Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information
No other studies concerning toxicity to reproduction are available.
Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07-11-2007 to 28-05-2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: OPPTS 870.3650
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: HanRcc: WIST(SPF)
Sex:
male/female
Details on test animals 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 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.
Details on mating procedure:
During the pairing period, females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronised timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually if the daily vaginal smear was positive for sperm, or a copulation plug was observed.
The day of mating was designated Day 0 post coitum.
Female that did not mate during the 14-day pairing period , was paired with a male of the same group which had already mated successfully.
All dams were allowed to give birth and rear their litters (F1 pups) up to Day 4 post partum. Day 0 post partum was designated as the day on which a female had delivered all her pups.
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 storage 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
Details on study schedule:
See table.
Remarks:
Doses / Concentrations:
0, 40, 100 and 250 mg/kg bw/day
Basis:
actual ingested
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:
No data
Parental animals: Observations and examinations:
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 faecal 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.
Oestrous cyclicity (parental animals):
During the pre-pairing period , cages with males were interspersed among those holding females to promote the development of regular estrous cycles.
Sperm parameters (parental animals):
The reproductive organs were given particular attention during necropsy. The testes and epididymides of all parental males were weighed as pairs. The prostate, seminal vesicles with coagulating gland, testes and epididymides were preserved from all parental males. The testes were examined by PAS-hematoxylin. Histopathological examination placed special emphasis on stages of spermatogenesis and histopathology of interstitial cell structure.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Litter size, live births and any gross anomalies, sex ratio, individual pup weights on Day 0 (when possible) and on Day 1 and Day 4 post partum.

GROSS EXAMINATION OF DEAD PUPS: Yes except those excessively cannibalised.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All animals sacrificed after treatment of at least 28 days, when no longer needed for assessment of reproductive effects or found dead.
- Maternal animals: All animals sacrificed on day 5 post partum or found dead.

GROSS PATHOLOGY: Specimens of abnormal tissue were fixed in neutral phosphate buffered 4 % formaldehyde solution.
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).
Ovaries from all parental females were preserved in neutral phosphate buffered 4 % formaldehyde solution.
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.
Postmortem examinations (offspring):
SACRIFICE: Day 4 post partum.

GROSS NECROPSY: Dead pups, except those excessively cannibalised, were examined macroscopically.
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.
Reproductive indices:
The following reproduction parameters were calculated: Fertility indices, mean precoital time, post-implantation losses, mean litter size, pup sex ratios and viability indices.
For reproduction data, group mean values were calculated both on a litter basis and on a percentage per group basis. Mean pup weights were calculated from individual weights both on a per group and on a per litter basis.
Offspring viability indices:
The following offspring viability indices were calculated: Mean litter size, pup sex ratios and viability indices. Mean pup weights were calculated from individual weights both on a per group and on a per litter basis.
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
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
Other effects:
not specified
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
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 FOOD CONSUMPTION (PARENTAL ANIMALS)
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.
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.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No data

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No data

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
All pairs mated. Mean pre-coital time, fertility index and conception rates 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.

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

GROSS PATHOLOGY (PARENTAL ANIMALS)
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 (PARENTAL ANIMALS)
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 receiving 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 (PARENTAL ANIMALS):
CLINICAL LABORATORY INVESTIGATIONS: The statistically significant alterations observed at 250 mg/kg/day were not considered to be adverse.
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on reproduction/developmental toxicity.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
VIABILITY (OFFSPRING)
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. 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.

CLINICAL SIGNS (OFFSPRING)
No abnormal pup was noted at any dose level, except at 250 mg/kg/day where mean pup weight development was reduced.

GROSS PATHOLOGY (OFFSPRING)
At necropsy of pups, there were no abnormal findings.

HISTOPATHOLOGY (OFFSPRING)
At necropsy of pups, there were no abnormal findings.

OTHER FINDINGS (OFFSPRING)
The sex ratio was also not affected.
Reproductive 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

