Amines, C14-16 (even numbered)-alkyldimethyl, N-oxides

Endpoint summary

Description of key information

No repeated dose toxicity studies have been performed using C14 -16 AO. Data are read across from category members C12 -14 AO and C12 -18 AO.

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)] performed using C12 -14 AO. 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

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

1974-05-08 to 1974-10-30

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

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.

Justification for type of information:

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
Please refer to the Amine Oxide Category justification attached in Section 13

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL
Please refer to the Amine Oxide Category justification attached in Section 13

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study 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 or test system 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: m & f at 0.5%: consumed less diet and a depression in the average body weight gain was observed by the end of the first week. Two m & 2 f in the 0.5% group developed moderate to severe bilateral cataracts.

Dose descriptor:

NOAEL

Effect level:

88 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: m & f at 0.5%: consumed less diet and a depression in the average body weight gain was observed by the end of the first week. Two m & 2 f in the 0.5% group developed moderate to severe bilateral cataracts.

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.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2011-11-23 to 2012-01-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

14-day exposure period, no formulation analysis and restricted examination parameters.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Charles River Laboratories Research Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany.
- Age at study initiation: Males: 7 weeks ; Females: 8 weeks
- Weight at study initiation: Males: 248.2 - 288.5 g; Females: 212.5 - 241.8 g
- Fasting period before study: No.
- Housing: MAKROLON cages (type III plus) with a basal surface of ca. 39 x 23 cm and a height of 18 cm with granulated textured wood as bedding.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 55 ± 15%
- Air changes (per hr): Not specified
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

IN-LIFE DATES: From: 2011-11-29 To: 2011-12-13

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Amount of vehicle : 10 mL/kg bw

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Not applicable

Duration of treatment / exposure:

14 days

Frequency of treatment:

Once daily

Dose / conc.:

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

Remarks:

Based on active substance

Dose / conc.:

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

Remarks:

Based on active substance

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Based on active substance

No. of animals per sex per dose:

5 per sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: On agreement with sponsor based on information on analogous substances.

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Before and after dosing
- Cage side observations: skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns

DETAILED CLINICAL OBSERVATIONS: Yes, mortality
- Time schedule: early morning and afternoon of each working day

BODY WEIGHT: Yes
- Time schedule for examinations: Time of group allocation, on the day of commencement of treatment and once a week thereafter on the same day of the week throughout the experimental period.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: The quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period. Food intake per rat (g/rat/week) was calculated using the total amount of food given to, and left by each rat in each group on completion of a treatment week.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: 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: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: monitored daily by visual appraisal

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to necropsy
- Anaesthetic used for blood collection: Yes isofurane ether
- Animals fasted: Yes
- How many animals: All
- Parameters checked: haemoglobin content, erythrocytes, leucocytes, differential blood count (relative and absolute), reticulocytes, platelets, haematocrit value, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy
- How many animals: All
- Parameters checked: albumin, globulin, albumin/globulin ratio, bilirubin (total), cholesterol (total), creatinine, protein (total), urea (in blood), calcium chloride, potassium, sodium, alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase,

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal, the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.
Organ weights: Kidneys, liver, spleen, mesenteric lymph, stomach

HISTOPATHOLOGY: Yes
kidney (2) and ureter
liver
lymph node (1, mesenteric)
spleen
stomach

Other examinations:

None

Statistics:

The test item groups 2 to 4 were compared to the control group 1 (group 1).

The following statistical methods were used:

Multiple t-test based on Body weight / food consumption /
DUNNETT, C. W. haematology / clinical biochemistry /
New tables for multiple relative and absolute organ weights
comparisons with a control (p =< 0.01)
Biometrics, 482-491
(September 1964) The following limits were used:
p = 0.01 ^ t = 3.39 for 16 degrees of freedom
p = 0.01 ^ t = 3.39 for 12 degrees of freedom
p = 0.01 ^ t = 3.45 for 11 degrees of freedom

Exact test of R. A. FISHER Histopathology
(p =< 0.05)

These statistical procedures were used for all data.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see details below

Mortality:

mortality observed, treatment-related

Description (incidence):

see details below

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see details below

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see details below

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

see details below

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

see details below

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

see details below

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see details below

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

see details below

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see details below

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Two of 5 male and 3 of 5 female animals treated with 300 mg N,N-dimethyldecylamine-N-oxide/kg b.w./day revealed pilo-erection from test day 8 onwards or on test day 3, respectively. In addition, all females revealed slightly to moderately reduced motility from test day 3 onwards. Furthermore, ptosis was noted for one of 5 females starting on test day 3.
All animals treated with 1000 mg N,N-dimethyldecylamine-N-oxide/kg b.w./day revealed slight salivation, slightly reduced motility and tonic convulsions on test day 2 and/or 3. Additionally,hunched posture was noted in one male of the high dose group on test day 3.

No deaths occurred in the low dose group.
One of 5 intermediate dosed males (no. 25) treated with 300 mg N,N-dimethyldecylamine-N-oxide/kg b.w./day was found dead in the morning of test day 10.Prior to death, no symptoms were recorded.
All high dosed group males and females, treated with 1000 mg N,N-dimethyldecylamine-N-oxide/kg b.w./day, died on test day 2 or 3, respectively. Prior to death, symptoms were noted in form of reduced motility, tonic convulsions and hunched posture. The deaths of these animals were considered to be test item-related.

BODY WEIGHT AND WEIGHT GAIN
The body weight of the male and female animals treated with 300 mg N,N-dimethyldecylamine-N-oxide/kg b.w./day was slightly below the body weight of the control group by up to 12% for the males and by up to 11% for the females in test weeks 1 and 2.
Body weight gain and body weight at autopsy were reduced accordingly.
No changes were noted for the animals treated with 1000 mg N,N-dimethyldecylamine-N-oxide/kg b.w./day during the 2 or 3 days of treatment until death compared to the control group.

FOOD AND WATER CONSUMPTION
A decrease in food intake by 22% for the males and by 23% for the females was noted in test week 1 at a dose of 300 mg N,N-dimethyldecylamine-N-oxide/kg b.w./day.
Afterwards, the food intake recovered to the values of the control group.
No test item-related influence was noted for the animals treated with 1000 mg N,N-dimethyldecylamine-N-oxide/kg b.w./day during the 2 or 3 days of treatment until death compared to the control group.
No test item-related influence was noted for the drinking water consumption at any of the tested dose levels.

HAEMATOLOGY
An increase in the percentage of neutrophilic granulocytes and a decrease in the percentage of lymphocytes was observed at 100 or 300 mg N,N-dimethyldecylamine-N-oxide/kg b.w./day.
Haematological examination of the animals treated with 1000 mg N,N-dimethyldecylamine-N-oxide/kg b.w./day was not possible due to the premature deaths of all high dosed animals on test day 2 or 3.

CLINICAL CHEMISTRY
Treatment with 100 or 300 mg N,N-dimethyl-decylamine-N-oxide/kg b.w./day resulted in a decrease in the plasma level of bilirubin and an increase in the plasma activity of ALAT.
Determination of biochemical parameters of the animals treated with 1000 mg N,N-dimethyl-decylamine-N-oxide/kg b.w./day was not possible due to the premature deaths of all high dosed animals on test day 2 or 3.

ORGAN WEIGHTS
The relative and absolute stomach weight was increased for the males (rel. up to 63%, abs. 51%) and females (rel. 51%, abs. 31%) starting at a dose of 100 mg N,N-dimethyl-decylamine-N-oxide/kg b.w./day.
Female animal no. 30 treated with 300 mg N,N-dimethyldecylamine-N-oxide/kg b.w./day revealed an increased relative and absolute liver weight (rel. 67%, abs. 9%).
Male animal no. 25 treated with 300 mg N,N-dimethyldecylamine-N-oxide/kg b.w./day that was found dead in the morning of test day 10 revealed an increased relative and absolute liver weight (rel. 61%, abs. 38%) compared to the control.
An increased relative and absolute stomach weight (males: rel. 85%, abs. 73%; females: rel. 64%, abs. 49%) and an increased relative and absolute liver weight (males: rel. 62%, abs. 50%; females: rel. 37%, abs. 24%) compared to the control was noted for the prematurely on test day 2 or 3 deceased males and females treated with 1000 mg N,N-dimethyldecylamine-N-oxide/kg b.w./day.

GROSS PATHOLOGY
Test item-related changes in form of a thickened mucosa of all areas of the stomach were noted in 3 of 5 male and all female animals treated with 300 mg N,N-dimethyl-decylamine-N-oxide/kg b.w./day.
Male animal no. 25 treated with 300 mg N,N-dimethyldecylamine-N-oxide/kg b.w./day that was found dead in the morning of test day 10 did not reveal any macroscopic changes.
Macroscopic inspection of the prematurely deceased 5 male and 5 female animals treated with 1000 mg N,N-dimethyl-decylamine-N-oxide/kg b.w./day on test day 2 or 3 revealed changes in the stomach (dilated, filled with liquid/ gas, fundus/cardia region mucosa reddened or red-stained discoloured, ulceration in fundus region).

HISTOPATHOLOGY: NON-NEOPLASTIC
A purulent gastritis with ulceration and marked inflammatory oedema of the subepithelial tissue was observed in the forestomach of all male and female rats treated with 1000 mg N,N-dimethyldecylamine-N-oxide/kg b.w./day and in one prematurely deceased rat treated with 300 mg N,N-dimethyldecylamine-N-oxide/kg b.w./day.
Furthermore, the fundus region of the glandular stomach showed a dilatation and a thin mucosa with focal atrophy, but without inflammatory reactions.
The test item appears to damage the squamous epithelium of the forestomach only.

Basis for effect level:

other: Test item-related mortality at 1000 mg AO/kg bw/day. Gastro-intestinal effects from 300 mg AO/kg bw/day.

Remarks on result:

not measured/tested

Critical effects observed:

not specified

Conclusions:

The aim of this study was to select the dose levels for possible repeated dose toxicity or reproduction/developmental studies of N,N-dimethyldecylamine-N-oxide by oral administration to rats.
One of 5 males treated with 300 mg N,N-dimethyldecylamine-N-oxide/kg b.w./day and all males and females treated with 1000 mg N,N-dimethyldecylamine-N-oxide/kg b.w./day died prematurely.
Starting at the low dose of 100 mg N,N-dimethyldecylamine-N-oxide/kg b.w./day changes in several haematological and biochemical parameters were noted. Additionally, the relative and absolute stomach weight was increased.
In addition, treatment with the intermediate dose of 300 mg N,N-dimethyldecylamine-N-oxide/kg b.w./day resulted in pilo-erection, reduced motility and ptosis. Weight, body weight gain and food intake were reduced. Macroscopic examination revealed a thickened mucosa of all areas of the stomach.
Histopathological examination revealed a purulent gastritis with ulceration and marked inflammatory oedema of the sub-epithelial tissue in the stomach of the animals treated with 1000 mg N,N-dimethyldecylamine-N-oxide/kg b.w./day and the prematurely deceased 300 mg/kg dosed animal. The test item appears to damage the squamous epithelium of the forestomach only.

Executive summary:

In a GLP 14 -day dose range-finding study performed in a similar manner to OECD Guideline 407, groups of Sprague-Dawley rats (5 per sex per dose) were dosed daily by oral gavage with 0 (vehicle, tap water only), 100, 300 and 1000 mg/kg bw/day of the test material. Doses were corrected for active material content.

The animals were examined during the exposure period for clinical signs of toxicity including mortality, food and water consumption, body weight and body weight gain. All animals were necropsied on Day 15 with examination of histopathology, blood chemistry, macroscopic findings including organ weights and microscopic findings.

One of 5 males treated with 300 mg N,N-dimethyldecylamine-N-oxide/kg b.w./day and all males and females treated with 1000 mg N,N-dimethyldecylamine-N-oxide/kg b.w./day died prematurely. Starting at the low dose of 100 mg N,N-dimethyldecylamine-N-oxide/kg b.w./day changes in several haematological and biochemical parameters were noted. Additionally, the relative and absolute stomach weight was increased. In addition, treatment with the intermediate dose of 300 mg N,N-dimethyldecylamine-N-oxide/kg b.w./day resulted in pilo-erection, reduced motility and ptosis. Weight, body weight gain and food intake were reduced. Macroscopic examination revealed a thickened mucosa of all areas of the stomach. Histopathological examination revealed a purulent gastritis with ulceration and marked inflammatory oedema of the sub-epithelial tissue in the stomach of the animals treated with 1000 mg N,N-dimethyldecylamine-N-oxide/kg b.w./day and the prematurely deceased 300 mg/kg dosed animal. The test item appears to damage the squamous epithelium of the forestomach only.

The findings of the 14 -day dose range finder are in line with previous sub-acute and sub-chronic oral dose studies in the rat performed with C12 -14 AO and C12 -18 AO. As the results of the existing studies are consistent read-across to the sub-acute and sub-chronic studies on the analogous AO species is considered to be justified.

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

07-11-2007 to 28-05-2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: HanRcc: WIST(SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd, Füllinsdorf, Switzerland.
- Age at study initiation: 11 weeks
- Weight at study initiation: Males, 294 to 330 g; females, 175 to 214 g.
- Housing: Individually in Makrolon type 3 cages with wire mesh tops and sterilised standard softwood bedding. During the pre-pairing period, cages with males were interspersed among those holding females to promote the development of regular estrus cycles.
- Diet: Pelletted standard mouse maintenance diet available ad libitum.
- Water: Community tap-water available ad libitum.
- Acclimation period: Under test conditions for 1 week after health examination.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 30 - 70 %
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 h light/dark

IN-LIFE DATES: From: 07-11-2007 To: 03-01-2008

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item was used as provided by the sponsor, adjusting the dosing formulations for its purity i.e. 30.27 %. The dosage formulations were prepared weekly using the test item as supplied by the Sponsor and using a correction factor of 3.3 due to the purity of 30.27 % of the test item. Lauramine oxide was weighed into a glass beaker on a tared precision balance and approximately 80 % of the vehicle was added (w/v). Using an appropriate homogeniser, a homogenous suspension was prepared. Having obtained a homogenous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration. Homogeneity of the test item was maintained during the daily administration period using a magnetic stirrer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first day of treatment, samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (4 h and 7 days). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 ºC) and delivered on dry ice to be stored at -20 ± 5 ºC until analysis.
The samples were analysed by HPLC coupled to an ELSD detector following an analytical procedure developed at RCC and quantified with the area under the peak. The test item was used as the analytical standard. Analysed samples were not discarded without written consent from the study director.
The analytical part of the study was conducted at RCC Ltd, Itingen, Switzerland under GLP-compliant conditions. The identity of Lauramine oxide was confirmed by its retention time, which was similar to that measured in the working standards. The test item content in all samples was found to be in the accepted range of ± 20 % of the nominal content. In addition, the homogenous distribution of Lauramine oxide in highly purified water was demonstrated. The results of the analytical phase confirmed the correct preparation and sotrage of application formulations during the conduct of this study.

Duration of treatment / exposure:

Lauramine oxide was administered to male rats for at least 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation period until the F1 generation reached day 4 post partum.

Frequency of treatment:

Daily

Dose / conc.:

0 other: mg AO/kg bw/day (actual dose received)

Dose / conc.:

40 other: mg AO/kg bw/day (actual dose received)

Dose / conc.:

100 other: mg AO/kg bw/day (actual dose received)

Dose / conc.:

250 other: mg AO/kg bw/day (actual dose received)

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on a previous dose range finding toxicity study in Han Wistar rats, RCC study No. B51592 in which dose levels of 30, 60, 120, 500 and 1000 mg/kg/day corrected for purity were tested. Both the 1000 and 500 mg/kg/day resulted in lethality after a single or two doses, respectively. The overall NOEL was 120 mg/kg/day (corrected for purity).
- Rationale for animal assignment: Computer-generated random algorithm was used, with body weights taken into consideration in order to ensure similar mean body weights in all groups.
10 mL/kg bw was administered.

