempt fur, alopecia, hyperactivity, arched spine, distended abdomen, cyanosis swelling in the genital or axillary area, labored respiration, clipper cut and sensitivity to touch.

BODY WEIGHT AND WEIGHT GAIN
The average body weights were comparable among the control and experimental groups and in the normal range for this species and strain.

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
A control vs. treatment group comparisons showed that the mid-dose group mean for liver weight was significantly different from the control in the female mice, however there was no indication of a dose related compound effect.

GROSS PATHOLOGY
The most frequent gross observations in the mice were redness, swelling, irritation, and laceration of the right ear due to the presence of the metal ear identification tag. Scabs or healing lesions of the right ear were also observed frequently. The tagged ear was scratched excessively by the animal, with additional skin lesions being made in the adjacent neck and upper shoulder skin.

HISTOPATHOLOGY: NON-NEOPLASTIC and NEOPLASTIC
Microscopic evaluations of hematoxylin and eosin stained sections of tissues from male mice which died during the first year of study failed to reveal evidence of compound effects . A variety of spontaneous lesions and incidental findings were observed without relationship to treatment.

Microscopic evaluations at terminal necropsy revealed compound related irritation in treated skin sections from high dose mice of both sexes. The epidermal response was characterized by diffuse acanthosis with the following incidence: Control males: 1/64, Control females: 4/75, Group 4 (0.26%) males: 43/69, Group 4 (0.26%) females: 43/74. In most instances, the acanthosis and hyperkeratosis ranged from slight to moderately severe and the findings were essentially comparable between sexes.

The neoplasms observed were of the usual type and incidence observed in mice of this age and strain, with no indication of compound effect.

Spontaneous disease lesions and incidental findings were essentially comparable between control and treated groups. A wide variety of spontaneous disease lesions and incidental findings were noted in various tissues examined and occurred without relationship to treatment. The most commonly observed neoplastic lesions were hepatocellular neoplasms in male mice, pulmonary neoplasms in mice of both sexes, and malignant lymphomas of various cell types which occurred primarily in female mice.

Compound related histomorphologic alterations were observed in treated skin sections from male and female high does mice consisting of diffuse acanthosis and hyperkeratosis which varied from slight to moderately severe. Spontaneous disease lesions including neoplastic lesions were of the usual type and number observed in mice of this age and strain and were essentially comparable in incidence and severity between control and treated groups.

HISTORICAL CONTROL DATA (if applicable)
N/A

OTHER FINDINGS: The incidence of urine stains, distended bladder, irritated penis, irritated scrotum, and enlargement of preputial gland in control and experimental male mice were normal for this strain. Clinical signs of urogenital disease were observed in some male mice. None of the female mice were affected.
Dose descriptor:
NOEL
Effect level:
0.26 other: % alkyl dimethyl amine oxide.
Sex:
male/female
Basis for effect level:
other: No skin carcinogenicity observed in the study.
Remarks on result:
other: Effect type: carcinogenicity (migrated information)

Occasionally compound residue was observed on the treated skin of rats in group 2, 3, 4.

Conclusions:
The study did not result in any carcinogenic response on the exposed skin or systemically. Microscopic evaluation at the exposure site revealed compound-related dermal irritation in treated skin sections from the high-dose mice of both sexes. The epidermal response was characterized by acanthosis that increase in incidence and severity with dose.
Executive summary:

The objective of this study was to evaluate the percutaneous carcinogenicity of alkyl dimethyl amine oxide in mice. Dermal application of 0.1 ml of an aqueous solution of C10 -C16 alkyldimethyl, N-oxide at 0.05%, 0.13%, and 0.26% (active ingredient) to the dorsal skin of mice, 3 times/week, for 2 years did not result in any carcinogenic response for the skin or systemically.

Animals were evaluated for mortality, twice daily and gross observations were made at least weekly. Individual body weights were recorded weekl for the first thirteen weeks and monthly thereafter.

At necropsy the liver , brain, heart, kidneys, and gonads were weighed. The following tissues were preserved in 10% neutral buffered formalin: skin from treatment area, gross lesions-including tissue masses/tumors and associated regional lymph nodes, mandibular lymph node, ileo-cecocolic lymph nodes, mammary gland (females), salivary gland (mandibular gland), thyroids, and parathyroids, esophagus, seminal vesicles, testes, ovaries, uterus, brain, liver, stomach-to include cardiac, fundic and pyloric regions, small intestine-to include the duodenum, jejunum and ileum, colon, cecum, rectum, kidneys, adrenal, urinary bladder, prostate, femur including marrow, thymus, trachea, lungs (both including mainstem bronchi, spleen, pancreas, heart, pituitary, eyes, sciactic nerve, thigh muscle, costochondral junction, gallbladder, spinal cord, larynx, blood smear, carcass, tissues from male mice which exhibit urogenital lesions, urethra, ureter, pulbourethral glands with bulbocavernosus muscles, preputial glands. Histopathology was performed on these tissues from the control and high dose group and selected tissues from the mid and low dose groups which died or were sacrificed during the first year. At study competion the above tissues from all control and high dose mice were sectioned and examined. All tumors and selected tissues form the mid and low dose animals were also examined histologically.