-

-

-

-

-

-

-

-

Reproduction data

Mean pre-coital time

-

-

-

-

Implantation rate

-

-

-

-

Conception rate

-

-

-

-

Gestation index

-

-

-

+

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

Microscopic Reproductive abnormalities

-

-

-

-

-

-

-

-

Litter data

Litter size

-

-

-

-

Mean no. of pups at first litter check

-

-

-

-

Sex ratio

-

-

-

-

Pup abnormalities

-

-

-

-

Pup death postnatal Day 4

-

-

-

+

Decreased Pup weight Day 4

-

-

-

+ ns

Pup necropsy findings

-

-

-

-

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

ns: not significant

Conclusions:
The Lauramine oxide 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. Lauramine oxide was administered to male rats for at least 28 days and to female rats for 14 days prior to pairing, through pairing and gestation period until the F1 generation reached Day 4 post partum.
Administration at 100 and 250 mg/kg bw /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 bw/day treatment with the test item resulted in a general reduction of body weight gain in males and females, statistically reduced locomotor activity in females and in statistically significantly uncreased 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 with an irregular surface. The histopathological data showed lesions in the forstomach and in the kidneys. At 100 mg/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/kg/day. The NOEL for reproduction/developmental toxicity was considered to be 100 mg/kg/day.
Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
One screening study (K=1) is available.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a combined repeat dose toxicity study with the reproduction/developmental toxicity screening test conducted under GLP according to OECD TG 422 the substance was administered via gavage to 10 animals/sex/group at 0, 40, 100 or 250 mg/kg bw/day. Males were dosed for at least 14 days prior to mating and during 14 days pairing and female rats for 14 days prior to pairing, through the pairing and gestation period until the offspring reached Day 4 post-partum. Litter size and mean number of pups at first litter check were not affected by treatment. The sex ratio was also not affected. No abnormal pup was noted at any dose level. At 250 mg AO/kg bw/day a statistically significant increase in pups 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. Mean pup weight development was reduced at 250 mg AO/kg bw/day. At necropsy, there were no findings in pups. The NOEL for reproduction/developmental toxicity was considered to be 100 mg/kg bw/day.


Short description of key information:
In a combined repeat dose toxicity study with the reproduction/developmental toxicity screening test conducted under GLP according to OECD TG 422 the substance was administered via gavage to 10 animals/sex/group at 0, 40, 100 or 250 mg/kg bw/day. The NOEL for reproduction/developmental toxicity was considered to be 100 mg/kg bw/day.

Justification for selection of Effect on fertility via oral route:
Only study available

Effects on developmental toxicity

Description of key information
In a prenatal developmental toxicity study in which pregnant female rats were dosed by gavage with 0, 25, 100 or 200 mg AO/kg bw on gestation days 6-19, the NOAEL for both maternal toxicity and developmental toxicity is 25 mg AO/kg/day.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
N/A
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Developmental and teratogenicity study. Study was well conducted and documented. Three dose and one control groups were tested (25 rats/sex/group). GLP.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
N/A
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina.
- Age at study initiation: Females: 65 days; Males: 42 days.
- Weight at study initiation: Females: 218 - 262 g; Males: 506 - 969 g.
- Fasting period before study: N/A
- Housing: Rats were individual housed except during cohabitation period. During cohabitation, each pair of male and female rats was housed in the male rat's cage.
- Diet (ad libitum): Certified Rodent Diet #5002 (Purina Nutrition International, St. Louis, Missouri) in individual feeders.
- Water (ad libitum): Local water that had been processed by passage through a reverse osmossis membrane (R.O. water) was available to the rats from an automatic watering system and/or individual water bottles. Chlorine was added to the processed water as a bacteriostat.
- Acclimation period: N/A


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 26
- Humidity (%): 30 to 70
- Air changes (per hr): 10/hr
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: N/A To: N/A
Route of administration:
oral: gavage
Vehicle:
other: Sterile Water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Solutions of the test substances were prepared weekly at the Testing Facility. Dosage calculations were adjusted for the 32% (w/v) concentration of the test substance. Prepared formulations were stored at room temperature and stirred continuously (magnetic stir plate with stir bar) during dosage administration.


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


VEHICLE
- Justification for use and choice of vehicle (if other than water): Sterile Water
- Concentration in vehicle: N/A
- Amount of vehicle (if gavage): 5 mL/kg
- 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
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: 5 days
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: N/A
- Further matings after two unsuccessful attempts: N/A
- Verification of same strain and source of both sexes: N/A
- Proof of pregnancy: sperm in vaginal smear and/or a copulatory plug observed in situ will be referred to as day 0 of pregnancy
- Any other deviations from standard protocol: N/A
Duration of treatment / exposure:
Days 6 through 19 of presumed gestation.
Frequency of treatment:
Daily
Duration of test:
Approximately 4 weeks.
Remarks:
Doses / Concentrations:
25, 100, and 200 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosages were selected on the basis of a dosage-range study (Angus Research Laboratories, Inc., Protocol 916-025P), in which dosage levels of 0, 32.5, 100, 325 and 650 mg/kg/day were evaluated.
Dosages of 325 and 650 mg/kg/day were excessively toxic to the dams and conceptuses. At the 32.5 and 100 mg/kg/day dosage levels, no developmental toxicity was observed. Excess salivation was observed at the 100 mg/kg/day dosage level, and slight, non-statistically significant decreases in maternal body weight gains and feed consumption values were observed at the 32.5 and 100 mg/kg/day dosage levels. Doses of 200, 100, and 25 mg akly dimethyl amine oxide/kg/day were chosen.
- Rationale for animal assignment (if not random): Unpon arrival, rats were assigned to individual housing on the basis of computer-generated random units. Female rats were assigned to four dosage groups (Groups I through IV), twentry-five rats per dosage group, using a computer-generated (weight-ordered) randomization procedure based on body weights recorded on DG 0.
- Other: N/A
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day
- Cage side observations checked were: mortality, moribundity, pertinent behavioral changes and other signs of overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly and Rats were also examined for clinical observations immediately before and approximately 60 minutes after dosage and on the day of sacrifice (DG 20).