Positive control:

None specified

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily.
- Cage side observations included: Viability and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to the first administration of the test item and then weekly thereafter, detailed clinical observations were performed outside the home cage. Animals were observed for the following: Changes in skin fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, ruffled fur, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes or bizarre behaviour were also reported. Additionally females were observed for signs of difficult or prolonged parturition and behavioural abnormalities during nesting and nursing.
At one time during the study (males shortly before the scheduled sacrifice and females on day 3 or 4 post partum) relevent parameters were performed with five P-generation males and five P-generation females randomly selected from each group. The Functional Observation Battery (FOB) assessment was conducted following the daily dosage administration.
Animals were observed for the following:
a) cage-side observations: Unusual body movements (e.g. tremours, convulsions) abnormal behaviour (e.g. circling, stereotypy) and posture as well as resistance to removal.
b) hand-held observations: Palpebral closure, pinna reflex, lacrimation, pupil size, pupil reactivity, salivation, muscle tone, extensor thrust response, righting reflex and reaction to handling.
c) Open field observations: Level of ambulatory activity, including rearing (one minute evaluation) responsiveness to sharp noise, paw pinch, gait evaluation, quantity of urine and fecal pellets voided.
d) Categorical observations (could be made at any time during the FOB): Hair coat, behaviour, respiration, muscle movements, eyes, hearing ability (Preyer's reflex) urine or faeces, soiling, general abnormalities, posture.
e) Measurements/counts: Hind limb/fore limb grip strength, landing food splay, rectal temperature.
Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151. Activity of the animals (based on beam count) was recorded for 6 minute intervals over a period of 30 min. These data and the total activity over 30 min were reported.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded daily from treatment start to day of necropsy.

FOOD CONSUMPTION:
- Food consumption:
Males, weekly during pre-pairing periods and after pairing periods.
Females, prepairing period days 1-8 and 8-14; gestation days 0-7, 7-14 and 14-21 post coitum, and days 1-4 post partum. No food consumption was recorded during the pairing period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were obtained on the day before or the day of the scheduled necropsy from 5 males (randomly selected) from each group. Blood samples for 5 lactating females (randomly selected) from each group were obtained on day 5 post partum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.
- Anaesthetic used for blood collection: Yes, isoflurane.
- Animals fasted: Yes, approximately 18 h before blood sampling but allowed access to water ad libitum.
- How many animals: 5 males (randomly selected) from each group. Blood samples for 5 lactating females (randomly selected) from each group were obtained on day 5 post partum.
- Parameters examined:
Complete blood cell count: Erythrocyte count, haemoglobin, hematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, haemoglobin concentration distribution width, leukocyte count (total), differential leukocyte count, platelet count.
Coagulation: Thrombin time (= thromboplastin time), activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were obtained on the day before or the day of the scheduled necropsy and for lactating females (randomly selected) from each group obtained on day 5 post partum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.
- Animals fasted: Yes, approximately 18 h before blood sampling but allowed access to water ad libitum.
- How many animals: 5 males (randomly selected) from each group. Blood samples for 5 lactating females (randomly selected) from each group were obtained on day 5 post partum.
- Parameters examined: Glucose, urea, creatinine, bilirubin (total) cholesterol (total) triglycerides, aspartate aminotrasferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transferase, bile acids, creatine kinase, sodium, potassium, chloride, calcium, phosphorous, protein (total) albumin, globulin, albumin/globulin ratio.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: See section above on clinical signs.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals sacrificed or found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4 % formaldehyde solution.
Dead pups, except those excessively cannibalised, were examined macroscopically.
For the parental animals, special attention was directed to the organs of the reproductive system. The number of implantation sites and corpora lutea were recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible haemorrhagic areas of implantation sites.

Organ weights.
The testes and epididymides of all parental males were weighed as pairs. In addition, from 5 males and females selected randomly from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight recorded.
Adrenal glands (weighed as pairs), brain, heart, kidneys (weighed as pairs), liver, thymus and spleen.
The following tissues from all parental males were preserved in neutral phosphate buffered 4 % formaldehyde solution or in Bouin's fixative:
Prostate, seminal vesicles with coagulating gland, testes (in Bouin's fixative) and epididymides (in Bouin's fixative).
The following tissues from all parental females were preserved in neutral phosphate buffered 4 % formaldehyde solution: Ovaries.
In addition, from 5 males and 5 females per group selected for organ weights, the following tissues were preserved in neutral phosphate buffered 4 % formaldehyde solution: Gross lesions, brain, spinal chord, small and large intestine (including Peyer's patches), stomach, liver, kidneys, adrenals, spleen, heart, thymus, thyroids and parathyroids (if possible) trachaea and lungs (preserved by inflation with fixative and then immersion) uterus with vagina, urinary bladder, lymph nodes (mesenterial, mandibular) peripheral nerve (sciatic) and bone marrow.


HISTOPATHOLOGY: Yes all organ and tissue samples were processed, embedded and cut at an approximate thickness of 2 - 4 μm and stained with hematoxylin and eosin. Additionally, the testes were examined by PAS-hematoxylin.
Slides of all organs and tissues listed above and collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the study pathologist. The same applied to all occurring gross lesions and to all animals which died spontaneously.
Special emphasis was placed on stages of spermatogenesis and histopathology of interstitial cell structure.
If test item-related morphological changes were detected in organs of any high-dose animal, those same organs from the mid- and low-dose group were examined to establish a no-effect level, if possible.
Histological examination of ovaries was carried out on any females that did not give birth.

Other examinations:

Litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were also weighed individually (without identification) on days 0 (when possible) 1 and 4 post partum.

Statistics:

The following statistical methods were used for food consumption, body weight, macroscopical findings, organ weights and reproduction data, locomotor activity, rectal temperature, landing foot splay, grip strength, haematology and clinical chemistry:
Mean and standard deviations of various data were calculated and included in the report.
The Dunnett-test (many to one t-test) based on pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
The Steel-test (many-one rank test ) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
Fisher's exact test was applied if the variables could be dichotomized without loss of information.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

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
At 250 mg/kg/day, one female was found dead at the beginning of the gestation period. This was not considered to be a test item-related effect, since no adverse clinical signs were noted before and it was a single case. At 100 and 250 mg/kg/day all males were noted to have reduced activity. Rales and salivation were noted occassionally at 250 mg/kg/day. In females, rales and salivation were noted in three females at 250 mg/kg/day during the gestation period.
Functional Observational Battery:
At 250 mg/kg/day, total locomotor activity was statistically significantly reduced in females.

BODY WEIGHT AND WEIGHT GAIN
Males at 250 mg/kg/day mean body weight and mean body weight gain were reduced throughout the study. At 100 mg/kg/day a decrease was noted during the pre-pairing period. In females at 250 mg/kg/day mean body weight and mean body weight gain were generally decreased for the whole study.

FOOD CONSUMPTION
Males at 250 mg/kg/day mean food consumption was dose-dependently reduced throughout the study treatment period. In females, at 100 and 250 mg/kg/day, mean food consumption was dose-dependently reduced during the pre-pairing period and gestation period. During the lactation period, at 100 mg/kg/day it recovered and at 250 mg/kg/day remained reduced.

CLINICAL LABORATORY INVESTIGATIONS
The statistically significant alterations observed at 250 mg/kg/day were not considered to be adverse.

ORGAN WEIGHTS
At 250 mg/kg/day, an increase in the absolute and relative liver weight was noted in males and females.

GROSS PATHOLOGY
At 250 mg/kg/day, at necropsy, the mucosa in the forestomach was thickened and with irregular surface and a thickened stomach was observed in 5 of 10 males.

HISTOPATHOLOGY:
Microscopically the test item-related lesions recorded were:
Spleen: Lymphoid depletion was noted in females (2/5) at 250 mg/kg/day.
Liver: Hepatocellular hypertrophy in males (2/5) and females (1/5) at 250 mg/kg/day.
Kidneys: An increase of tabular basophilia and hyaline droplets was recorded in treated males. However, this increase in incidence was considered below the scope of concern in males receiveing 40 or 100 mg/kg/day since they showed the same severity grade as control males. An increase in severity grade of both findings was only recorded in males treated at 250 mg/kg/day.
Forestomach: Hyperkeratosis, parakeratosis, squamous cell hyperplasia and submucosal inflammation were recorded in all males and females at 100 and 250 mg/kg/day.
Submucosal oedema was recorded in males (3/5) and in females (3/5) at 250 mg/kg/day and in females at 100 mg/kg/day.
Erosions were recorded in males (3/5) and females (3/5) at 250 mg/kg/day.
Ulcerations were recorded in females at 100 (1/5) and 250 mg/kg/day (1/5).
Pustules were recorded in males at 250 mg/kg/day (2/5) and females at 100 (1/5) and 250 mg/kg/day.
Microscopic examination of the reproductive organs of males and females treated at 250 mg/kg/day failed to find any abnormality.

OTHER FINDINGS
Reproduction data: All pairs mated. Mean pre-coital time, fertility index and conception rate were not affected by the treatment with the test item. At 250 mg/kg/day gestation index was reduced since two dams did not deliver any pups and one dam delivered only one dead pup.
Implantation rate was unaffected. At 250 mg/kg/day, a statistically significant increase of post-implantation loss was observed.
Litter size, sex ratio and abnormalities at first litter check, postnatal loss Day 0 - 4 post partum: Litter size and mean number of pups at first litter check were not affected by the treatment with the test item. The sex ratio was also not affected. No abnormal pup was noted at any dose level. At 250 mg/kg/day, a statistically significant increase in pup death was observed on postnatal days 0 - 4. Although the higher postnatal loss was in the range of historical control data, this resulted in a reduced number of pups.
Pup weights to Day 4 post partum and macroscopical findings: At 250 mg/kg/day, mean pup weight development was reduced. At necropsy of pups, there were no abnormal findings.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: At doses of ≥100 mg AO/kg bw/day: reduced activity, body weight gain and food consumption noted; forestomach lesions observed. At 250 mg AO/kg bw/day: increased liver weights and microscopic changes in liver and kidney.

Critical effects observed:

not specified

Summary of results:

Parameter	Administration dose	Control group		Low dose group		Medium dose group		High dose group
	mg/kg	0		40		100		250
	Sex	M	F	M	F	M	F	M	F
	No. of animals	10	10	10	10	10	10	10	10
Mortality		0	0	0	0	0	0	1*	0
Tolerability
Reduced activity		-	-	-	-	+	-	+	-
Rales and salivation		-	-	-	-	-	-	+	+ (gestation period)
FOB
Total locomotor activity		-	-	-	-	-	-	-	+
Food consumption
Reduction		-	-	-	-	+	+ (pre-pairing & gestation)	+	+ (pre-pairing & gestation)
Recovery		-	-	-	-	-	+	-	-
Body weight		-	-	-	-	-	-	+	+
Body weight gain		-	-	-	-	-	-	+	+
Clinical Laboratory Investigation		-	-	-	-	-	-	-	-
Organ weights
Liver weight increase Absolute		-	-	-	-	-	-	+	+
Liver weight increase Relative		-	-	-	-	-	-	+	+
Macroscopic findings and histopathology
Thickened mucosa in the forestomach (n)		-	-	-	-	-	-	5	-
Microscopic lesions
Spleen: Lymphoid depletion (n)		-	-	-	-	-	-	-	2
Liver: Hepatocellular hypertrophy (n)		-	-	-	-	-	-	2	1
Kidney: Increased incidence of tubular basophilia and hyaline droplets		-	-	+ ns	-	+ ns	-	+	-
Forestomach: Hyperkeratosis		-	-	-	-	+	+	+	+
Forestomach: Parakeratosis		-	-	-	-	+	+	+	+
Forestomach: Squamous cell hyperplasia		-	-	-	-	+	+	+	+
Forestomach: Submucosal inflammation		-	-	-	-	+	+	+	+
Forestomach: Submucosal edema (n)		-	-	-	-	-	2	3	3
Forestomach: Erosions (n)		-	-	-	-	-	-	3	3
Forestomach: Ulcerations (n)		-	-	-	-	-	1	-	1
Forestomach: Pustules (n)		-	-	-	-	-	1	2	1

*Not considered to be a test item-related effect.

ns: not significant

Conclusions:

The test item was administered in highly purified water as vehicle, at dosages of 40, 100 and 250 mg/kg/day, corrected to 30.27 % purity and controls received the vehicle only over a number of consecutive weeks. The test item was adminsitered to male rats for at least 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation period until the F1 generation reached Day 4 post partum.
Administration at 100 and 250 mg/kg/day caused a reduction in activity and of the body weight gain in males during the pre-pairing period and a dose-dependent reduction of food consumption in males and females.
At 250 mg/kg/day treatment with the test item resulted in a general reduction of body weight gain in males and females, in statistically reduced locomotor activity in females in statistically significantly increased post-implantation and postnatal loss and in decreased pup body weight development. An increase of liver weights (absolute and ratios) was observed and correlated with hepatocellular hypertrophy noted during the histopathological examination. At necropsy, the mucosa in the forestomach was thickened and with an irregular surface. The histopathology data showed lesions in the forestomach and in the kidneys.
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/kg/day.
The NOEL for reproduction/developmental toxicity was considered to be 100 mg/kg/day.

Reference 4

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

23-03-2010 to 19-05-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

As not every positive mating sign results in pregnancy, the mating period was extended until a positive mating sign was noted for all females (up to 17 days). This additional mating period was conducted to guarantee at least 8 pregnant females per group.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Research Models and Services Germany GmbH
- Age at study initiation: 10 weeks
- Weight at study initiation: Males 327.9 - 424.0 g; females 212.7 - 280.0 g
- Housing: Except during the mating period, the animals were kept singly in MAKROLON cages (type III) with a basal surface of approximately 39 cm x 23 cm and a height of approximately 15 cm.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: The animals were held for 13 days for adaptation.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C  3°C (maximum range)
- Humidity (%): Relative humidity of 55% - 15% (maximum range)
- Photoperiod (hrs dark / hrs light): The rooms were alternately lit (from 150 lux at 1.5 m room height) and darkened for periods of 12 hours.

IN-LIFE DATES: From: 23-03-2010 To: 19-05-2010

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was suspended in the vehicle (tap water) to the appropriate concentrations and was administered orally at a constant application volume of 10 mL/kg b.w./day. The test item-diet mixture was freshly prepared every day.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg b.w./day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At study initiation:
Analysis of stability and concentration: Immediately after preparation of the mixtures as well as 8 and 24 hours after storage of the test item preparations at room temperature (3 samples/dose level group). Total number of samples: 9.
Analysis of Homogeneity: At the start of administration, during (middle) administration and before administration to the last animal of each dose level group (3 samples/dose level group). Total number of samples: 9.

At study termination:
Analysis of concentration: During treatment with the test item always before administration to the last animal/dose level group (1 sample/dose level group). Total number of samples: 3.
Test item-vehicle mixtures (in total 21 vials), 4 further vials containing the test item or vehicle of test days 1 and 42 were dispatched on dry ice by courier for analysis.

Duration of treatment / exposure:

Main study males (mated):
50 % of the main study males were dosed for 31 days and the other 50 % of the main study males were dosed for 36 days, depending if they were sacrificed on test day 32 or 37. This dosing period includes the pre-mating period (2 weeks), the mating period (1 to 17 days) and the post mating period up to and including the day before sacrifice (terminal sacrifice was conducted on test day 32 or 37.

Main study females (mated):
The main study females were dosed for 41 to 56 days. This period includes 2 weeks prior to mating and continuing up to, and including, day 3 post-partum or the day before sacrifice.

Satellite animals (not mated animals):
The satellite animals were treated for 41 days followed by a recovery period of 16 days. The animals were not mated. Treatment started on the same day as of the main study animals and was conducted up to the day before the first scheduled sacrifice of the main study dams on test day 42.

Frequency of treatment:

Once daily

Dose / conc.:

40 other: mg AO/kg bw/day

Dose / conc.:

100 other: mg AO/kg bw/day

Dose / conc.:

200 other: mg AO/kg bw/day

No. of animals per sex per dose:

Main Study:
80 animals (40 males and 40 females)

Recovery period: 20 animals (10 males and 10 females) were allocated to groups 1 and 4 for a 14-day recovery period. These animals were not mated.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on available toxicological data and a preliminary dose-range finding study (LPT study no. 25122). No mortality was noted in this dose-range-finding study no. 25122. Signs of systemic intolerance were noted in the animals of both sexes starting at 40 mg AO/kg b.w./day. A slightly to severely reduced mean food consumption and mean body weight were noted in the male rats starting at 100 mg AO/kg b.w./day or at 250 mg AO/kg b.w./day, respectively. The females were not affected.
Necropsy revealed a thickened cardiac region of the stomach in one male rat treated with 250 mg AO/kg b.w./day as well as in all high dosed male and female rats treated with 400 mg AO/kg b.w./day.