A wide variety of spontaneous disease lesions (including neoplasms) were noted in the various tissues examined and were of the type and incidence commonly observed in mice of this age and strain. Compound-related histomorphologic changes were observed in treated skin sections from male and female high dose mice. The epidermal responses consisting of diffuse acanthosis and hyperkeratosis were the result of test substance related irritation. The severity ranged from slight to moderately severe.

The study did not result in any carcinogenic response on the exposed skin or systemically. Microscopic evaluation at the exposure site revealed compound-related dermal irritation in treated skin sections from the high-dose mice of both sexes. The epidermal response was characterized by acanthosis that increase in incidence and severity with dose. The NOEL for dermal carcinogenicity was determined to be the high dose of 0.26% dodecyl dimethyl amine oxide, which is equivalent to 2.6 mg/ml. 0.1ml of the 2.6 mg/ml dose solution was applied to mice three times per week (7 days), resulting in a total delivered dose of 0.111 mg/day. Using a default body weight for mice of 0.028 kg, this results in an applied dose of 3.98 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
One study available (K=2) performed using a method similar to OECD TG 451

Additional information

Two reliable animal studies are available. In the key study [International Research and Development Corporation (1983)] which was conducted in methods comparable to OECD guideline 451 "Carcinogenicity Studies" groups of 60 male and 60 female rats (Charles River CD) were offered diets containing the test substance at concentrations of 0.01, 0.1 and 0.2% for up to 2 years.Haematological, biochemical and urinalysis tests were conducted on all rats (pre-designated at study initiation) that were sacrificed at 52 weeks of study (10 males and 10 females). Haematological and biochemical tests were conducted for all surviving rats at study termination. No test substance-related trends in physical appearance and behaviour, survival or food (with compound) consumption were established. In treated males, dosage-related reductions were noted in body weights, whereas in females, significant reductions were only seen at the high dose level. The few haematological and biochemical values that reached statistically significant differences from the control values were not considered biologically meaningful. There were no notable features in the urinalysis results. There were no compound-related macroscopic changes observed among animals in this study. No neoplastic or non-neoplastic treatment related effects were identified. The NOAEL for the test substance was determined to be 0.2% in diet or 2000 mg AO/kg diet. Using a food consumption factor of 0.045kg diet/kg bw/day for rats, this translates into a dose of 90 mg AO/kg/day.

In the supporting study [Hazelton Laboratories Inc. (1982)] which was conducted in methods comparable to OECD guideline 451 "Carcinogenicity Studies" the dermal application of 0.1 ml of an aqueous solution of test substance at 0.05%, 0.13%, and 0.26% (active ingredient) was applied to the dorsal skin of mice (ICR Swiss mice, CD-1), 3 times/week, for 2 years. Histopathology was performed on an extensive list of tissues from the control and high dose group and selected tissues from the mid and low dose groups which died or were sacrificed during the first year. At study completion tissues from all control and high dose mice were sectioned and examined. All tumours and selected tissues form the mid and low dose animals were also examined histologically.A wide variety of spontaneous disease lesions (including neoplasms) were noted in the various tissues examined and were of the type and incidence commonly observed in mice of this age and strain. Compound-related histomorphologic changes were observed in treated skin sections from male and female high dose mice. The epidermal responses consisting of diffuse acanthosis and hyperkeratosis were the result of test substance related irritation. The severity ranged from slight to moderately severe.The study did not result in any carcinogenic response on the exposed skin or systemically. Microscopic evaluation at the exposure site revealed compound-related dermal irritation in treated skin sections from the high-dose mice of both sexes. The epidermal response was characterized by acanthosis that increase in incidence and severity with dose. The NOEL for dermal carcinogenicity was determined to be the high dose of 0.26% test substance, which is equivalent to 2.6 mg/ml. 0.1ml of the 2.6 mg/ml dose solution was applied to mice three times per week (7 days), resulting in a total delivered dose of 0.111 mg/day. Using a default body weight for mice of 0.028 kg, results in an applied dose of 3.98 mg/kg/day.


Justification for selection of carcinogenicity via oral route endpoint:
Only study availlable

Justification for selection of carcinogenicity via dermal route endpoint:
Only study available (K=2)

Justification for classification or non-classification

In an oral carcinogenicity study in rats [International Research and Development Corporation (1983)] conducted in methods comparable to OECD TG 451, rats (Charles River CD) were fed diets containing 0.01, 0.1 and 0.2 % C12 -14 AO for up to 2 years. Based on findings, the NOAEL for systemic toxicity was 0.2 % in diet, equivalent to 90 mg AO/kg bw/day. There were no macroscopic changes observed and no neoplastic or non-neoplastic treatment related effects were identified.

In a dermal carcinogenicity study in mice [Hazelton Laboratories Inc. (1982)] conducted in methods comparable to OECD TG 451, C12 -14 AO was applied to the skin of mice (ICR Swiss mice, CD-1) at 0.05 %, 0.13 % and 0.26 %, 3 times/week for two years. A wide variety of spontaneous lesions, including neoplasms, were noted in the various tissues and were of the type and incidence commonly observed in mice of this age and strain. Substance related irritation was noted. The study did not result in any carcinogenic response in the exposed skin or systemically.

On the basis of these studies, the substance should not be classified as carcinogenic.