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during acclimation, on DG 0, daily during the dosage period and on DG 20.

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

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: N/A

OTHER: N/A
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: A late resorption was defined as one in which the occurrence of organogenesis was grossly evident. A live fetus was defined as a term fetus that responded to stimuli. Nonresponding term fetuses are considered to be dead (there were no dead fetuses). Dead fetuses and late resorptions are differentiated by the degree of autolysis present; marked to extreme autolysis indicated that the fetuses was a late resorption.
Fetal examinations:
- External examinations: Yes: half per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
Clinical observation and other proportion data were analyzed using the Variance Test for Homogeneity of the Bionomial Distribution.
Continuous data (e.g., body weights, body weight changes, feed consumption values, organ weights and litter averages for percent male fetuses, percent resorbed conceptuses, fetal body weights and feal anomaly data) were analyzed using Bartletts' Test of Homogeneity of Variances and the Analysis of Variance, when appropriated [i.e., Bartlett's Test was not significant (pCount data obtained at Caesarean-sectioning of the dams were evaluated using the procedures described above for the Kruskal-Wallis Test.
Indices:
N/A
Historical control data:
N/A
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
MORTALITY:
Two rats in the 200 mg/kg/day dosage group died; one of these deaths was attributed to the test substance, the other was the result of an intubation error. All other rats survived until scheduled sacrifice.
A single rat in the 200 mg/kg/day dosage group was found dead on day 19 of gestation (DG 19) after 13 daily dosages. This dam lost body weight after DG 9 and had reduced feed consumption values throughout the study. Adverse clinical observations included excessive salivation (DGs 8 to 9, 14 and 17 to 18), gasping (DGs 9 to 11 and 17 to 18), labored breathing (DGs 10 to 13 and 16 to 18), ungroomed coat (DGs 10 to 13), brown perianal substances (DGs 12 to 13), urine-stained abdominal fur (DGs 12 to 14 and 16 to 18), brown perivaginal substance (DGs 12 to 15), chromorhinorrhea (DG 15), rales (DG 16) and emaciation (DGs 17 to 18). No gross lesions were revealed by necropsy; all tissues appeared normal for moderate degree autolysis. There were 17 early resorptions in utera. This death was attributed to effects of the test substance because similar observations occurred in surviving rats in this dosage group.
A single rat in the 200 mg/kg/day dosage group was found dead on DG 18 after 12 daily dosages. This death was attributed to an intubation accident. This dam lost body weight after DG 13 and had reduced feed consumption values throughout the study. Adverse clinical observations included rales (DGs 10 to 11 and 13), excessive salivation (DGs 13 to 14), gasping (DGs 13 to 14), labored breathing (DGs 13 to 17), red perioral substance (DGs 15 to 17), urine-stained abdominal fur (DGs 15 to 17), dehydration (DG 16) and emaciation (DG 17). External observations at necropsy included red substance on fur of the nose, forepaws and forelimbs. Gross necropsy revealed a tear in the esophagus; all other tissues appeared normal for slight degree of autolysis. There were 14 fetuses in utero. The viability of the fetuses could not be determined because of the maternal death.

CLINICAL OBSERVATIONS:
Adverse clinical observations occurred at increased incidences in the 100 and 200 mg/kg/day dosage group rats. Excessive salivation, rales, urine-stained abdominal fur, brown or red perioral substance, labored breathing and gasping occurred in 100 mg/kg/day dosage group rats and in significantly increased (pAll other clincal observations were considered unrelated to the test substance because: 1) the incidences were not dosage-dependent; and/or 2) the observations occurred in only one or two rats. These clinical observations included one 200 mg/kg/day dosage group dam with an axillary mass attributed to an intubation error, and localized alopecia (limbs and underside) in one or two rats in the 0 (Vehicle), 25, and 200 mg/kg/day dosage groups.