- Rationale for animal assignment: Random. At commencement of the study, the weight variation of animals used was minimal and did not exceed 20% of the mean weight of each sex.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily throughout the test period.
- Cage side observations included: Behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality. Any signs of illness or reaction to treatment were recorded for each individual animal. Cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded. Additionally, once before the first exposure (to allow for within-subject comparisons) and once a week thereafter, detailed clinical observations were made in all animals. Detailed clinical observations were made in all male main study animals until terminal sacrifice in test week 5 or 6, respectively, in all female main study animals until the day of parturition and in all male and female satellite animals until terminal sacrifice in test week 8. These observations were made outside the home cage in a standard arena at the same time, each time. Signs noted included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.
Mortality: Further checks were made early in the morning and again in the afternoon of each working day to identify dead or moribund animals.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily throughout the test period.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of dosing, weekly thereafter and at study termination. During gestation, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum) and on day 4 post-partum during lactation. Body weights were recorded individually for each adult animal. Live pups were weighed individually within 24 hours of parturition (day 0 or 1 post-partum) and on day 4 post-partum during lactation.

FOOD CONSUMPTION:
The quantity of food left by individual animals was recorded on a weekly basis through-out the experimental period with the execution of the mating period. Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment week.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Drinking water consumption was monitored daily by visual appraisal throughout the study.

OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the pre-mating period and at the end of the recovery period.
- Anaesthetic used for blood collection: Yes, light ether anaesthesia
- Animals fasted: Yes, fasted overnight
- How many animals:
At the end of the pre-mating period: 5 male and 5 female main study animals randomly selected of each group
At the end of the recovery period: All satellite animals
- Parameters checked: Haemoglobin content (HGB), erythrocytes (RBC), leucocytes (WBC), reticulocytes, platelets, differential blood count (relative), differential blood count (absolute), haematocrit value, thromboplastin time, activated partial thromboplastin time, mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the pre-mating period and at the end of the recovery period.
- Animals fasted: Yes, fasted overnight
- How many animals:
At the end of the pre-mating period: 5 male and 5 female main study animals randomly selected of each group
At the end of the recovery period: All satellite animals
- Parameters checked: Albumin, globulin, albumin/globulin ratio, bile acids, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), urea (blood urea), calcium, chloride, potassium, sodium, alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase.

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: See clinical signs

OTHER:
Neurological screening:
Screening of sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli) as well as the assessment of grip strength and motor activity assessment were conducted in five males and five females randomly selected from each main study group. The tests were conducted towards the end of the main study, 1 - 2 hours after dosing and before any blood sampling for labo-ratory examinations.

Observational screening
Males: Shortly before scheduled sacrifice (test day 29)
Females: During lactation, shortly before scheduled kill (test days 42, 43 or 56)
Righting reflex:
The animal was grasped by its tail and flipped in the air (approx. 60 cm) above the cart surface so that it turned head over heels. The normal animal should land squarely on its feet. If it landed on its side, 1 point was scored; if it landed on its back, 2 points were scored. This test was repeated five times and the total scores were recorded.
Body temperature:
An electronic probe thermometer (with a blunt probe) was used to take a rectal tem-perature, being allowed to equilibrate for 30 seconds before the reading was recorded.
Salivation:
Discharge of clear fluid from mouth, most frequently seen as beads of moisture on lips in rats. Normal state was to see none, in which case the score sheet space was left blank. If present, a plus sign was recorded in blank.
Startle response:
With the animal on the cart, the metal cage was struck with the blunt probe. The normal animal should exhibit a marked but short-lasting response, in which case the space on the scoring sheet was left blank. If present, a plus sign was entered.
Respiration:
While at rest on the cart, the animal's respiration cycle was observed and evaluated in terms of a scale from 1 (reduced) to 5 (increased), with 3 being normal.
Mouth breathing:
Rats are normally obligatory nose-breathers. Each animal was observed, whether it was breathing through its mouth or not (if it was, a check was placed in the appropriate box).
Urination:
When an animal was removed from its cage, the pan beneath the animal's cage was examined while returning the animal to its cage. The signs of urination were evaluated on a scale of 0 (lacking) to 5 (polyuria).
Convulsions:
If clonic or tonic convulsions were observed to occur, they were graded on intensity (from 1 (minor) to 5 (marked)) and the type and intensity were recorded.
Pilo-erection:
The fur of the animal's back was observed, whether it was raised or elevated. In the normal case (no pilo-erection) the space blank is left. If pilo-erection was present, a plus sign was entered in the blank.
Diarrhoea:
In examining the pan beneath an animal's cage, it was noted if there were any signs of loose of liquid stools. Normal state was for there to be none (0), in case of diarrhoea the intensity was recorded on a scale of 1 (slight) to 5 (much increased).
Pupil size:
The pupils were determined if they were constricted or dilated and were graded on a scale of 1 (constricted) to 5 (dilated), respectively, 3 being normal.
Pupil response:
The beam of light from the pen light was played across the eyes of the animal and the changes in pupil size were noted. In the normal animal, the pupil was constricted when the beam was on it and then dilated back to normal when the light was removed. It was noted if there was no response (in which case a minus sign was recorded in the blank space).
Lacrimation:
The animal was observed for the secretion and discharge of tears. In rats the tears contain a reddish pigment. No discharge was normal and in this case the box was left blank. If the discharge was present, a plus sign was entered.
Impaired gait:
The occurrence of abnormal gait was evaluated. The most frequent impairments were waddling (W), hunched gait (H), or ataxia (A, the inability of all the muscles to act in unison). The extend of any impairment was recorded on a scale of 1 (slight) to 5 (marked).
Stereotypy:
Each animal was evaluated for stereotypic behaviour (isolated motor acts or partial sequences of more complex behavioural patterns, occurring out of context and with an abnormal high frequency). These were graded on a scale of 0 to 5 if such signs were present.
Toe pinch:
The blunt probe was used to bring pressure to bear on one of the digits of the hindlimb. This should evoke a response from the normal animal, graded on a scale from 1 (ab-sent) to 5 (exaggerated).
Tail pinch:
The procedure detailed above was utilized with the animal's tail instead of its hindlimb and was graded on the same scale.
Wire manoeuvre:
The animal was placed on the metal rod suspended parallel to the cart approx. 60 cm above it. Its ability to move along the rod was evaluated. If impaired, a score of from 1 (slightly impaired) to 5 (unable to stay on wire) was recorded.
Hind leg splay:
Using an ink pad, the hind paws were marked with ink. The rat was then held 30 cm above a sheet of blotting paper on the cart. The animal was dropped and the distance between the prints of the two hind paws was measured.
Positional passivity:
When placed in an awkward position (such as on the edge of the top of the wire bot-tomed cage) on the cart surface, it was examined whether the animal immediately moved into a more normal position. If not, a score was recorded on a scale of 1 (slightly impaired) to 5 (cataleptic).
Tremors:
Periods of continued fine movements, usually starting in the limbs (and perhaps limited to them). The normal case is to have none, in which case no score was recorded. If present, they were graded on a scale of 1 (slight and infrequent) to 5 (continuous and marked).
Positive geotropism:
The animal was placed on the inclined (at an angle of approx. 30°) top surface of the wire cage with its head facing downward. It should turn 180° and face 'uphill', in which case the space on the form should be left blank. If this did not occur, a negative sign was recorded.
Limb rotation:
One of the animal's hindlimbs was taken and moved through its normal plane of rota-tion. In the normal state, it should rotate readily but there should be some resistance. The variations from normal were from no resistance (1) to markedly increased resis-tance or rigidity (5), with 3 being normal.
Auditory function:
Each animal was placed into a container and observed for Preyer's reflex (twitching of the pinna) in response to a high frequency sound stimulus. The stimulus was repeated, if necessary, up to 3 times.

Functional tests
Grip strength:
Prior to testing, the gauge (Chatillon, Modell DPP - 1.0 kg) was calibrated with a set of known weights and the apparatus adjusted for the size of the animal (about 1 cm clearance on both sides of the animal). After the strain gauge was zeroed and set in the record mode, the animal was placed into the trough with the forepaws inside the triangular grasping ring. Using one hand, the animal was grasped about 2.5 cm of the way up toward the base of the tail and steadily pulled (approx. 2.5 cm/sec) away from the ring until the grip was broken. The animal continued to be pulled along the trough until the hindlimbs grasped the T-bar. The trial was completed when grip of the hindlimbs was broken. Three successive readings were taken for each animal with an inter-trial interval long enough to record the data and zero both meters for the next trial.

Locomotor activity
The rats were individually placed into a motility system with microprocessor control and automatic statistical evaluation. Each unit had 2 individually adjustable channels so that slight static movements and active moving could be differentiated and separately scored (TSE Systems, Bad Homburg).
The motility meter created a magnetic field, movement of the animals interfered with the magnetic field. Two separate magnetic fields allowed two sensitivity levels to be chosen to distinguish between two types of movements:
High sensitivity: stereotype, static movement
(slight movements) (e.g. grooming, a stationary movement of the animal without leaving its own position)

Low sensitivity: active locomotion
(active moving)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
50 % of the male main study animals were sacrificed in a randomised way after a total dosing period of 31 days on test day 32, the other half of the male main study animals were sacrificed after a total dosing period of 36 days on test day 37. Dams with offspring were sacrificed on day 4 post-partum, or shortly thereafter. Females which did not deliver were sacrificed on the fourth or sixth day after the calculated day of delivery.
Dissection of all animals allocated to the recovery period was performed on test day 58.
At the time of sacrifice or death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
Apparently non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium.
All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection, all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination.
The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory re-productive organs were recorded.
The weights of the following organs of all adult male animals were determined before fixation: Epididymis (2), testicle (2)
The weights of the following organs of in total 20 adult males and 20 adult females (5 animals/sex/main study group; randomly selected of each main study group, including not pregnant animals) and all satellite animals were determined before fixation: Adrenal gland (2), kidney (2), spleen, Brain, liver, thymus, heart, ovary.
Adrenal glands, gonads and kidneys were weighed individually and identified as left or right.

HISTOPATHOLOGY: Yes
The following organs or parts of organs of the in section 5.2 mentioned randomly selected 20 male and 20 female animals (5 animals/sex/main study group) and all satellite animals were preserved in 7% Formalin:
Adrenal gland (2)
Bone marrow (os femoris)
Brain (cerebrum, cerebellum, brain stem)
Gross lesions observed
Heart (right and left ventricle, septum)
Intestine, large (colon, rectum)
Intestine, small (duodenum, jejunum,
ileum, incl. Peyer's patches, Swiss roll
method)
Kidney and ureter (2)
Liver
Lungs (with mainstem bronchi and
bronchioles [preserved by inflation
with fixative and then immersion])
Lymph node (cervical) (1)
Lymph node (mesenteric) (1)
Nerve (sciatic)
Oesophagus
Seminal vesicle
Spinal cord (3 sections)
Spleen
Stomach
Thymus
thyroid (incl. parathyroids)
Tissue masses or tumours
(including regional lymph nodes)
Tongue (incl. base)
Trachea (incl. larynx)
Urinary bladder
In addition, the following organs or parts of organs of the reproductive system of all adult male and female animals were preserved in 7% Formalin; the testes and epididymides were preserved in Bouin's fixative:
Epididymides (2)
Mammary gland
Ovary (2)
Prostate
Testicle (2)
Uterus (incl. Cervix and oviducts)
Vagina
The afore-listed organs of the randomly selected parental animals of groups 1 and 4 (5 male and 5 female animals per group) and all satellite animals (in total 20 satellite animals) were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining.
The afore-listed organs of the reproductive system of all main study animals of groups 1 and 4 (in total 40 main study animals) and all satellite animals (in total 20 satellite animals) were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining.
Any other organs displaying macroscopic changes were also preserved.
Detailed histopathologic examination was performed on the ovaries, testes and epididymides (with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure) of the animals of the highest dose group (group 4) and the control group (group 1) following H.& E. and PAS staining.
In addition, frozen sections of the heart, liver and one kidney were made and stained with scarlet R. Parathyroids cannot always be identified macroscopically. They were examined microscopically if in the plan of section and in all cases where they were noted as grossly enlarged.
Due to test item-related changes, the following organs of 5 male and 5 female animals of the low and intermediate dose level groups (groups 2 and 3) were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining: Forestomach, lymph node (1, mesenteric)

Statistics:

The test item-treated groups 2 to 4 were compared with the control group 1.
STUDENT's t-test: All numerical functional tests (p ≤ 0.01)
The following limits were used:
p ≤ 0.01 ≙ t = 2.878 (18 degrees of freedom)
p ≤ 0.01 ≙ t = 2.921 (16 degrees of freedom)
p ≤ 0.01 ≙ t = 2.947 (15 degrees of freedom)
p ≤ 0.01 ≙ t = 3.355 (8 degrees of freedom)

Multiple t-test based on DUNNETT New tables for multiple comparisons with a control: Body weight / food consumption / haematology / clinical biochemistry /organ weights (absolute and relative) (p ≤ 0.01).
The following limits were used:
p ≤ 0.01 ≙ t = 3.09 (36, 33, 32 and 31 degrees of freedom)
p ≤ 0.01 ≙ t = 3.36 (8 degrees of freedom)
p ≤ 0.01 ≙ t = 3.39 (16 degrees of freedom)

For all numerical values (body weight, food consumption and organ weight data) gen-erated from the start of the mating period onwards homogeneity of variances was tested by using the BARTLETT chi-square test. If the variances were homogeneous, the DUNNETT test (p ≤ 0.01) was used to compare the experimental groups with the control group.
In case of heterogeneity of variances, the STUDENT's t-test was carried out, limit of significance was p ≤ 0.01.

Exact test of R.A. FISHER: Histopathology (p ≤ 0.05)
For the comparison of classification measurements (for example the fertility index) the FISHER's exact test, n < 100 or chi 2-test with Yates' correction for continuity, n ≥ 100 (p ≤ 0.05) were employed.
These statistical procedures were used for all data. Significantly different data are indicated in the tables.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Main study / satellite animals: No test item-related mortality was noted. No signs of systemic toxicity were noted for the male and female animals treated with 40 mg AO/kg b.w./day. Starting at 100 mg/kg b.w./day increased salivation was noted for a very few male and female animals at 100 mg AO/kg b.w./day and most animals at 200 mg AO/kg b.w./day during all periods of the study (pre-mating, mating, post-mating, gestation and/or lactation period, respectively) starting on test day 1. Further, at 200 mg AO/kg b.w./day laboured breathing was observed for one male and rough fur was noted sev-eral females during the pre-mating, mating, gestation and/or lactation period, respectively.

Functional observations (carried out 1 to 2 hours after administration):
Main study animals: No influence was noted on the parameters of the functional observations at any of the tested dose levels for the males.
Females treated with 200 mg AO/kg b.w./day revealed salivation and pilo-erection.
In addition, hindlimb grip strength of the females was reduced (statistically significant at p ≤ 0.01) by up to 71 % starting at 40 mg AO/kg b.w./day, though no dose-response relationship was noted. A slight but not significant reduction was also observed for the males at 100 and 200 mg AO/kg b.w./day.

BODY WEIGHT AND WEIGHT GAIN
Main study / satellite animals: No influence was noted on the body weight of main study and satellite animals during the entire study after treatment with 40, 100 or 200 mg AO/kg b.w./day.

FOOD CONSUMPTION/WATER CONSUMPTION
Main study / satellite animals:No test item-related influence was observed on the food consumption of the male and female animals treated with either 40 or 100 mg AO/kg b.w./day during the pre-mating, gestation and/or lactation period, respectively.
Treatment with 200 mg AO/kg b.w./day resulted in a food intake statistically sig-nificant reduced by 11% in test week 2 (pre-mating period) for the male main study animals and for the female main study animals by 10% in test week 1 (pre-mating period) and by 13% on gestation day 7 (gestation period) compared to the control.
The food intake of the female satellite animals treated with 200 mg AO/kg b.w./day was statistically significant reduced by 20% in test week 1 compared to the control. No influence was noted on the visual appraisal of the drinking water consumption at any of the tested dose levels.

HAEMATOLOGY
Main study animals: No test item-related changes in haematological parameters were noted at the end of the pre-mating period (test day 15) of male and female rats treated with 40 or 100 mg AO/kg b.w./day compared to the control. Changes in haematological parameters were noted for the animals treated with 200 mg AO/kg b.w./day: Increased neut (relative and absolute). This was statistically significant (p ≤ 0.01) for neut absolute in females.

CLINICAL CHEMISTRY
Main study animals:
No test item-related changes were noted on bio-chemical parameters on test day 15 of female rats treated with 40 mg AO/kg b.w./day compared to the control.
For the male and/or female animals treated with 40, 100 or 200 mg AO/kg b.w./day the following changes in biochemical parameters were observed: Increased ALAT statistically significant (p ≤ 0.01) for both males and females.

ORGAN WEIGHTS
Main study animals: No test item-related influence was noted.

GROSS PATHOLOGY
Main study animals: Macroscopic inspection at necropsy revealed no test item-related changes in the organs or tissues after treatment with either 40, 100 or 200 mg AO/kg b.w./day.
Recovery period: Satellite animals: An increased salivation was still noted for 2 of 5 males previously treated with 200 mg AO/kg b.w./day on test days 42 and 43. No test item related influence was noted at the end of the recovery period on test day 58.

HISTOPATHOLOGY:
The forestomach revealed a squamous cell hyperplasia with submucosal inflammatory reac-tion and hyperkeratosis/parakeratosis in the stra-tum corneum of the animals treated with 200 mg AO/kg b.w./day. These lesions were completely reversible within the 16-days recovery period. Further, a dose dependent increase of macro-phages with vacuolization in the mesenteric lymph nodes was observed in the animals treated with 100 or 200 mg AO/kg b.w./day. The effect was still noted at the end of the recovery period.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Histopathological changes noted in the forestomach and the mesenteric lymph nodes and signs of systemic toxicity (salivation) observed at dose levels of 100 and/or 200 mg AO/kg bw/day

Critical effects observed:

not specified

Conclusions:

The test item was assessed for repeated dose toxicity according to OECD guidline 422. No test item-related mortality was noted. Starting at 100 mg AO/kg b.w./day increased salivation was noted for a very few male and female animals at 100 mg AO/kg b.w./day and most animals at 200 mg AO/kg b.w./day during all periods of the study (pre-mating, mating, post-mating, gestation and/or lactation period, respectively) starting on test day 1. Further, at 200 mg AO/kg b.w./day laboured breathing was observed for one male and rough fur was noted several females during the pre-mating, mating, gestation and/or lactation period, respectively. Due to histopathological changes noted in the forestomach and the mesenteric lymph nodes and signs of systemic toxicity (salivation) observed at dose levels of 100 and/or 200 mg AO/kg b.w./day, p.o., the no-observed-effect level (NOEL) was 40 mg AO/kg b.w./day, p.o. via gavage for the F0 generation.

Reference 5

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1976-08-05 to 1976-09-02

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

28-day oral toxicity study in which only one dose level was tested and only body weight, food and water consumption were evaluated. No gross or histopathology, hematology, clinical chemistry, or detailed clinical examinations. Pre-GLP.

Qualifier:

no guideline followed

Principles of method if other than guideline:

N/A

GLP compliance:

no

Remarks:

Pre-GLP

Limit test:

no

Species:

rat

Strain:

other: Charles River Sprague Dawley derived

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: N/A
- Age at study initiation: N/A
- Weight at study initiation: 288 g
- Fasting period before study: N/A
- Housing: The rats were housed individually in cages with wire mesh bottoms.
- Diet (e.g. ad libitum): Purina rat chow ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): N/A
- Humidity (%): N/A
- Air changes (per hr): N/A
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark


IN-LIFE DATES: From: N/A To: N/A

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was prepared fresh daily in water.


DIET PREPARATION
- Rate of preparation of diet (frequency): N/A
- Mixing appropriate amounts with (Type of food): N/A
- Storage temperature of food: N/A


VEHICLE
- Justification for use and choice of vehicle (if other than water): N/A
- Concentration in vehicle: N/A
- Amount of vehicle (if gavage): 5 ml
- 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:

N/A

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

The test substance was administered daily by stomach tube

Remarks:

Doses / Concentrations:
86 mg commercial amine oxide daily
Basis:
actual ingested

No. of animals per sex per dose:

10 male rats

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose selected for the study was chosen to be equivalent to the amount consumed by rats fed a diet containing 0.5 % commercial amine oxide from a previous study for comparison of growth rate and feed consumption of the different routes.
- 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: No data
- Time schedule: N/A
- 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: weekly


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal was determined weekly: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data


FOOD EFFICIENCY:
- Body weight gain in grams (average weekly weight gain)/100 g of diet consumed (average weekly food consumption): Yes


WATER CONSUMPTION: Yes
- Time schedule for examinations: daily


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


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: No data
- 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: Yes
- Time schedule for collection of urine: daily
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined: urine volume


NEUROBEHAVIOURAL EXAMINATION: No data
- 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: No data
HISTOPATHOLOGY: No data

Other examinations:

N/A

Statistics:

Student's t-test

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

effects observed, treatment-related

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

no effects observed

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY- N/A


BODY WEIGHT AND WEIGHT GAIN- The growth of the rats dosed with the test substance was significantly depressed from that of control rats. On a weekly basis, the weight gain for the test substance treated rats significantly differed from controls only during weeks 1 and 4; the greatest effect on growth was during the fourth week.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)- The food consumption of the test substance treated group was depressed significantly only during the first week of treatment. Thereafter, their food consumption was similar to that of the control group.


FOOD EFFICIENCY- The weekly feed efficiency was lowered in the test substance group during the first and fourth week of treatment, but only the latter was significantly lowered than that of the water treated group.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)- After the first week of treatment, the rats dosed with the test substance consumed more drinking water than the control group, however, they also excreted a greater volume of urine. The net difference between water intake and urine output was similar for both groups.


OPHTHALMOSCOPIC EXAMINATION- N/A


HAEMATOLOGY- N/A


CLINICAL CHEMISTRY- N/A


URINALYSIS- After the first week of treatment, the rats dosed with the test substance consumed more drinking water than the control group; however, they also excreted a greater volume of urine. The net difference between water intake and urine output was similar for both groups.


NEUROBEHAVIOUR- N/A


ORGAN WEIGHTS- N/A


GROSS PATHOLOGY- N/A


HISTOPATHOLOGY: NON-NEOPLASTIC- N/A


HISTOPATHOLOGY: NEOPLASTIC (if applicable)- N/A


HISTORICAL CONTROL DATA (if applicable)- N/A


OTHER FINDINGS- N/A

Dose descriptor:

NOAEL

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Critical effects observed:

not specified

Gonadal tissues were not examined for gross pathology or histopathology.

Conclusions:

Commerical amine oxide was administered daily by oral gavage for 4 weeks to male rats. The test substance treated group showed a slight, but statistically significant growth depression which was reflected in a significantly decreased feed efficiency during the fourth week of treatment. When 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.

Executive summary:

A four week repeated dose toxicity study was conducted where adult male Charles River Sprague Dawley derived rats were dosed 86 mg of commercial amine oxides (containing approx 27% amine oxide) in 5 ml of water daily by stomach tube for four weeks. 10 animals were assigned to the test substance group and 10 were assigned to the water control group, which was treated on the same regimen as the test substance group. The body weight and feed consumption of each animal was determined weekly and the water consumption and urine volume were measured daily. The weekly feed efficiency for each group was calculated from the average weekly weight gain and the average weekly feed consumption.

The test substance treated rats showed a slight (approximately 8 %), but statistically significant growth depression which was reflected in a significantly decreased feed efficiency during the fourth week of treatment. When the results of the 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.

No NOAEL was determined.

Reference 6

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1976-03-17 to 1977-06-16

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study was conducted according to methods similar to OECD 408 with the following deviations: (1) rabbits were used rather than rat or mouse; (2) hematology did not include an assessment of platelet count or blood clotting time; (3) all animals of the high dose group and 5/sex/dose of the low and mid-dose groups and control were sacrificed and examined at 13-weeks and the remaining animals of the low and mid-dose group and control were sacraficed and examined at 32 or 33 weeks.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study 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:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Bunnyville Farms, Littlestown, PA
- Age at study initiation: N/A
- Weight at study initiation: 2280-3475 grams for males and 2295-3455 grams for females
- Fasting period before study: N/A
- Housing: Individual, elevated cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): N/A
- Humidity (%): N/A
- Air changes (per hr): N/A
- Photoperiod (hrs dark / hrs light): N/A


IN-LIFE DATES: From: 1976- 03-17 To: 1976-11-03

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was administered in specially prepared feed pellets by Ralston Purina Company that consisted of 0.1, 0.5,a nd 1.0% DDAO.

DIET PREPARATION
- Rate of preparation of diet (frequency): Two shipments (prior to study and week 15) sent to the lab. 500 grams of feed with appropriate dose placed into the animals feeder. Feeder was filled at least weekly.
- Mixing appropriate amounts with (Type of food): Purina Laboratory Rabbit Chow prepared with appropriate doses
- Storage temperature of food: 40 deg F


VEHICLE
- Justification for use and choice of vehicle (if other than water): N/A
- Concentration in vehicle: N/A
- Amount of vehicle (if gavage): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The diets were analyzed to confirm the desired activity prior to shipment to the lab. The diets were sent weekly to be analyzed during the course of the study to check for stability of the test substance in the feed.

Duration of treatment / exposure:

13-weeks initially for all dose groups and control. Low and mid-dose groups and controls were exposed for an additional 19 weeks for a total of 32 weeks.

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0.1, 0.5, and 1.0% DDAO
Basis:
nominal in diet

No. of animals per sex per dose:

25

Control animals:

yes, plain diet

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: Daily
- Cage side observations checked in table [No.?] were included. N/A


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily


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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: 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: 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: Week 1, 6, 9, 13, 18, 22, 26, and 32
- Dose groups that were examined: all surviving


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 0, 13, and 32
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10 animals/sex/group - Weeks 0 and 32, and 5 animals/sex/group - Week 13
- Parameters checked in table [No.?] were examined. hematocrit, hemoglobin, erythrocyte counts, total and differential leukocyte counts, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 0, 13, and 32
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10 animals/sex/group - Weeks 0 and 32, and 5 animals/sex/group - Week 13
- Parameters checked in table [No.?] were examined. fasting glucose, blood urea nitrogen, total serum protein, total serum bilirubin, serum albumin, serum sodium, serum chloride, carbon dioxide, serum calcium, serum alkaline phosphatase, serum glutamic-oxaloacetic transaminase, and serum potassium

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


NEUROBEHAVIOURAL EXAMINATION: No data
- 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 - brain, pituitary, cervical lymph nodes, mesenteric lymph nodes, eyes, lungs, heart, liver, spleen, kidney, adrenal, stomach, urinary bladder, testes, prostate, ovaries with fallopian tubes, uterus, ear, skin, neck, and absence of fat.
HISTOPATHOLOGY: Yes - tissues were preserved in 10% neutral buffered formalin: eyes, brain (with meninges), thoracic spinal cor, tongue, pituitary, mandibular salivary gland, thyroids, trachea, esophagus, adrenals, heart, lungs, spleen, liver, kidneys (with ureters), stomach, small intestine, large intestine, pancreas, ovaries with fallopian tubes, uterus with cervix, vagina, testes with epididymides, prostate, seminal vesicles, salivary glands, mesenteric and pulmonary lymph nodes, urinary bladder (with urethra), gallbladder, nerve with muscle, mammary glands (right inguinal), bone marrow smear, bone (left tibia), skin, and any unusual lesions.

Other examinations:

Organs weighed from each animal sacrificed at Week 13 and 32/33 included pituitary, adrenal, ovaries, testes with epidiymides, heart, liver, and kidneys.

Statistics:

Statistical analyses of the clinical laboratory, terminal body weight, and organ weight data were performed by Bartlett's test for homogeneity of variances and the one-way classification analysis of variances. When differences were noted in the analysis of variances, Scheffe's method for judging all contrasts was utilized. Statistical analyses of the body weights were performed by the F-test and Student's t-test. When variances differed significantly, Student's t-test was appropriately modified and Cochran's approximation utilized.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

effects observed, treatment-related

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

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

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:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
Anorexia was noted in 1 mid-dose animal and in 5 high-dose animals during Week 1. Thereafter, anorexia was noted sporadically in all groups but at a greater incidence in the high-dose rabbits than among rabbits of the other 3 groups. Other clinical signs were noted occasionally in all groups during the first 13 weeks but at greater incidence in the high-dose group included listlessness, hyperpnea, alopecia, wheezing, thinness, and rough furcoat.

A treatment related effect on survival was observed in the high-dose group as compared to the control group during the first 13 weeks. The mortality rates of the animals in the other treated groups were comparable to those of the control group during the full 32 weeks.

BODY WEIGHT AND WEIGHT GAIN
Evaluations of the Week 13 sacrifice data revealed significantly lower mean terminal body weights for the high-dose males (statistically significant) and females when compared to the control group. Evaluations of the Week 32 sacrifice data revealed a statistically significantly elevated liver/body weight ratio for the mid-dose males as compared to control males.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The mean food consumption of the males and females of the high-dose group were lower than those of the control group through the first 13 weeks of the study. Slightly lower mean food consumption values were consistently noted in the low and mid-dose groups during the first 13 weeks. Food consumption values during the last 19 weeks of the study were generally comparable between the dosed and control groups. Not considered a toxicological significance.


FOOD EFFICIENCY
The efficiency of food utilization reflected the markedly lower food consumption and body weight values of the high dose group.

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

OPHTHALMOSCOPIC EXAMINATION
A few animals were observed to have the formation of mild cataracts but they were equally interspersed among the groups and no one particular test group had any more incidences of cataract formation than the other groups. The test substance did not have a statistically significant cataractogeniceffect on the rabbit lens.

HAEMATOLOGY
The mean hematocrit and hemoglobin values of the high-dose animals, and the erythrocyte and mean corpuscular hemoglobin values of the high-dose females were significantly lower than the respective control values at Week 13. No consistent differences were noted in the low and mid-dose groups when compared to the control group. The mid-dose males had generally lower hemogram (hematocrit, hemoglobin and erythrocyte) values as compared to the control males at Weeks 13 and 32. These differences were also present pretreatment and are not considered to be of toxicological significance.

CLINICAL CHEMISTRY
Dose-related increases in the total bilirubin values at Week 13 with marked increases in the high-dose males and mid and high-dose females. Significantly lower mean alkaline phophatase values as compared to the respective control values were noted in teh high-dose males and females at Week 13 and in the mid-dose males at Week 32 (statistically significant).

URINALYSIS
N/A

NEUROBEHAVIOUR
N/A

ORGAN WEIGHTS
Evaluations of the week 32 sacrifice data revealed a statistically significant higher liver/body weight ratio for the mid-dose males as compared to the control males.

GROSS PATHOLOGY
At Week 13 sacrifice, gross findings that were noted at a greater incidence in the high-dose animals and considered treatment-related consisted of enlarged cervical and mesenteric lymph nodes, the presence of purulent material (including abcesses) in the lungs, and the absence of body fat. The remaining findings at Week 13 and 32 occurred at comparable incidences in the test and control groups and were considered incidental.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic pathology exams revealed findings attributable to the administration of the high-dose level of the test substance consisting 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.

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:

32 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Critical effects observed:

not specified

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

Conclusions:

The NOAEL was considered to be 0.1% in the diet based on increased liver/body weight ratio at mid dose and on treatment-related effects on mortality, body weight, hematology, clinical chemistry, and gross and histopathology relative to control at high dose.