NECROPSY OBSERVATIONS:
All necropsy observations were considered unrelated to the test substance because they were single events. These observations included slight dilation of the pelvis of the right kidney of one vehicle group dam and one 200 mg/kg/day dosage group dam. A single dame also had a distended urinary bladder with ten calculi and thickened and red bladder walls. One 100 mg/kg/day dosage group dam had large adrenals, four dark red areas on the fundic mucosa and numerous raised tan areas on the pyloric mucosa of the stomach, the intestines and stomach were distended with gas and the left lateral lobe of the liver was mottled. One 200 mg/kg/day dosage dam had a mass in the left axilla in-life; at necropsy, a clear tan gelatinous fluid was located subcutaneously in the area of the ventral neck, left axilla and leateral chest, the esophagus had a thickened area and the plyoric folds of the stomach were thickened. These observation were presumed to be the sequelae of a presumed intubation error.

MATERNAL BODY WEIGHTS, GRAVID UTERINE WEIGHTS AND BODY WEIGHT CHANGES:
Rats in the 100 and 200 mg/kg/day dosage groups had significantly reduced (pThe gravid uterine weight and the corrected DG 20 maternal body weight (DG 20 body weight minus the gravid uterine weight) were significantly reduced (pBody weights and body weights gains were unaffected at dosages of 25 mg/kg/day of the test substance. Gravid uterine weights were unaffected by the 25 and 100 mg/kg/day dosages of the test substance.

MATERNAL ABSOLUTE (g/day) and RELATIVE (g/kg/day) FEED CONSUMPTION VALUES:
Absolute (g/day) feed consumption values for the entire dosage period (calculated as DGs 6 to 20) and the entire gestation period (DGs 0 to 20) were significantly reduced (p
CAESAREAN-SECTIONING:
Caesarean-sectioning observations were based on 24, 25, 24 and 22 pregnant rats in the 0 (vehicle), 25, 100 and 200 mg/kg/dosage groups, respectively. Male and female fetal body weights were significantly reduced (pNo other Caesarean-sectioning or litter parameters were affected by dosages of the test substance as high as 200 mg/kg/day. The litter averages for corpora lutea, implantations, late resorptions, percent resorbed conceptuses (calculated excluding the completely resorbed litter in the 200 mg/kg/day dosage group), and percent live male fetuses were comparable among the four dosage groups and did not significantly differ. There were no dead fetuses.
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
FETAL ALTERATIONS:
Fetal alterations were defined as: 1) malformations (irreversible changes that occur at low incidences in this species and strain); or 2) variations (common findings in this species and strain and reversible delays or accelerations in development). Litter averages were calculated for specific fetal ossification sites as part of the evaluation of the degree of fetal ossification.
Fetal evaluations were based on 339, 375, 348, and 297 Ceasarean-delivered live fetuses in 24, 25, 24, and 21 litters in the 0 (vehicle), 25, 100 and 200 mg/kg/day dosage groups, respectively. Each fetus was examined for gross external alterations, approximately one half of the fetuses in each litter were examined for soft tissue alterations and the remaining fetuses were examined for skeletal alterations and the number of ossification sites.
The percentages of fetuses and litters with alterations in the 200 mg/kg/day dosage group were significantly increased (pAll other gross external, soft tissues or skeletal alterations (malformations or variations) were considered unrelated to the test substance because 1) the litter and fetal incidences were not dosage-dependent; 2) the alteration occurred in only one fetus; or 3) the incidences were within the ranges observed historically at the Test Facility.

FETAL ALTERATIONS:
In groups I through IV, litters with fetuses with alterations numbered 8 (33.3%), 11 (44.0%), 8 (33.3%), and 15 (71.4%), respectively. The numbers of fetuses with any alterations were 11 (3.2%), 25 (6.7%), 14 (4.0%) and 32 (10.8%) respectively. The percentages of fetuses with any alteration per litter were 3.6%, 6.4%, 4.5% and 11.5% in the four respective dosage groups.
The significant increases in the percentages of fetuses and litters with alterations in the 200 mg/kg/day dosage group were considered to reflect delays in skeletal ossification, related to the significantly reduced (p
FETAL GROSS EXTERNAL ALTERATIONS:
One vehicle group fetus had whole body edema (anasarca); this fetus also had an absent innominate artery at soft tissue examination. One 25 mg/kg/day fetus had a thread-like tail; at skeletal evaluation, this fetus had fused arches of the 3rd sacral vertebra and no caudal vertebrae. One 200 mg/kg/day group fetus had a kinked tail, the 4th and 5th digits of the lift hindlimb were fused and a skin tab located to the left of its tail. At skeletal evaluation of this fetus, further evaluation of the skin tab revealed two bones (possibly a femur and fibula) fused together on the left side of the pelvis. An extra paw appeared to be attached to the underside of the left paw; also the centra of the 6th and 11th thoracic vertebrae were bifid. One 200 mg/kg/day fetus had a micrognathia; soft tissue evaluation of this fetus revealed a small tongue.