Executive summary:

The purpose of this study was to evaluate the cataractogenic potential of dodecyl dimethyl amine oxide (DDAO) in New Zealand White rabbits. A 32 -week repeated dose toxicity study was conducted where the test substance was administered in Purina Laboratory Rabbit Chow at dosage levels of 0.1, 0.5, and 1.0% active DDAO in diet to three groups of rabbits (25/sex/group) and the fourth group were also fed the Purina Laboratory Rabbit Chow and served as the control. An interim sacrifice was performed after 13 weeks on all surviving high-dose animals and 5 animals/sex in the control, low and mid-dose groups. The remaining animals in the control, low and mid-dose groups were maintained on study through 32 weeks.

The criteria evaluated for compound effect included mortality, appearance and behavior, body weights, food consumption, food efficiency; hematology (hematocrit, meoglobin, erythrocyte and total and differential leukocyte counts, mean corpuscular hemoglobin and corpuscular hemoglobin concentration and mean corpuscular volume), and blood chemistry (fasting glucose, blood urea nitrogen, total serum chloride, carbon dioxide, serum calcium, serum alkaline phasphatase, serum glutamic oxaloacetic transaminanse and serum potassium), ophthalmology, terminal body weights, organ weights, organ/body weight ratios (pituitary, adrenals, ovaries, testes with epididymidis, heart liver, kidney), gross and microscopic pathology (eyes, brain, thoracic spinal cord, tongue, adrenals, pituitary, mandibular salivary gland, thyroids, trachae, esophagus, heart, lung, spleen, liver, kidney, stomach, small and large intestines, pancrease, ovaries, uterus with fallopian tubes, vagina wiht cervix, testes with epididymides, prostate, seminal vesicles, salivary glands, mesenteric and pulmonary lymph nodes, urinary bladder, gall bladder, nerve, mammary glands, bone marrow smear, bone (left tibia), skin, and any unusual lesions).

Treatment related effects attributable to the administration of the high-dose (1% DDAO 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, hemoglobin, 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 abscessess) 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.

A dose-related increased bilirubin levels at Week 13 and the decreased alkaline phosphatase level and increased liver/body weight ratio Week 32 occurred at the mid-dose (0.5% DDAO in diet). No findings attributable to the administration of any dose of the test substance were apparent in results of the ophthalmologic exams.

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

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 C10 -C16 alkyl dimethyl amine, N-oxide was 0.1% in diet or 1000 mg DDAO/kg diet. Using a food consumption factor of 0.032 kg diet/kg BW/day for rabbits, this translates to a dose of 32 mg/kg BW/day.

Reference 7

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1978-06-09 to 1978-09-11

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Subchronic feeding study in rats similar to repeated dose oral study (OECD 407-Repeated dose 28-Day Oral Toxicity Study in Rodents). Test substance was not homogenously distributed in the food.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study 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 or test system and environmental conditions:

TEST ANIMALS
- Source: N/A
- Age at study initiation: In utereo
- Weight at study initiation: N/A
- Fasting period before study: N/A
- Housing: Individual stainless steel wire mesh cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23
- Humidity (%): 45-55
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 1978-06-21 To: 1978-09-25

Route of administration:

oral: feed

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: For the first week of preparation, P0475 was taken from the refrigerator and 75 grams was weighed out. 10 milliliters was reserved for analytical analysis. 12.5 grams of test substance solution was mixed wih 987.5 grams of feed for the desired 0.35% active level in the feed. For the second week preparation on, the preparation was altered due to the test substance concentration not being on target. Test substance was taken from the refrigerator and 75 grams was weighed out. 10 milliliters was reserved for analytical analysis. 12.95 grams of test substance solution was mixed wih 987.05 grams of feed for the desired 0.35% active level in the feed.


DIET PREPARATION
- Rate of preparation of diet (frequency): once a week
- Mixing appropriate amounts with (Type of food): Purina lab chow
- Storage temperature of food: refrigerated



VEHICLE
- Justification for use and choice of vehicle (if other than water): Distilled water
- Concentration in vehicle: N/A
- Amount of vehicle (if gavage): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test substance Analysis: Weekly throughout the study, representative 10 milliliter samples were collected from each prepared test diet. The 10 milliliter samples were analyzed by the Sponsor each week for test substance analysis.
Diet Analysis: Weekly throughout study, 10 gram sample of chow was collected and submitted to the Sponsor each week for analysis.

Duration of treatment / exposure:

P0475 in diet was administered for 6 weeks

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0.35% DDAO
Basis:
nominal in diet

No. of animals per sex per dose:

15

Control animals:

yes, concurrent no treatment

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: The study was extended beyond the original designated termination to allow the lenticular lesions to progressand another three weeks to allow for development of histopathological examinations.
- 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 (no data reported)
- Cage side observations checked in table [No.?] were included. N/A


DETAILED CLINICAL OBSERVATIONS: N/A
- Time schedule: N/A


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, weekly
- 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: Yes, weekly
- 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
- Time schedule for examinations: N/A


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Initiation and 2 week intervals
- Dose groups that were examined: All rats


HAEMATOLOGY: No data
- Time schedule for collection of blood: N/A
- Anesthetic 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: No data
- 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: No data
- 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: No data
- 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: No data
HISTOPATHOLOGY: Yes, only of the eyes specifically to evaluate the lenticular lesions was performed.

Other examinations:

N/A

Statistics:

Data from this study were analyzed by analysis of variance and distribution-free techniques.

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

effects observed, treatment-related

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

not specified

Ophthalmological findings:

effects observed, treatment-related

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY: N/A

BODY WEIGHT AND WEIGHT GAIN: Weekly cumulative body weight was statistically analyzed after Week 3 and Week 6. At week 3, statistically significant differences were noted between the average cumulative weight gain of treated males as compared to controls. However, there were no significant differences in the average weekly weight gain of treated males versus controls. The average weekly weight gain and the average cumulative weight gain of the females did not differ significantly from the female controls. At week 6, statistically significant differences were noted between the average weekly weight gain and the average cumulative weight gain of treated males as compared to controls. The average weekly weight gain and the average cumulative weight gain of the females did not differ significantly from the female controls.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Weekly cumulative food consumption was statistically analyzed after Week 3 and Week 6. At week 3, statistically significant differences were noted between the average cumulative food consumption of treated males as compared to controls. However, there were no significant differences in the average weekly food consumption of treated males versus controls. The average weekly food consumption and the average cumulative food consumption of the females did not differ significantly from the female controls. At week 6, statistically significant differences were noted between the average cumulative food consumption of treated males as compared to controls. However, there were no significant differences in the average weekly food consumption of treated males versus controls. The average weekly food consumption and the average cumulative food consumption of the females did not differ significantly from the female controls.

FOOD EFFICIENCY: Weekly feed efficiencies were statistically analyzed after Week 3 and Week 6. At week 3, statistically significant differences were noted between the average cumulative food efficiency of treated males as compared to controls. However, there were no significant differences in the average weekly food efficiency of treated males versus controls. The average weekly food efficiency and the average cumulative food efficiency of the females did not differ significantly from the female controls. At week 6, statistically significant differences were noted between the average weekly food efficiency and the average cumulative food efficiency of treated males as compared to controls. The average weekly food efficiency and the average cumulative food efficiency of the females did not differ significantly from the female controls.

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

OPHTHALMOSCOPIC EXAMINATION: Four of the 6 lenticular lesions observed in animals fed P0475 were opacities that extended from the hyaloid membrane to the posterior cortex of the lens. Two of the animals fed P0475 exhibited bilateral lenticular opacities.
Ophthalmocopic evaluations of animals through the termination of the study revealed the following incidences of lenticular lesions:
Controls: Males: 0/15, Females 0/15
P0475: Males: 4/15, Females 2/15

HAEMATOLOGY: N/A

CLINICAL CHEMISTRY: N/A

URINALYSIS: N/A

NEUROBEHAVIOUR: N/A

ORGAN WEIGHTS: N/A

GROSS PATHOLOGY: N/A

HISTOPATHOLOGY: NON-NEOPLASTIC: Eyes only. No information was obtained upon the examination of the lenticular opacities pertaining to the hyaloid membrane because these lesions were so minute that it could not be discerned in routine sectioning of the eye issue. The bilateral cataracts appeared histologically normal.

HISTOPATHOLOGY: NEOPLASTIC (if applicable): N/A

HISTORICAL CONTROL DATA (if applicable): N/A

OTHER FINDINGS: N/A

Dose descriptor:

NOAEL

Basis for effect level:

other: Lenticular lesions were observed in 4/15 males and 2/15 females exposed to P0475 in diet (0.35% commercial DDAO in diet) as compared with 0/15 males and 0/15 females on control diet.

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Critical effects observed:

not specified

Gonadal tissues were not evaluated grossly or histopathologically.

The primary lenticular lesions observed in this study (opacities that extend from the hyaloid membrane to the cortex)

were noted as not those previously seen with this test substance in previous studies. Previous studies have primarily shown bilateral lenticular opacities and this was only observed in 2/6 lesions associated with P0475 (total 6/30 animals exhibited lenticular lesions).

Conclusions:

The cataractogenic activity of 0.35% commercial dimethyl dodecyl amine oxide in feed was demonstrated.

Executive summary:

This subchronic rat feeding study was conducted to investigate the cataractogenic producing potential of various forms of the test substance in ground Purina Laboratory chow. Four groups of 30 rats (15 males and 15 females) were fed 0.35% commercial dimethyl dodecyl amine oxide (from P0475) for a period of 6 weeks. The original anticipated study duration was 8 weeks. At the end of week 6, the other test materials were depleted so at this time all rats were fed control diet until the completion of the ophthalmoscopic examinations at the termination of the study. The study was extended 2 weeks beyond designated termination to allow for lenticular lesions to progress and another 3 weeks for developmental of assays for histopathogical exams.

Body weight and food consumption were measured weekly while rats were on the test diet and observed daily for mortality and gross signs of toxicity. Ophthalmocopic examinations were performed on all rats prior to the initiation of the study and at approximately 2 -week intervals during the study. Two additional biomicroscopic exams were done to allow the lenticular lesions to progress to a point where they could be differentiated from the norm.

Body weight gain, feed consumption, and feed efficiency were statistically analyzed twice, once at week 3 and once at week 6.

Analysis of the male rat's third week cumulative data indicates that the rats receiving test diet had statistically significantly lower values than animals receiving control diet. However, statistical analysis of female rat's growth data for the same time period is not as well defined. There is no significant difference among the female rat's cumulative feed consumed. Statistical analysis of the sixth week growth data showed that the rats had no significant changes from week 3.

Weekly cumulative body weight, food consumption, and feed efficiencies were statistically analyzed after Week 3 and Week 6. At week 3, statistically significant differences were noted between the weekly cumulative weight gain, food consumption, and feed efficiencies of males fed P0475 versus controls. No statistical change versus control was noted in females for any of the parameters. At week 6, there were no significant changes from week 3.

Ophthalmoscopic exams revealed that incidences of lenticular lesions occurred for: Controls 0/15 males and 0/15 females, P0475 4/15 males and 2/15 females. Four of the 6 lenticular lesions in rats fed P0475 were opacitites that extended from hyaloid membrane to the posterior cortex of the lens. These lesions were not typical with test substance in previous studies. Only 2/6 rats fed P0475 exhibited bilateral lenticular opacitities typical of observed previously.

No gonadal examination information was available.

No histopathological information was obtained upon the exam of lenticular opacities pertaining to the hyaloid membrane because the lesions were so minute that it could not be discerned in routine sectioning of the eye tissue. The bilateral cataracts appeared histopathogically normal.

No NOAEL was identified in this study.

Reference 8

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1975-12-15 to 1976-09-02

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Special study for cataractogenic potential of DDAO. Two study phases were included in the report. Only cataractogenic effects, body weight, feed consumption, and feed efficiency were recorded. Not GLP. No no effect level was identified.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

N/A

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: Charles River CD, Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: N/A
- Age at study initiation: N/A
- Weight at study initiation: 40 to 60 grams Phase I and 149 to 288 grams Phase II
- Fasting period before study: N/A
- Housing: individually in stainless steel cages with wire mesh bottoms
- Diet: Purina Chow, ad libitum
- Water: fresh tap water, ad libitum
- Acclimation period:N/A


ENVIRONMENTAL CONDITIONS
- Temperature (°C): N/A
- Humidity (%): N/A
- Air changes (per hr): N/A
- Photoperiod (hrs dark / hrs light): N/A


IN-LIFE DATES: N/A

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:


DIET PREPARATION
- Rate of preparation of diet (frequency):two week intervals
- Mixing appropriate amounts with (Type of food): Purina Chow
- Storage temperature of food:N/A


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:

no

Duration of treatment / exposure:

30-weeks for Phase I
16-weeks for Phase II

Frequency of treatment:

daily, 7 days for 16 weeks Phase II and 30-weeks Phase I

Remarks:

Doses / Concentrations:
0.12%, 0.35%, 0.5% C-DDAO
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
0.35%, 0.4%, & 0.5% L-DDAO
Basis:
nominal in diet

No. of animals per sex per dose:

25 males and 25 females (control and C-DDAO) Phase I & II
20 males and 20 females (L-DDAO) Phase I
15 males and 15 females (L-DDAO) Phase II

Control animals:

yes, concurrent no treatment

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: weekly
- Cage side observations: gross abnormalities

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations:weekly

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

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

WATER CONSUMPTION AND COMPOUND INTAKE: No
- Time schedule for examinations:No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:4 week intervals throught study
- Dose groups that were examined: all, 0.12%, 0.35%, 0.5% C-DDAO and 0.35%, 0.4%, 0.5% L-DDAO

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

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

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

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

OTHER:N/A

Sacrifice and pathology:

GROSS PATHOLOGY: No
HISTOPATHOLOGY: No

Other examinations:

N/A

Statistics:

N/A

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

effects observed, treatment-related

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

not specified

Ophthalmological findings:

effects observed, treatment-related

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY: No mortalities reported, but no other data


BODY WEIGHT AND WEIGHT GAIN:
Mean body weight gain relative to controls at 16 wks:
0.35% Commercial DDAO = 71%
0.50% Commercial DDAO = 45%
0.35% Pure DDAO = 75%


FOOD CONSUMPTION AND COMPOUND INTAKE:
Mean amine oxide consumed (mg) in 112 days:
0.35% Commercial DDAO = 6236
0.50% Commercial DDAO = 8685
0.35% Pure DDAO = 7913


FOOD EFFICIENCY:
Calculated % food efficiency (males/females)
Control = 13.6/7.5
0.35% Commercial DDAO = 10.1/6/1
0.50% Commercial DDAO = 9.0/4.5
0.35% Pure DDAO = 11.1/4.0


WATER CONSUMPTION AND COMPOUND INTAKE:No data


OPHTHALMOSCOPIC EXAMINATION:
Incidence of cataracts relative to controls at 16 wks:
0.35% Commercial DDAO = 46%
0.50% Commercial DDAO = 66%
0.35% Pure DDAO = 10%


HAEMATOLOGY: No data


CLINICAL CHEMISTRY: No data


URINALYSIS: No data


NEUROBEHAVIOUR: No data


ORGAN WEIGHTS: No data


GROSS PATHOLOGY:No data


HISTOPATHOLOGY-NON-NEOPLASTIC: No data


HISTOPATHOLOGY- NEOPLASTIC: No data


HISTORICAL CONTROL DATA: No data


OTHER FINDINGS: No data

Dose descriptor:

NOAEL

Basis for effect level:

other: see 'Remark'

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Critical effects observed:

not specified

Gonadal tissues were not examined for gross pathology or histopathology for this study.

Conclusions:

No NOAEL/ LOAEL was identified since all doses affected body weight gain, food consumption, and feed efficiency.

Executive summary:

Two study phases were included in the report. Two levels of commericial DDAO and one level of laboratory prepared DDAO in diet were tested in Phase I for 30 weeks. Phase II included two levels of commercial DDAO (0.5% and 0.35% in feed) and one level of laboratory prepared pure DDAO (0.35% in feed) dosed for 16 weeks. In addition, a group of adult rats was treated with commercial DDAO in phase II at 0.5% in feed. Only cataractogenic effects, body weight, feed consumption, and feed efficiency were recorded. No NOAEL/ LOAEL was identified since all doses affected body weight gain, food consumption, and feed efficiency.