MALFORMATIONS - SOFT TISSUE EVALUATION:
One 200 mg/kg/day fetus had a small tongue; micrognathia was identified at gross external evaluation.

VARIATIONS - SOFT TISSUE EVALUATION:
One control group fetus and one 25 mg/kg/day dosage group fetus had an absent innominate vessel; whole body edema (anasarca) was identified at gross external evaluation for the control fetus, also an additional fetus had the umbilical artery descending to the left of the urinary bladder.

MALFORMATION - SKELETAL ALTERATIONS:
One control group fetus had fused arches of the 4th cervical vertebra; additional variations in ossification occurred in this fetus (unossified 1st sternal centra, incompletely ossified pubes).
One 25 mg/kg/day dosage group fetus had fused arches of the 3rd sacral vertebra; this fetus also had no caudal vertebrae (thread-like tail was noted at gross external examination).

VARIATIONS - SKELETAL ALTERATIONS:
A cervical rib was present at the 7th vertebra in one 25 mg/kg/day fetus. This fetus had no other skeletal alterations.
A bifid centrum in the thoracic vertebrae occurred in 1, 6, 5 and 9 fetuses from 1, 3, 5 and 8 litters in the 0 (vehicle), 25, 100 and 200 mg/kg/day dosage groups respectively. One fetus in the 25 mg/kg/day dosage group also had incompletely ossified 1st and 2nd sternal centra, a fetus in the 100 mg/kg/day dosage group also had a bifid centrum of the 1st lumbar vertebra and a fetus in the 200 mg/kg/day dosage group also had incompletely ossified pubes.
An incompletely ossified arch in the 6th lumbar vetrebra occurred in one 100 mg/kg/day dosage group fetus; this fetus also had incompletely ossified ischia and pubes.
The significant increase (pDelayed sternal ossification (incompletely ossified and/or not ossified 1st and/or 2nd sternebrae) occurred in 4, 8, 3 and 11 fetuses from 4, 6, 2 and 7 litters in the 0 (vehicle), 25, 100 and 200 mg/kg/day dosage groups, respectively. The fetal incidence of only incompletely ossified 1st sternal centra was also significantly increased (pThe significant increases (pThe pubes and/or ischia were incompletely ossified in 8, 12, 7 and 16 fetuses from 6, 4, 3 and 6 litters in the 0 (vehicle), 25, 100 and 200 mg/kg/day dosage groups, respectively. The fetal incidenc of only incompletely ossified pubes was significantly increased (pThe significant increase (pThe litter averages for ossified caudal vertebrae, sternal centers and metacarpals per fetus were significantly decreased (pAnalyses of the average numbers of fetal ossification sites per fetus did not reveal any other statistically significant differences among the four dosage groups. Ossification of the hyoid, vertebrae (cervical, thoracic, lumbar, and sacral) ribs, sternum (manubrium and xiphoid), forelimbs (carpals and phalanges) and hindlimbs (tarsals, metatarsals and phalanges) occurred at similar incidences in litter in all dosage groups.
Abnormalities:
not specified
Developmental effects observed:
not specified

N/A              

Conclusions:
On the basis of these data, the maternal no-observable-adverse-effect-level (NOAEL) for the test substance (alkyl dimethyl amine oxide) is 25 mg/kg/day. The 200 mg/kg/day dosage caused mortality in one dam, the 100 and 200 mg/kg/day dosages caused adverse clinical observations, reductions in body weight gain and reduced feed consumption values. Reductions in relative feed consumption values were also noted in the 25 mg/kg/day dose group; however, no concomitant adverse effects were noted at this dosage level. The developmental NOAEL was 25 mg/kg/day; the 200 mg/kg/day dosage caused reduced fetal body weights and associated delays in skeletal ossification and the 100 mg/kg/day dosage also caused delays in skeletal ossification.
Executive summary:

One-hundred Crl:CDBR VAF/Plus presumed pregnant female rats were randomly assigned to four dosage groups (Groups 1 through IV), 25 rats per group. The test substance preparations for dosing were corrected for purity. The test substance, was administered orally (via gavage) once daily to these female rats on days 6 through 19 of presumed gestation (DGs 6 through 19), at dosages of 0 (Vehicle), 25, 100, and 200 mg alkyl dimethyl amine oxide/kg/day. The dosage volume was 5 mL/kg, adjusted daily on the basis of the individual body weights recorded before intubation. The rats were intubated at approximately the same time each day.

The rats were observed for viability at least twice a day and for general appearance weekly during acclimation and on DG 0. The rats were also examined for clinical observations of effects of the test substance, abortions, premature deliveries and deaths immediately before and approximately 60 minutes after dosage and on the day of sacrifice (DG 20). Body weights were recorded weekly during acclimation, on DG 0, daily during the dosage period and on DG 20. Feed consumption values were recorded on DGs 0, 6, 9, 12, 15, 18, and 20.

All surviving rats were sacrificed on DG 20, and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed. Caesarean-sectioning and subsequent fetal observations were conducted without knowledge of dosage group in order to minimize bias. The number of corpora lutea in each ovary was recorded. The uterus of each rat was excised and examined for pregnancy, number and distribution of implantations, live and dead fetuses and early and late resorptions. The gravid uterus was weighed. Each fetus was removed from the uterus and subsequently weighed and examined for sex and gross external alterations. Approximately one-half of the fetuses in each litter were examined for soft tissue alterations using a variation of the microdissection techniques. The remaining fetuses in each litter were examined for skeletal alterations.

Two rats in the 200 mg/kg/day dosage group died; one of these deaths was attributed to the test substance, the other was the result of an intubation error. All other rats survived until scheduled sacrifice.

Excessive salivation, rales, urine-stained abdominal fur, brown or red perioral substance, labored breathing and gasping occurred in 100 mg/kg/day dosage group rats and in significantly increased numbers of 200 mg/kg/day dosage group rats. Additionally, chromorhinorrhea occurred in one and two rats in these two dosage groups, respectively. Observations of brown or red perivaginal substance, emaciation, brown perianal or perinasal substance, dehydration, ungroomed coat and soft or liquid feces occurred in one or two rats in the 200 mg/kg/day dosage group. All necropsy observations were considered unrelated to the test substance.

Rats in the 100 and 200 mg/kg/day dosage groups had significantly reduced body weight gains for the entire dosage period (calculated as days 6 to 20 of gestation) and body weight gains were significantly reduced in the 200 mg/kg/day dosage group for the entire gestation period (DGs 0 to 20). Body weights were significantly reduced in the 200 mg/kg/day dosage group on DGs 11 through 20. The gravid uterine weight and the corrected DG 20 maternal body weight (DG 20 body weight minus the gravid uterine weight) were significantly reduced in the 200 mg/kg/day dosage group.

Absolute (g/day) and relative (g/kg/day) feed consumption values for the entire dosage period (calculated as DGs 6 to 20) and the entire gestation period (DGs 0 to 20) were significantly reduced in the 200 mg/kg/day dosage group. Relative feed consumption values for the entire dosage period were also significantly reduced in the 100 mg/kg/day dosage group, while values for the gestation period were significantly reduced in the 25, 100 and 200 mg/kg/day dosage groups.

Male and female fetal body weights were significantly reduced in the 200 mg/kg/day dosage group. Live litter size was decreased and the number of early resorptions was increased in the 200 mg/kg/day dosage group, but apparently as the result of one dam in this dosage group that had a litter consisting of 16 early resorptions.

The percentages of fetuses and litters with alterations in the 200 mg/kg/day dosage group were significantly increased and reflected delays in skeletal ossification related to the significantly reduced fetal body weights in this group. These delays in ossification included significant increases in the fetal and/or litter incidences of bifid thoracic vertebrae centra, incompletely and/or not ossified 1st or 2nd sternal centra, incompletely ossified pubes and significant decreases in the numbers of ossified caudal vertebrae, sternal centers and metacarpals. Additionally, delays in ossification occurred in the 100 mg/kg/day dosage group and included a significant increase in the litter incidence of bifid thoracic vertebrae centra.

NOAEL for both maternal toxicity and developmental toxicity is 25 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
25 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
There is one prenatal developmental toxicity study (K=1) and one screening study (K=1) available.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The key study is a prenatal developmental toxicity study performed under GLP according to EPA OTS 798.4900 [York RG (1999). In this study presumed pregnant female rats (Crl:CDBR VAF/Plus; 25 animals/group) were dosed by oral gavage with 0, 25, 100 or 200 mg AO/ kg bw daily on days 6 through 19 of presumed gestation (DG 6-19). The rats were observed for viability at least twice a day and for general appearance weekly during acclimation and on DG 0. The rats were also examined for clinical observations of effects of the test substance, abortions, premature deliveries and deaths immediately before and approximately 60 minutes after dosage and on the day of sacrifice (DG 20). Body weights were recorded weekly during acclimation, on DG 0, daily during the dosage period and on DG 20. Feed consumption values were recorded on DGs 0, 6, 9, 12, 15, 18, and 20.