Reference 9

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1978-10-23 to: 1980-03-30

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented non-guideline study, evaluating one dose with 15 animals/sex. Not GLP.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Rats were dosed for 10 weeks via feed and evaluated for ocular toxicity. General toxicity parameters such as mortality clinical signs and body weight and food consumption were also evaluated.

GLP compliance:

no

Remarks:

N/A

Limit test:

no

Species:

rat

Strain:

other: Sprague-Dawley C.D.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratory, Wilmington, MA
- Age at study initiation: 28 days
- Weight at study initiation: 80-100 g
- Fasting period before study: N/A
- Housing: Housed individually during quarantine in wire mesh cages (59 inches long x 11 ¾ inches deep x 6 ¾ inches high with wire lid). After randomization, animals were individually housed in wire mesh compartmented cages, 5 males and 5 females per rack. Two empty compartments separated the sexes. The rack holders were equipped with an automatic water system. Cage racks and rack holders were cleaned weekly; drain pans were removed and cleaned daily. Animal rack holders were repositioned weekly on alternate sides of the room to minimize environmental variations in light intensity and exposure.
- Diet: Purina Rat Chow (Ralston Purina Company, St. Louis, MO) ad libitum
- Water: ad libitum
- Acclimation period: 14 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 70 ± 3C
- Humidity (%): 46 ± 9 %
- Air changes (per hr): N/A
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1978-10-23 To: 1979-01-05

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: 73.3 grams of the test substance was mixed with 7.9267 kg of Purina Rat Chow in a Hobart Blender for 5 minutes and then transferred to a 16 quart capacity Patterson-Kelly twin shell blender equipped with an intensifier bar and mixed for 8 minutes. Diets were mixed weekly and appropriate decontamination and cleaning procedures were maintained between batch preparations. Six kilogram quantities of the test diet were dispensed into feed cups (200 grams each) and were stored covered at room temperature. The remaining 2.0 kg of unused diet was stored refrigerated 6 +/- 2 degrees C in glass gallon containers. Feed samples were immediately placed into one-pint glass jars and stored at -10 to -20 degrees C.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Purina Rat Chow Meal (lot number September 25-782)
- Storage temperature of food: see above

VEHICLE
- Justification for use and choice of vehicle (if other than water): N/A
- Concentration in vehicle: N/A
- Amount of vehicle (if gavage): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A pre-study preparation of the test substance feed formulation was performed and a 100 gram sample of the test substance was taken and submitted to the sponsor for analysis. A 100 gram sample of the diet was removed from the top, middle and bottom portions of a 16 quart Patterson-Kelly twin shell blender and was sent to Procter and Gamble for analysis. Similar feed sampling procedures were performed for the test diet prepared for use during Study Weeks 1 and 2. Triplicate 100 gram diet samplings were taken from the middle blender position for each treatment diet prepared for Study Week 9. These samples and a 100 gram quantity of the test substance were sent to the sponsor. Additionally, a 100 gram sample obtained from the middle blender position was analyzed by the sponsor for each treatment diet prepared for Study Weeks 3 through 8 and 10 through 11. Control diet samples (100 gram) were submitted with all weekly test diet samples except during the pretest period when no control diet was prepared.

Duration of treatment / exposure:

10 weeks

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0.25 %
Basis:
nominal in diet

No. of animals per sex per dose:

15/sex/dose group

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: N/A
- Rationale for animal assignment (if not random): Randomized by weight and sex. Homogeneity of the groups was assured by using a computerized randomization procedure.
- 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
- Cage side observations checked: Viability checks were performed twice daily and animal displaying any symptoms of abnormality were recorded.

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 weekly diet consumption and reported in mean grams per week per sex: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: 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: Food efficiency was reported, but it was not reported in the study on how it was calculated.

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to study on quarantine day 13 and during study at weeks 6 and 10.
- Dose groups that were examined: all groups examined

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: N/A
- 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: N/A

Sacrifice and pathology:

GROSS PATHOLOGY: No; In general gross pathology was not performed. However if an animal was found dead or in a moribund condition it was euthanized and necropsied.
HISTOPATHOLOGY: No

Other examinations:

N/A

Statistics:

Statistical analyses were performed separately from each sex on weekly body weights, feed consumption, body weight gains and feed efficiency. Differences between groups were tested b y one-way analysis of variance (ANOVA). Specific treatments versus control group differences were determined by the least significant differences test (LSD). When significant heterogeneity of variance was shown across groups by the Bartlett test, overall group comparisons were made using the Kruskal-Wallis test, a non-parametric equivalent to the ANOVA. In this case, multiple treatments versus control group comparisons were made with a t-test using separate variance estimates for each group.
All analyses were performed on Battelle’s CDC 6500 and Cyber 73 computer systems using a set of packaged programs. The 95 percent level of significance was used in all test procedures.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

effects observed, treatment-related

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

not specified

Ophthalmological findings:

effects observed, treatment-related

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY-Observed abnormalities recorded for rats during the 10-week study were categorized into 4 major classifications as follows:
-ocular discharge: control-1 male (24 hour period) and 1 female (< 7 days); test substance-1 male (>30 days)and 2 females (<7 days and >7 days)
-ear tag inflammation: control-1 male and 2 females (all controls <7days); test substance- 1 male (<7 days) and 2 females (> 7 days)
-nasal discharge: control-0; test substance 3 males (2 for a 24 hour period and 1 for <7 day period)
-miscellaneous abrasions: contorl-0; test substance- 2 males (>7 days) and 1 female (<7days)
* numbers in parentheses above indicate the duration of abnormal observation.
Inflammation of short duration resulted from rat identification tags being caught on the wire mesh cages. The incidence and severity of the ocular and nasal discharge noted were not considered test substance related effects. Rats noted as having injuries or self inflicted trauma such as catching their teeth in the wire cages were classified as miscellaneous abrasions. One test substance female died as a result of cervical dislocation when a cage compartment cover was inadvertently closed on the animal. No other clinical observations were reported.

BODY WEIGHT AND WEIGHT GAIN- male mean body weights for the test substance group were significantly lower compared to controls by Study Week 1 and remained significantly reduced for the entire 10 week test period. Test substance females consistently displayed significantly lower average body weights compared to controls from Week 6 through Week 10. Sporadic decreases in average body weight gain were observed for male test substance group animals throughout the test period. A statistically significant decrease in total body weight gain compared to control was observed in the test substance males at study completion. Compared to controls, significantly lower body weight gains were displayed in the test substance females at Weeks 6. The test substance females experienced a negative mean body weight gain. At Week 7, the test substance females displayed a significantly increased mean body weight gain. Compared to the males, the females were less affected with respect to body weight gain. Compared to the controls, the percentage body weight reductions for the male test substance group at Week 10 was 7.53 % and the females was 7.69 %

FOOD CONSUMPTION AND COMPOUND INTAKE - Feed consumption estimates for the test substance groups were decreased at Week 7 compared to controls. Test substance males had significantly decreased mean feed consumption estimates compared to control at Week 8. There was a general reduction of weekly feed consumption of the test substance females compared to the control. Depressions in body weight or significant variations in body weight gain did not correlate closely with the over all feed consumption patterns displayed for either male or female test substance animals. A general trend in male feed consumption were not evident by graphic interpretation.

FOOD EFFICIENCY- Males had significantly lower feed efficiency values at weeks 1, 2 and 6 when compared to the controls. Females had significantly lower feed efficiency values at weeks 6 and 7 when compared to the controls. The sporadically occurring incidence of group significance with respect to feed efficiency was primarily due to minor fluctuations in the weekly mean body weight gain for control animals. An example of this was seen during Weeks 6 and 7 in the female groups. During the study some animals repeatedly spilled their feed cups and others wasted large quantities of their weekly ration. Feed consumption estimates for these animals were excluded from the data on those weeks when excessive feed loss was noted. Due to the fluctuations of the feed consumed data, no definitive conclusion regarding test substance effects upon feed efficiency could be presented.

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

OPHTHALMOSCOPIC EXAMINATION- Mild to moderate dacryoadenitis was the predominate abnormality observed in the animals at the 6-week examination period. Dacryoadenitis was not considered a test substance effect. Comments with respect to embryonal, fetal or nuclear opacities were not considered test substance related abnormalities. It was concluded from the results that ophthalmic lesions were not produced by the test substance during the first 6 weeks. At the 10 week examination, no treatment related ophthalmologic abnormalities were observed for rats fed the control diet. Ophthalmopathy (lenticular opacity) was observed for 2 females in the test substance group. These ocular abnormalities were considered to be treatment related effects.

HAEMATOLOGY-N/A

CLINICAL CHEMISTRY-N/A

URINALYSIS-N/A

NEUROBEHAVIOUR-N/A

ORGAN WEIGHTS-N/A

GROSS PATHOLOGY-N/A

HISTOPATHOLOGY: NON-NEOPLASTIC-N/A

HISTOPATHOLOGY: NEOPLASTIC (if applicable)-N/A

HISTORICAL CONTROL DATA (if applicable)-N/A

OTHER FINDINGS-N/A

Dose descriptor:

LOEL

Effect level:

0.25 other: %

Sex:

male/female

Basis for effect level:

other: Based on reductions in body weight for both sexes and lenticular opacities in female animals.

Critical effects observed:

not specified

N/A

Conclusions:

Sprague-Dawley rats were evaluated for 10 weeks to evaluate the ophthalmic effects induced by C10 -C16 alkyldimethyl, N-Oxide when fed in a diet containing nominal 0.25% test substance (TSIN=P0543). Rats were ophthalmoscopically examined prior to the study initiation and at study Weeks 6 and 10. Body weight, body weight gain, and feed consumption were measured and statistically analyzed as well. 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 gainds in both sexes.

Executive summary:

Sprague-Dawley C.D. rats, 15 males and 15 females per group (28 days of age at study initiation and weighing 80 -100 g), were evaluated in this 10 week oral feed study to evaluate the ophthalmic effects induced of C10 -C16 alkyldimethyl, N-Oxide. The dose groups consisted of a plain diet control and a diet with 0.25% the test substance . Rats were ophthalmoscopically examined prior to the study initiation and at study Weeks 6 and 10. Body weight, body weight gain, and feed consumption were measured and statistically analyzed as well.

Diets were prepared, sampled and analyzed weekly to assure proper concentrations and mixing of the test substance. Values obtained were within normal limits.

Male mean body weights for the test substance group were significantly lower compared to controls by Study Week 1 and remained significantly reduced for the entire 10 week test period. Females dosed with the test substance consistently displayed significantly lower average body weights compared to controls from Week 6 through Week 10. Sporadic decreases in average body weight gain were observed for male test substance group throughout the test period. A statistically significant decrease in total body weight gain compared to control was observed in the test substance males at study completion. Compared to controls, significantly lower body weight gains were displayed in the test substance females at Weeks 6. The test substance females experienced a negative mean body weight gain. At Week 7, the test substance females displayed a significantly increased mean body weight gain. Compared to the males, the females were less affected with respect to body weight gain. Compared to the controls, the percentage body weight reductions for the male test substance group at Week 10 was 7.53 % and the females was 7.69 %.

Feed consumption estimates for the test substance groups were decreased at Week 7 compared to controls. Test substance males had significantly decreased mean feed consumption estimates compared to control at Week 8. There was a general reduction of weekly feed consumption of the test substance females compared to the control. Depressions in body weight or significant variations in body weight gain did not correlate closely with the over all feed consumption patterns displayed for either male or female test substance animals. A general trend in male feed consumption were not evident by graphic interpretation.

Males had significantly lower feed efficiency values at weeks 1, 2 and 6 when compared to the controls. Females had significantly lower feed efficiency values at weeks 6 and 7 when compared to the controls. The sporadically occurring incidence of group significance with respect to feed efficiency was primarily due to minor fluctuations in the weekly mean body weight gain for control animals. An example of this was seen during Weeks 6 and 7 in the female groups. During the study some animals repeatedly spilled their feed cups and others wasted large quantities of their weekly ration. Feed consumption estimates for these animals were excluded from the data on those weeks when excessive feed loss was noted. Due to the fluctuations of the feed consumed data, no definitive conclusion regarding test substance effects upon feed efficiency could be presented.

Mild to moderate dacryoadenitis was the predominate abnormality observed in the animals at the 6-week examination period. dacryoadenitis was not considered a test substance effect. Comments with respect to embryonal, fetal or nuclear opacities were not considered test substance related abnormalities. It was concluded from the results that ophthalmic lesions were not produced by the test substance during the first 6 weeks. At the 10 week examination, no treatment related ophthalmologic abnormalities were observed for rats fed the control diet. Opththalmopathy (lenticular opacity) was observed for 2 females in the test substance group. These ocular abnormalities were considered to be treatment related effects.

Based on the results the LOEL was determined to be 0.25 % alkyl dimethyl amine oxide in diet based on decreased body weight and body weight gain and lenticular opacities in female animals.

Reference 10

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1979-10-19 to 1980-05-02

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

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 or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. Wilmington Mass.
- Age at study initiation: four weeks old
- Weight at study initiation: males ranged from 167.6 to 231.1 grams, females ranged from 124.2 to 173.5 grams.
- Fasting period before study: N/A
- Housing: housed individually in elevated stainless steel wire-mesh cages
- Diet: ad libitum ( NIH-07 powdered diet from Ziegler Bros. Inc.
- Water: automated system; ad libitum
- Acclimation period: N/A


ENVIRONMENTAL CONDITIONS
- Temperature (°C): N/A
- Humidity (%): N/A
- Air changes (per hr): N/A
- Photoperiod (hrs dark / hrs light): N/A


IN-LIFE DATES: From: June 29, 1978 To: October 4, 1978

Route of administration:

oral: feed

Vehicle:

other: feed: NIH-07 powdered diet from Ziegler Bros. Inc.

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The appropriate amount of test substance needed to obtain the desired dose level was added to the basal diet on a weight per weight basis in the following manner: The amount of test substance required for each test level was weighed on a Toledo scale and premixed in a Hobart mixer with 2 kg of the basal diet for five minutes. The premix was then sifted, using a fine mesh screen, to remove the lumped compound. The retrieved compound was then ground into a fine powder in a Waring blender for one minute and then placed in the Twin-Shell blender fitted with an intensifier bar. The test substance was mixed for one minute per kg. Fresh diets were prepared weekly (from the same batch of test substance and the same lot of feed) and were stored at 40 degree F +- 4 degree F in a one gallon glass jar.


DIET PREPARATION
- Rate of preparation of diet (frequency): once a week
- Mixing appropriate amounts with (Type of food): NIH-07 powdered diet purchased from Ziegler Bros. Inc.
- Storage temperature of food: 40 degree F +- 4 degree F


VEHICLE
- Justification for use and choice of vehicle (if other than water): N/A
- Concentration in vehicle: The appropriate amount of test substance needed to obtain the desired dose level was added to the basal diet on a weight per weight basis.
- 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:

Prior to initiation of the study, full size batches of feed containing the appropriate concentrations of test substance were prepared. After preparation, two 100 gram aliquots of prepared test substance were sampled from the top, middle, and bottom of each batch, placed in sealed glass containers, and sent, packed in dry ice, immediately to the sponsor for homogeneity analysis and verification of the test substance. The remainder of these batches was stored at 40 degree F +- 4 degree F. After 1, 2, 4, 8, and 12 weeks, the batches were again sampled in duplicate 100 g and one set of the duplicate diet samples were sent to the sponsor in sealed glass containers for stability analyses.

After initiation of the study, a sample (50 grams) of all freshly prepared diets were sent to the sponsor in sealed glass containers for verification of the test substance. The sponsor analyzed randomly chosen feed samples prepared for each study group for test substance concentration.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

continuous

Remarks:

Doses / Concentrations:
0, 0.1, 0.2, 0.4 % AO
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
0, 88, 176, 352 mg AO/kg bw/day
Basis:
nominal in diet

No. of animals per sex per dose:

twenty rats/sex/dose

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: once in the morning and once in the evening on weekdays, and twice daily on weekends.
- Cage side observations checked in table [No.?] were included. N/A


DETAILED CLINICAL OBSERVATIONS: N/A
- Time schedule:


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, weekly
- 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: Yes, weekly
- 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
- Time schedule for examinations: N/A


OPHTHALMOSCOPIC EXAMINATION: N/A
- Time schedule for examinations: N/A
- Dose groups that were examined: N/A


HAEMATOLOGY: Yes
- Time schedule for collection of blood: weeks 7 and 13
- Anesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all rats
- Parameters checked in table [No.?] were examined. N/A


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: weeks 7 and 13
- Animals fasted: No data
- How many animals: all rats
- Parameters checked in table [No.?] were examined. N/A


URINALYSIS: Yes
- Time schedule for collection of urine: at week 6 and 13: Urine was collected every thirty minutes for a period of six hours until a sufficient quantity of urine was collected from each animal to perform all the urinalyses. If insufficient urine was collected during the sic hour period urinalysis was not conducted for that animal.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters checked in table [No.?] were examined. N/A


NEUROBEHAVIOURAL EXAMINATION: No data
- 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:
Organ weights were determined for the following tissues: brain, heart, liver, kidneys, ovaries, testes with epididymides.

The following tissues were removed, trimmed of excess fat and preserved in neutral buffered formalin: skin, gross lesions, mandibular lymph node, mesenteric lymph node, mammary gland, salivary gland, stomach, small intestine, liver, pancreas, spleen, kidneys, adrenals, urinary bladder, prostate separate from bladder (males), right femur, thymus, colon, cecum, rectum, thyroid, parathyroid, esophagus, seminal vesicles, testes (male), ovaries (females), uterus (females), brain, treches, lungs, and mainstream bronchi, heart, pituitary, eyes, sciatic nerve, thigh muscle, costochondral junction, spinal cord, and larynx.

HISTOPATHOLOGY: Yes:
The preserved tissues from the control animals, the mid-dose females, and ten rats from from the high dose group (both sexes) were embedded in paraplast, sectioned, stained with Hematoxylin and Eosin, and examined microscopically.

Other examinations:

N/A

Statistics:

Statistical analysis using both the analysis of variance and the distribution-free techniques were performed separately for each sex on the following values: body weight, food consumption, weight gain, feed efficiency, hematology, clinical chemistry, organ weights and organ to body weight ratios. Provided that Bartlett's test of homogeneity of variance was not significant, comparisons with the control group were based upon the least significant difference criterion. If Bartlett's test was significant, Wilcoxon's rank sum test was used. Regression analysis, using dose as the independent variable, was performed. All statistical tests were based upon 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:

effects observed, treatment-related

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

not examined

Ophthalmological findings:

effects observed, treatment-related

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: One mid-dose male died during week 9 of the study. This animal appeared normal prior to death. No compound-related clinical signs were noted in the animals that survived the study.

BODY WEIGHT AND WEIGHT GAIN: Inspection of the mean body weight data revealed lower mean body weights for all compound-treated male Groups and for the mid and high dose females from week 1 through week 13 when compared to the respective control groups. These lower body weights appear to be dose-related. Statistical analysis revealed statistically significant lower mean body weights for the high dose males and females from weeks 1 through 13; and for the mid dose females from weeks 3 to 5 and 9 to 13.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Inspection of the food consumption data of the mid and high dose males revealed a lower mean food consumption at weeks 3 through 13 when compared to control, which appears to be dose-related. Statistical analysis revealed statistically significant lower mean food consumption for low dose males at weeks 6 and 13, mid dose males at week 2, 6, 11, 12, and 13, and high dose males at weeks 8, 9, 11, 12, and 13. In addition, a lower mean food consumption in females was noted for the mid dose group at weeks 2 through 7 and weeks 9 through 13 when compared to control. Statistical analysis revealed statistically significant lower mean food consumption for the mid dose females at weeks 3, 9, 11, and 12, and high dose females at weeks 11 and 12.

FOOD EFFICIENCY: Statistical analysis revealed statistically significant lower mean feed efficiencies for high dose males at weeks 1, 2, and 8. Statistical analysis revealed statistically significant lower mean feed efficiencies for the low dose females at weeks 1 and 7, mid dose females at weeks 1, 9, and 12, and high dose females at weeks 1, 2, 9, and 12.

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

OPHTHALMOSCOPIC EXAMINATION: An ophthalmoscopic examination was performed (two days after randomization) on all animals and on animals used for replacements. Fourteen of the randomized animals had unusual or abnormal ocular lesions and were discarded on the recommendation of the ophthalmologist. The discarded animals were replaced with retained animal that had normal eyes. Lenticular opacities pertaining to the posteror cortex of the lens were present in 3/20 males and 3/20 females at 6 weeks and 12/20 males and 8/20 females in the high dose group. Lenticular opacities pertaining to the posteror cortex of the lens were present in 1/20 males at 6 weeks and 3/19 males at 13 weeks.

HAEMATOLOGY: Week 7 hematology values were not compared to the week 13 values since the blood samples were collected by different methods at each interval. In week 7, there were higher mean erythrocyte counts in the Low, mid and high dose males and in the low and mid dose males (statistically significant mid dose females only) and lower mean leukocytes counts in the low and high dose males (statistically significant in high dose males only) and in the low, mid and high dose females (statistically significant in low and high dose females only), when compared to the respective control values. Although there were instances of increased and decreased mean hematology values, all changes were minimal and all values were within acceptable laboratory limits. Therefore, no biological significance was attributed to these alterations.

CLINICAL CHEMISTRY: In week 7 there were lower glutamic pyruvic transaminase levels in the low, mid and high dose males (statistically significant high dose males only). For the females at week 7, lower mean serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase levels were noted in all dose groups (statistically significant the mid and high dose groups only); a lower mean blood urea nitrogen level was noted in the mid dose group; and higher mean fasting glucose levels were noted in the low, mid and high dose groups (statistically significant mid dose females only); lower serum glutamic oxaloacetic transaminase (statistically significant mid dose males and high dose females only) and serum glutamic pyruvic transaminase (statistically significant high dose males and all female dose groups). In addition, at week 13, higher mean alkaline phosphates levels were noted in all male dose groups and the mid and high female dose groups (statistically significant high dose females); a higher mean blood urea nitrogen level was noted in the the low, mid and high dose males and in the mid and high dose females (statistically significant high dose males and mid dose females only) when compared to the respective control values. As this elevated enzyme level was not corroborated by increased liver weight or histopathology it is not considered toxicologyically relevant.

URINALYSIS: Urinalysis was unremarkable at weeks 6 and 13.

NEUROBEHAVIOUR: N/A

ORGAN WEIGHTS: Inspection of the terminal body weight and organ weight data of the compound-treated males compared to the control males revealed a lower mean terminal body weight in the high dose males; a higher mean relative testes weight in the high dose (statistically significant mid and hogh dose males); a higher mean relative brain weight in the high dose (statistically significant mid and high dose males); and a higher mean relative kidney weight in the high dose(statistically significant Groups low and high dose males).

For the females, inspection of the terminal body weight and organ weight data revealed higher mean relative heart weight in the high dose (statistically significant Group mid and high dose females).

Differences in the organ weight data of the treated groups when compared to the control were noted only in mid- and high-doses, and were not always consistent between sexes. Therefore, the biological significance of these findings cannot be determined.

GROSS PATHOLOGY: No distinct compound-related gross pathological findings were noted in either the compound-treated males or females. Gross pathological findings noted in the mid dose male that died during Week 9 were a few small dark red areas on all lobes of the lungs and a smooth stomach lining.

HISTOPATHOLOGY: NON-NEOPLASTIC: microscopic examination of tissues from the control rats and mid dose female rats failed to reveal any compound-induced alterations. Spontaneous disease lesions observed in the sixty rats examined were of the usual numbers and type seen in rats of this age and strain and were essentially comparable in frequency in control and treated groups.

Microscopic examination of tissues from ten male and ten female rats in the high dose group failed to reveal any compound-induced alterations. Spontaneous disease lesions observed in these twenty rats were of the usual type seen in rats of this age and strain and were essentially comparable in frequency in the control and treated groups.

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: No significant decreases in body weight or lenticular lesions at this dose level.

Dose descriptor:

NOAEL

Effect level:

88 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Dose descriptor:

LOAEL

Effect level:

0.2 mg/kg diet

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Lenticular lesions in high dose females and mid and high dose males and significantly decreased body weights in mid and high dose females and high dose males.

Dose descriptor:

LOAEL

Effect level:

176 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Critical effects observed:

not specified

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

Wasting of feed was noted for the high-dose males and females during the initial weeks of the study and for two low dose females at week 8 and one mid dose female at week 11.

Conclusions:

The test substance induced decreased body weights and lenticular lesions at dose levels of 0.2% and above in the diet when administered to the rat for 91 days. The NOAEL was determined to be 0.1% in the diet.

Executive summary:

The objective of this study was to establish the dose levels of alkyl dimthyl amine oxide that would likely to be maximum tolerated levels throughout a two-year chronic feeding study in rats.

One hundred sixty cesarean derived, Sprague-Dawley rats were randomly assigned to four study groups (20 rats/sex/group). Group 2, 3, and 4 received 0.1%, 0.2%, and 0.4%, respectively, of the test substance blended in NIH-07 powdered diet. Group 1 received NIH-07 powdered diet only, and served as the control group. The appropriate diet and water were available to the rats ad libitum until sacrifice.

Criteria evaluated for compound-effect were appearance, behavior, mortality, moribundity, body weights, food consumption, feed efficiency, terminal body weights, organ weights, and organ/body weight ratios. In addition, the following clinical laboratory studies were performed: Hematology (weeks 7 and 13) - hematocrit, hemoglobin, erythrocyte counts, and total and differential leukocyte counts; Clinical Chemistry (weeks 7 and 13) - alkaline phosphatase, blood urea nitrogen, fasting glucose, glutamic pyruvic transaminase, and glutamic oxaloacetic transaminase; Urinalysis (weeks 6 and 13) - appearance and color, pH, specific gravity, glucose, bilirubin, occult blood, albimin, and microscopic examination of the sediment. In addition, ophthalmoscopic examinations were performed by a DVM ophthalmologist contracted by the sponsor.

The NIH-07 basal feed and the laboratory water were analyzed for contaminants by outside laboratories. The diet was analyzed for homogeneity, stability, and verification of the test material by the sponsor.

The sponsor statistically analyzed the following data: body weight changes, total food consumption, feed efficiency, hematology and clinical chemistry data, organ weights, and organ/body weight ratios.

Inspection of the mean body weight data revealed lower body weights for all compound-treated male groups and for the mid and high dose, when compared to the respective control group. These lower body weights in the compound-treated groups appear to be dose-related. Lower mean food consumption values, which appear to be dose-related, were noted in the mid and high dose males when compared to the control group from week 3 through week 13. In addition, the mean food consumption values of the low dose males for weeks 1 and 2, weeks 5 through 8, and weeks 11 through 13 were lower than the control values. For the females, there was a higher mean food consumption in the high dose group at weeks 1 though 8 when compared to the control, which could be attributed to the wasting of feed. In addition, lower mean food consumption values were noted for mid dose females at weeks 2 through 7 and weeks 9 through 13 when compared to control. At week 6, the mean food consumption values for all males and female groups (control and compound-treated) were decreased from the respective values of the previous week, probably due to the stress caused by the ophthalmoscopic examination and the blood and urine collections. In addition, between weeks 5 and 6, the mean body weight gains were much less for all male and female groups than for the preceding weeks, probably as a result of the lower food consumed due to stress. Between weeks 6 and 7, and weeks 7 and 8, the mean body weight gains increased somewhat for all male and female groups, but subsequently reached plateaus and declined throughout the remaining weeks of study.

Decreased feed efficiencies were noted for all male and female groups at week 6 and are also probably related to the ophthalmoscopic examination and the blood and urine collections. The feed efficiency values increased somewhat for all male and female groups at weeks 7 and 8, but reached plateaus and declined until week 13.

Blood was collected for the hematology and clinical chemistry determination by segmental tail amputation at week 7 from the abdominal aorta at terminal sacrifice at week 13. Because the collection methods differed, the week 7 data were not compared to the week 13 data. However, comparisons were made at each collection interval between the mean values of the compound-treated groups and the control. Elevated alkaline phosphatase values were noted in all treatment males and the mid and high dose females at weeks 7 and 12, and may be compound-related. However there were no gross or histopathological findings to support any toxicological significance to these findings. No other differences noted between the treated and control males and females in the clinical chemistry or hematology date are considered to be of biological significance.

Urinalyses were unremarkable at week 7 and week 13.

Gross pathological findings noted in the mid dose male that died during Week 9 were a few small dark red areas on all lobes of the lungs and a smooth stomach lining. No distinct compound-related gross pathological findings were noted for any of the remaining compound-treated animals (male or female).

Differences were noted in the organ weight data for the mid and high dose males compared to control data and may be attributed to the lowercontrol food consumption values of the groups.

Differences observed in the organ weight data of the mid and high dose were considered to be incidental. No distinct differences were noted in the low dose males or females compared to the control. The biological significanc of the organ weight changes could not be determined.

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 and above when administered to the rat for 91 days. The NOAEL was determined to be 0.1% in the diet (presumed based on active level) 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/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

Reference 1

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.

Qualifier:

equivalent or similar to guideline

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 or test system 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. 

Reference 2

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Preliminary toxicity studies to obtain skin irritation and mortality data on the test substance. In the first study, four dose levels were tested for each test substance with 15 males and 15 females at each dose level. Animals received 5 to 7 applications. Based on the results of the first study, three lower dose levels (n=15/sex/dose) were tested in the second study. Animals in the second study received 20 applications during a 4-week period. Only clinical observations, body weight, and mortality were recorded. Not GLP.

Qualifier:

no guideline followed

Principles of method if other than guideline:

N/A

GLP compliance:

no

Limit test:

no

Species:

mouse

Strain:

other: Charles River ICR Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: N/A
- Age at study initiation: N/A
- Weight at study initiation: 23 to 29.7 grams
- Fasting period before study: N/A
- Housing:N/A
- Diet (e.g. ad libitum): N/A
- Water (e.g. ad libitum):N/A
- Acclimation period:N/A


ENVIRONMENTAL CONDITIONS
- Temperature (°C): N/A
- Humidity (%): N/A
- Air changes (per hr): N/A
- Photoperiod (hrs dark / hrs light): N/A


IN-LIFE DATES: N/A

Type of coverage:

not specified

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST SITE
- Area of exposure: N/A
- % coverage: N/A
- Type of wrap if used: N/A
- Time intervals for shavings or clipplings: once prior to study and once weekly for 4 weeks


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):0.5%, 1.0%, 2.0%, 5%, 10%, 15%, and 20%
- Constant volume or concentration used: yes
- For solids, paste formed: no


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


USE OF RESTRAINERS FOR PREVENTING INGESTION: no

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

N/A

Duration of treatment / exposure:

4 weeks / dermal for both studies

Frequency of treatment:

daily, for 5 days each week for both studies

Remarks:

Doses / Concentrations:
0.5%, 1.0%, 2.0%, 5%, 10%, 15%, and 20% UDL-1379
Basis:
other: percent DDAO in solution

Remarks:

Doses / Concentrations:
0.5%, 1.0%, 2.0%, 5%, 10%, 15%, and 20% UDL-1380
Basis:
other: percent DDAO in solution

No. of animals per sex per dose:

15 males and 15 females

Control animals:

yes, concurrent no treatment

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: daily
- Cage side observations checked:skin irritation, unkempt fur, arched spine, scaling, and scabs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

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

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

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

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: No

WATER CONSUMPTION: No
- Time schedule for examinations:No

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

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

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

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

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

OTHER:N/A

Sacrifice and pathology:

GROSS PATHOLOGY: No
HISTOPATHOLOGY: No

Other examinations:

N/A

Statistics:

N/A

Clinical signs:

effects observed, treatment-related

Dermal irritation:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY: Study 1- After five dermal applications all of the animals at the 5% and 10% dose levels of both test substances UDL-1379 and UDL-1380 showed slight to moderate irritation. The skin irritation was so severe at dosage levels of 15% and 20% for both test substances that the dermal applications were discontinued. After two additional dermal applications (total of 7 applications) all of the animals at the 5% and 10% dose levels of both test substances showed severe irritation and other signs of compound toxicity including arched spine and unkempt fur. Due to the severity and high incidence of the observations, dermal applications were discontinuted in these groups. Scar tissue was observed on 7 animals at 10%, 21 at 15%, and 19 at 20% for test substance UDL-1379 and 3 at 5%, 20 at 10%, 23 at 15%, and 23 at 20% for test substance UDL-1380.