All surviving rats were sacrificed on DG 20, and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed. Caesarean-sectioning and subsequent fetal observations were conducted without knowledge of dosage group in order to minimize bias. The number of corpora lutea in each ovary was recorded. The uterus of each rat was excised and examined for pregnancy, number and distribution of implantations, live and dead fetuses and early and late resorptions. The gravid uterus was weighed. Each fetus was removed from the uterus and subsequently weighed and examined for sex and gross external alterations. Approximately one-half of the fetuses in each litter were examined for soft tissue alterations using a variation of the microdissection techniques. The remaining fetuses in each litter were examined for skeletal alterations.

Two rats in the 200 mg AO/kg/day dosage group died; one of these deaths was attributed to the test substance, the other was the result of an intubation error. All other rats survived until scheduled sacrifice. Excessive salivation, rales, urine-stained abdominal fur, brown or red perioral substance, laboured breathing and gasping occurred in 100 mg AO/kg/day dosage group rats and in significantly increased numbers of 200 mg AO/kg/day dosage group rats. Additionally, chromorhinorrhea occurred in one and two rats in these two dosage groups, respectively. Observations of brown or red perivaginal substance, emaciation, brown perianal or perinasal substance, dehydration, ungroomed coat and soft or liquid feces occurred in one or two rats in the 200 mg AO/kg/day dosage group. All necropsy observations were considered unrelated to the test substance.

Rats in the 100 and 200 mg AO/kg/day dosage groups had significantly reduced body weight gains for the entire dosage period (calculated as days 6 to 20 of gestation) and body weight gains were significantly reduced in the 200 mg AO/kg/day dosage group for the entire gestation period (DGs 0 to 20). Body weights were significantly reduced in the 200 mg AO/kg/day dosage group on DGs 11 through 20. The gravid uterine weight and the corrected DG 20 maternal body weight (DG 20 body weight minus the gravid uterine weight) were significantly reduced in the 200 mg AO/kg/day dosage group.

Absolute (g/day) and relative (g/kg/day) feed consumption values for the entire dosage period (calculated as DGs 6 to 20) and the entire gestation period (DGs 0 to 20) were significantly reduced in the 200 mg AO/kg/day dosage group. Relative feed consumption values for the entire dosage period were also significantly reduced in the 100 mg AO/kg/day dosage group, while values for the gestation period were significantly reduced in the 25, 100 and 200 mg AO/kg/day dosage groups.

Male and female fetal body weights were significantly reduced in the 200 mg AO/kg/day dosage group. Live litter size was decreased and the number of early resorptions was increased in the 200 mg AO/kg/day dosage group, but apparently as the result of one dam in this dosage group that had a litter consisting of 16 early resorptions.

The percentages of fetuses and litters with alterations in the 200 mg AO/kg/day dosage group were significantly increased and reflected delays in skeletal ossification related to the significantly reduced fetal body weights in this group. These delays in ossification included significant increases in the fetal and/or litter incidences of bifid thoracic vertebrae centra, incompletely and/or not ossified 1st or 2nd sternal centra, incompletely ossified pubes and significant decreases in the numbers of ossified caudal vertebrae, sternal centra and metacarpals. Additionally, delays in ossification occurred in the 100 mg AO/kg/day dosage group and included a significant increase in the litter incidence of bifid thoracic vertebrae centra.

NOAEL for both maternal toxicity and developmental toxicity is 25 mg AO/kg/day.

In a combined repeat dose toxicity study with the reproduction/developmental toxicity screening test conducted under GLP according to OECD TG 422 the substance was administered via gavage to 10 animals/sex/group at 0, 40, 100 or 250 mg/kg bw/day. Males were dosed for at least 14 days prior to mating and during 14 days pairing and female rats for 14 days prior to pairing, through the pairing and gestation period until the offspring reached Day 4 post-partum. Litter size and mean number of pups at first litter check were not affected by treatment. The sex ratio was also not affected. No abnormal pup was noted at any dose level. At 250 mg AO/kg bw/day a statistically significant increase in pups 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. Mean pup weight development was reduced at 250 mg AO/kg bw/day. At necropsy, there were no findings in pups. The NOEL for reproduction/developmental toxicity was considered to be 100 mg/kg bw/day.

No data from prenatal developmental toxicity studies in a second species, typically the rabbit, are available for the amine oxides.

As discussed in the toxicokinetics section, amine oxides are extensively metabolised after oral administration as shown by at least 10 metabolites characterised in urine. In addition a substantial proportion of the dose is completely bio transformed to CO2and therefore eliminated by exhalation. There were several different pathways of metabolism proposed:

·        Oxidative degradation of the alkyl side chain by ω-oxidation to form a carboxylic acid followed by the loss of two-carbon fragments by sequential β-oxidation. This route of metabolism is frequently observed in similar substances to the test substance.