None of the control group died during the study. The following mice died: 1 at 5%, 7 at 10%, 9 at 15%, and 11 at 20% for test substance UDL-1379 and 3 at 5%, 8 at 10%, 7 at 15%, and 7 at 20% for test substance UDL-1380.

Study 2- After four weeks of the dermal applications the control group, three out of thirty animals were observed with unkempt fur. For the 0.5% UDL-1379 test group two out of thirty animals were observed with unkempt fur. Scabs on the dorsal skin and unkempt fur were observed in the remaining test groups (1.0% & 2.0% UDL-1379 and 0.5%, 1.0% & 2.0% UDL-1380).

None of the control group or 0.5% and 1.0% test groups for UDL-1379 and UDL 1380 died during the study. The following animals died: 1 at 2.0% for test substance UDL-1379 and 4 at 2.0% for test substance UDL-1380.

BODY WEIGHT AND WEIGHT GAIN: Study 1- After the first week the negative control group gained weight and all of the test groups lost weight. Over the next two weeks there was a slight weight gain in the negative control and test groups.

Study 2- After the first week the negative control group and test groups gained weight except for the male animals in group 6 (1.0% UDL-1380). Over the next three weeks all groups had a slight weight gain.

FOOD CONSUMPTION: No data

FOOD EFFICIENCY: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: No data

CLINICAL CHEMISTRY: No data

URINALYSIS: No data

NEUROBEHAVIOUR: No data

ORGAN WEIGHTS: No data

GROSS PATHOLOGY: No data

HISTOPATHOLOGY- NON-NEOPLASTIC: No data

HISTOPATHOLOGY - NEOPLASTIC: No data

HISTORICAL CONTROL DATA: No data

OTHER FINDINGS: No data

Dose descriptor:

LOEL

Effect level:

2 other: %

Sex:

male/female

Basis for effect level:

other: UDL 1379 - induced minimal effects on the dorsal skin and possibly the death of one animal. UDL1380 - caused mortality in 4 mice and some dermal and systemic effects in 10/30 mice

Dose descriptor:

NOAEL

Effect level:

1 other: %

Sex:

male/female

Basis for effect level:

other: UDL 1379 and UDL 1380 - Scabs on the dorsal in 2/30 animals were identified from each group but this was not considered significant.

Critical effects observed:

not specified

Gonadal tissues were not examined for gross pathology or histopathology for this study.

Conclusions:

Based on the observations of compound effects induced at dosage levels of 5%, 10%, 15%, and 20% of UDL-1379 and UDL-1380 it was concluded that a second study would be performed using lower concnetrations of each compond. The second study indicated that UDL-1379 at concentrations of 2.0% induced minimal effects in the treatment area of dorsal skin. The death of one animal in this group may have been caused by the compound. UDL-1380 at concentrations of 2.0% caused mortality in 4 mice and some dermal and systemic effects in 10 of 30 mice. UDL 1379 - induced minimal effects on the dorsal skin and possibly the death of one animal. A LOEL of 2% and a NOAEL of 1% was determined for the study.

Executive summary:

This preliminary study was conducted to obtain toxicity and mortality data on two test substances (UDL-1379 and UDL-1380, dodecyl dimethyl amine oxide) for use in setting dose levels in a subsequent chronic study. In the first study the test substances were administered dermally to four groups of Charles River ICR Swiss mice at dosage levels of 5%, 10%, 15%, and 20% dodecyl dimethyl amine oxide for three weeks. Based on the severity and high incidence observations from the first study a second study was conducted with three groups of Charles River ICR Swiss mice at dosage levels of 0.5%, 1.0%, and 2.0% for four weeks.

The parameters monitored were body weight, mortaility, and clinical observations. In the first study, after the first week the negative control group gained weight and all of the test groups lost weight. Over the next two weeks there was a slight weight gain in the negative control and all of the test groups. After five dermal applications all of the animals at the 5% and 10% dose levels of both test substances UDL-1379 and UDL-1380 showed slight to moderate irritation. The skin irritation was so severe at dosage levels of 15% and 20% for both test substances that the dermal applications were discontinued. After two additional dermal applications (total of 7 applications) all of the animals at the 5% and 10% dose levels of both test substances showed severe irritation and other signs of compound toxicity including arched spine and unkempt fur. Due to the severity and high incidence of the observations, dermal applications were discontinuted in these groups. Scar tissue was observed at dosage levels 10%, 15%, and 20% for test substance UDL-1379 and all dosage levels for test substance UDL-1380. None of the control group died during the study. Several mice died at all dosage levels for both test substances.

In the second study, after the first week the negative control group and test groups gained weight except for the male in group 6 (1.0% UDL-1380). Over the next three weeks all groups had a slight weight gain. After four weeks of the dermal applications the control group, three out of thirty animals were observed with unkempt fur. For the 0.5% UDL-1379 test group two out of thirty animals were observed with unkempt fur. Scabs on the dorsal skin and unkempt fur were observed in the remaining test groups (1.0% & 2.0% UDL-1379 and 0.5%, 1.0% & 2.0% UDL-1380). None of the control group or 0.5% and 1.0% dose UDL-1379 and UDL 1380 test groups died during the study. One animal died at 2.0% for test substance UDL-1379 and 4 at 2.0% for test substance UDL-1380. The second study indicated that UDL-1379 at concentrations of 2.0% induced minimal effects in treatment area of dorsal skin. The death of one animal in this group may have been caused by the compound. UDL-1380 at concentrations of 2.0% caused mortality in 4 mice and some dermal and systemic effects in 10 of 30 mice.

A LOEL of 2% and a NOAEL of 1% dodecyl dimethyl amine oxide was determined for the study.

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.

Qualifier:

equivalent or similar to guideline

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 or test system 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:

No repeated dose oral toxicity studies are available for C14 -16 AO. Data are read across from studies performed using category members C12 -14 AO and C12 -18 AO.

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 (equivalent to 440 mg AO/kg bw/day) 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. Regardless, 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 an administered dietary dose of 88 mg AO/kg bw/day.

In a supporting study (004) [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 (in 23/79 animals) were observed at dose levels of 0.2% in the diet (equivalent to 176 mg AO/kg bw/day) 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 an administered dietary dose of 88 mg AO/kg bw/day.

In a supporting study (005) [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 single dose level of 0.25% for 10 weeks. Rats were evaluated for general toxicity and ophthalmoscopically examined prior to the study initiation and at study Weeks 3, 6 and 10. The administered dose was the LOEL ( 0.25 % in diet, approximately equivalent to 200 mg AO/kg bw/day) based on lenticular opacities observed ophthalmologically in 2/30 (both female) animals and decreased body weights and body weight gains in both sexes.

In a supporting study (007) [Procter & Gamble (1978b)] 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 (approximately equivalent to 300 mg AO/kg bw/day) in feed was demonstrated in 6/30 animals. No NOAEL was identified in this study.

In a supporting study (008) [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. Ophthalmology examinations were performed at 16 weeks in animals receiving the commercial grade material, and 85/150 animals exhibited cataracts at dosages of≥ 0.35% in diet (approximately equivalent to ≥ 300 mg/kg/day). No NOAEL was identified since all doses affected body weight gain, food consumption, and feed efficiency.

In a supporting study (006) [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 haemogram (haematocrit, 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. Ophthalmology was evaluated in control, low and mid dose animals that survived to 32 weeks.  In the mid dose group of 0.5% (equivalent to 160 mg AO/kg bw/day), the incidence of cataracts was the same as in the control group. The incidence in the low dose group was less than in controls. 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 subacute study (002) [Ceccatelli R (2008) ] rats (HanRcc: WIST(SPF)) were dosed by oral gavage with the test substance (an aqueous solution of C12 -14 AO) at levels of 40, 100 and 250 mg AO/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 AO/kg bw/day, total locomotor activity was statistically significantly reduced in females. At 100 mg AO/kg bw/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 bw/day. The NOEL for reproduction/developmental toxicity was considered to be 100 mg AO/kg/day. No observations of lenticular lesions or cataracts were noted in this oral gavage study in rats.

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. No observations of lenticular lesions or cataracts were noted in this oral gavage study in rats.

Repeated dose toxicity dermal:

There are two repeated dose dermal toxicity studies available for category member C12 -14 AO. 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 classification or non-classification

The critical effects identified in the oral dietary repeat dose toxicity studies were reduced body weight gain in rats and rabbits, and eye lesions in rats. Additional details on the eye lesions are detailed in the table below.

Study Description	Methods	Results	NOAEL (mg/kg bw/day	LOAEL (mg/kg bw/day)
Subchronic 13-week oral (dietary) study in rats 0, 0.02, 0.1 and 0.5% AO (nominal in diet), equivalent to 0, 17.6, 88 and 440 mg AO/kg bw/day (actual ingested Hazelton Laboratories, Inc. (1974) 001 Key study	n = 20/sex/group 5/sex/group sacrificed/evaluated at 4 weeks. Ophthalmology performed at 13 weeks on all remaining 15/sex animals in control and high dose groups only. Histopathology (including eyes) was performed on some of the remaining 15/sex/group animals.	Depression in average body weight gain observed at high dose. 4 high dose rats exhibited cataracts. No cataracts detected at any other dose level. Incidence of eye findings by: Ophthalmology Control: 0/10 males, 0/10 females 17.6 mg/kg/day: Not examined 88 mg/kg/day: Not examined 440 mg/kg/day: 2/15 males, 2/15 females Described as “moderate to severe bilateral cataracts”   Histopathology Control: 0/10 males, 0/10 females 17.6 mg/kg/day: 0/5 males, 0/5 females 88 mg/kg/day: 0/5 males, 0/5 females 440 mg/kg/day: 2/11 males, 2/11 females	88	440
Combined repeated dose and reproduction/developmental screening oral (gavage) study in rats 0, 40, 100, and 250 mg AO/kg bw/day (actual ingested) Ceccatelli R, Flade D, & Romeo L (2008)   002 Supporting study	n = 10/sex/group No ophthalmology was performed. Histopathology (including eyes) was performed.	Stomach lesions observed via histopathology in both sexes at doses ≥100 mg/kg/day and decreased locomotor activity observed in females at 250 mg/kg/day. No cataractsdetected in histopathology.	40	100
Subacute 4-week oral (gavage) study in rats 0 or 86 mg/kg/day (actual ingested) The Procter & Gamble Company (1979b)   003 Supporting study	n = 10/sex/group Only body weight, food and water consumption, and urine output were evaluated.	Minor body weight loss occurred in some treated animals.  No mention of cataracts in study.	86	NA
Subchronic 91-day oral (dietary) study in rats 0, 0.1, 0.2, and 0.4% AO (nominal in diet) 0, 88, 176, and 352 mg AO/kg bw/day (nominal in diet) Hazelton Laboratories, Inc. (1980)   004 Supporting study	n = 20/sex/group Ophthalmology was performed in all rats. Histopathology (including eyes) was performed on 10 rats/sex in control and high dose.	Significantly decreased body weights in mid and high dose animals. 20 high dose rats and 3 mid dose rats exhibitedlenticular lesions.No lenticular lesions detected at the low dose. Incidence of eye findings by: Ophthalmology Control: 0/20 males, 0/20 females 88 mg/kg/day: 0/20 males, 0/20 females 176 mg/kg/day: 3/19 males, 0/20 females 352 mg/kg/day:12/20 males,8/20 females Detailed descriptionsof lenticular lesions,but no severity grades.   Histopathology No compound-induced alterations noted.	88	176
Subchronic 10-week (dietary) study in rats 0.25% AO (nominal in diet) Approximately 200 mg AO/kg bw/day Battelle (1980)   005 Supporting study	n = 15/sex/group Animals were co-dosed with NH4AE3S to evaluate whether it would mitigate lenticular effects of AO. Animals were evaluated for general toxicity (mortality, clinical signs, body weights, and food consumption) and Ophthalmology was performed on all rats after 3, 6 and 10 weeks of dosing.	Body weight reductions were observed in both sexes at 10 weeks. Ophthalmology identified cataracts in 2 animals (0/15 males and 2/15 females) after 10 weeks, compared to 0/15 males and 0/15 females in the control group. No cataracts were observed in any animals at 3 or 6 weeks.	NA	200
Subacute 6-week (dietary) study in rats 0.35% AO (nominal in diet) Approximately 300 mg/kg bw/day The Procter & Gamble Company (1978b)   007 Supporting study	n = 15/sex/group Animals were dosed with branched isomers of AO (present in commercial AO) to evaluate their cataractogenic potential.  Animals were evaluated for general toxicity (body weights and food consumption) and Ophthalmology was performed on all rats.	Ophthalmology identified lenticular lesions in 6 treated animals (4/15 males and 2/15 females) after 6 weeks, compared to 0/15 males and 0/15 females in the control group.	NA	300
Subchronic 30-week (Phase I) and 16-week (Phase II) (dietary) study in rats 0, 0.35, and 0.5% AO (nominal in diet) dosed to weanling rats 0 and 0.5% AO dosed to adult rats 0.35 and 0.5% are Approximately 300 and 440 mg AO/kg bw/day Gibson WB (1979)   008 Supporting study	n = 25/sex/group Animal (adults and weanlings) were dosed with commercial AO to evaluate any differences in cataractogenic potential. Animals were evaluated for general toxicity (body weights and food consumption). Ophthalmology was performed only in Phase II at 16 weeks.	All doses affected body weights and food consumption. Ophthalmology identified cataracts in 23 weanling rats at low dose and 33 weanling rats at high dose and 29 adult rats at high dose. Ophthalmology Control weanlings: 0/25 males, 0/25 females 300 mg/kg/day weanlings: 11/25 males, 12/25 females 440 mg/kg/day weanlings: 14/25 males, 19/25 females 440 mg/kg/day adults: 18/25 males, 11/25 females	NA	300
Subchronic 32-week and 13-week (dietary) study in rabbits 0, 0.1, 0.5, and 1.0% AO (nominal in diet) Approximately 0, 32, 160, and 320 mg/kg/day Hazelton Laboratories (1977) (1977)   006 Supporting study	All animals exposed for 13 weeks. Low and mid dose animals were exposed an additional 19 weeks for a total of 32 weeks.Ophthalmology was performed only in low and mid dose animals at 32 weeks. (Excessive mortality occurred in the high dose group by 13 weeks).	Significant increased mortality rates, decrease body weight and food consumption, increased incidence of clinical signs and clinical chemistry parameters, gross histopathologic changes were observed at the high dose, and increased liver to body weight ratio were observed at the mid dose. 7 animals exhibited cataracts in the study, including control animals. Incidence of eye findings were:   Ophthalmology at 32 weeks: Control: 2/14 males, 1/16 females 0.1% in diet (~32 mg/kg/day): 0/20 males, 1/19 females 0.5% in diet (~160 mg/kg/day): 2/15 males, 1/16 females   The incidence of cataracts in control animals (3/30) was higher than in low dose animals (1/39). Therefore, low dose was considered the NOAEL.	32	160

* doses were calculated using established biological default values (EPA, 1988).

In dietary studies, the eye lesions were observed only at higher doses of ≥176 mg/kg/day. 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) ] a 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. The critical effect, eye lesions, observed in these dietary studies were also observed in other supporting dietary studies with the registered substance. However, the data set clearly demonstrates a consistent dose threshold for this effect, with a NOAEL of 88 mg/kg/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.

Further, in the available carcinogenicity/chronic key study also provided in this dossier (see Carcinogenicity section), eye lesions were detected by ophthalmoscopic examination intermittently in the study. However, at the conclusion of the study, both consulting veterinary ophthalmologists concluded that there were no apparent treatment related ophthalmic changes that were distinguishable from normal aging changes and that there was insufficient evidence to support a contention of compound related pathology.