·        Hydroxylation of the alkyl side chain at a position four or five carbons from the end of the chain.

·        Reduction of the amine oxide group to the parent amine.

The metabolism of amine oxides is considered to be likely to be similar between different mammalian species including the rabbit and the rat.

 

A review of the literature has shown that some developmental toxicity data are available for amines and it is considered appropriate to read across to this class of chemicals as they are a relevant intermediate in the metabolic pathway of amine oxides.

In a study performed using cis-9-octadecenylamine (CAS No. 112-90-3) New Zealand White rabbits (22 artificially-inseminated females/group) were administered the test substance in corn oil via gavage at doses of 0, 3, 10 or 30 mg/kg bw/day for days 6 through 18 of gestation [US EPA (2010)]. Two females died in the high-dose group. Clinical signs (irritation of the lips and chin, laboured breathing and rales) of toxicity were observed at 10 and 30 mg/kg bw/day. Dose-dependent body weight losses or reduced gains and reduced food consumption occurred at the 10 and 30 mg/kg bw/day (significant only in the high dose group). There were no local effects on the gastrointestinal tract of the does. There were no treatment-related effects on the foetuses examined. The NOAEL (maternal toxicity) was 3 mg/kg bw/day and the NOAEL (developmental toxicity) was 30 mg/kg bw/day (highest dose tested).

In a study performed using N-methyl-N-octadecyl-1-octadecanamine (CAS No. 4088-22-6) pregnant New Zealand White rabbits (16/dose) were administered the test substance in corn oilviagavage at doses of 0, 50, 250 or 1000 mg/kg bw/day for days 6 through 18 of gestation [US EPA (2010)]. No treatment-related effects were found on pregnancy rate, mortality, abortions or gestation length. There was a high post implantation loss seen in the high dose group but this was not significant given the unusually high loss also observed in controls. No treatment-related effects were found on foetal size, foetal sex or mortalities; however slight reductions in foetal weight at 250 and 1000 mg/kg bw/day were observed and possible embryolethality at 1000 mg/kg bw/day cannot be disregarded. The NOAEL (maternal toxicity) was 50 mg/kg bw/day and the NOAEL (developmental toxicity) was 250 mg/kg bw/day.

The absence of developmental toxicity effects at dose levels below those at which maternal systemic toxicity was observed in studies performed in rats using C12-14 AO and in rabbits using cis-9-octadecenylamine or N-methyl-N-octadecyl-1-octadecanamine (surrogates for the metabolic intermediates of amine oxides) indicates that there is no concern for developmental toxicity from amine oxides.

Justification for selection of Effect on developmental toxicity: via oral route:
This study is the only prenatal developmental toxicity study available.

Toxicity to reproduction: other studies

Additional information

No other studies concerning toxicity to reproduction are available.

Justification for classification or non-classification

In the prenatal developmental toxicity study effects were seen in pups from 100 mg/kg bw/day. These effects included delayed ossification and reduced body weight (significant at 200 mg/kg bw/day). In addition, live litter size was decreased and the number of early resorptions increased in the 200 mg/kg bw/day group (results skewed by one dam with a high level of resorptions). These effects were seen at levels where maternal toxicity was evident. The maternal effects noted included excessive salivation, rales, urine-stained abdominal fur, brown or red perioral substance, labored breathing and gasping starting from 100 mg/kg bw/day and significant at 200 mg/kg bw/day, significantly reduced body weight gain in both groups and significantly reduced body weight at 200 mg/kg bw/day and mortality (one female) at 200 mg/kg bw/day. On this basis it is considered that the effects seen in pups at the high dose are asecondary non-specific consequence of maternal toxicity.

In the OECD TG 422 study total locomotor activity in females was statistically significantly reduced at 250 mg/kg bw/day. At 100 mg/kg bw/day lesions in the forestomach were noted at necropsy and histopathology. There were no effects on litter size, mean number of pups at first litter check, sex ratio or pup abnormality. At 250 mg AO/kg bw/day there was a statistically significant increase in pup death on postnatal days 0-4. The higher postnatal loss was in the range of historical controls. Mean pup weight development was reduced at 250 mg AO/kg bw/day. At necropsy there were no findings in pups. The NOEL for reproductive/developmental toxicity was considered to be 100 mg/kg bw/day in this study. The NOAEL for maternal toxicity was established at 40 mg AO/kg/day. In this study it is considered that the effects seen in pups at the high dose are asecondary non-specific consequence of maternal toxicity.

Based on the findings in these two studies it is concluded that any effects seen in pups are a secondary non-specific consequence of maternal toxicity and therefore no classification for reproductive toxicity is warranted.