Amines, C12-16-alkyldimethyl, N-oxides

Endpoint summary

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:

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

Adequacy of study:

key study

Study period:

23-03-2010 to 19-05-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP 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 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.

Details on mating procedure:

Sexually mature male and female main study rats were randomly paired. Mating was monogamous: 1 male and 1 female were placed in one cage during the dark period (1:1 mating) until copulation occurred or a maximum of 2 weeks had elapsed. If a positive mating sign was not observed during that time, an additional mating period was carried out with the same partner until a positive mating sign was noted for all females to guarantee at least 8 pregnant dams available for each group as not every positive mating sign results in pregnancy. Each morning, the females were examined for presence of sperm in the vaginal lavage or for the presence of a vaginal plug. The day on which sperm was found was considered as the day of conception and was defined as day 0 of gestation. If findings were negative, mating was repeated.
The satellite animals were not mated and, consequently, were not used for the assessment of reproduction/developmental data.

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) ad-ministration 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

Details on study schedule:

The duration of gestation was recorded and was calculated from day 0 of gestation. The females were allowed to litter normally. The duration of gestation in days was evaluated.

Remarks:

Doses / Concentrations:
40, 100 and 200 mg AO/kg bw/day
Basis:
nominal in water

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 Aromox B-W 500/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/kg b.w./day or at 250 mg/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/kg b.w./day as well as in all high dosed male and female rats treated with 400 mg Aromox B-W 500/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.

Parental animals: Observations and examinations:

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.

OTHER:
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.

Oestrous cyclicity (parental animals):

Each morning, the females were examined for presence of sperm in the vaginal lavage or for the presence of a vaginal plug. The day on which sperm was found was considered as the day of conception.

Sperm parameters (parental animals):

Adrenal glands and gonads were weighed individually and identified as left or right. Detailed histopathologic examination was performed on 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.

Litter observations:

The duration of gestation was recorded and was calculated from day 0 of gestation. The females were allowed to litter normally. The duration of gestation in days was evaluated.
Live pups were counted and the sex was determined. Each litter was examined as soon as possible after delivery to establish the number of stillbirths, live births, runts (pups were considered as runts if their weight was less than 70% of the mean litter weight) and the presence of gross abnormalities.
Evaluation / parameters
-Number of pregnant females
-Pre-coital time
-Gestation length calculated from day 0 of pregnancy

Corpora lutea
-number per dam
-absolute number per group
-mean per group

Implantations
-number per dam
-distribution in the uterine horns
-absolute number per group
-mean per group

Number of pups absolute
-at birth (alive and dead)
-after 4 days of life

Number of pups per dam
-at birth
-after 4 days of life

Number of male and female pups
-at birth
-after 4 days of life

Number of stillbirths
-absolute
-per dam

Number of pups with malformations
-absolute
-per dam

Postmortem examinations (parental animals):

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 se-lected 20 male and 20 female animals (5 animals/sex/main study group) and all satel-lite 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
L ungs (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 epidi-dymides (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 his-tologically after preparation of paraffin sections and haematoxylin-eosin staining: Forestomach, lymph node (1, mesenteric)

Postmortem examinations (offspring):

Dead pups and pups killed at day 4 post-partum, or shortly thereafter, were carefully examined externally for gross abnormalities.

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.

Reproductive indices:

For each group the following reproductive indices were determined:

Gestation Index = (number of litters with live pups/number pregnant) x 100


Fertility Index = (number pregnant/number of females evaluated for fertility) x 100


For each litter and group the following reproductive indices were determined:

Birth Index = (Total number of pups born (live + dead)/Number of implantation scars) x 100


Live Birth Index = (Number of pups born alive on day 0/1 / Total number born (live + dead) x 100


Viability Index =number of pups alive on day 4/number of pups live on day 0/1) x 100


Pre-implantation loss [%] = (corpora lutea - implantations/ corpora lutea) x 100


Post-implantation loss [%] = (implantations - no. pups born alive/implantations) x 100

Offspring viability indices:

Live pups were counted and the sex was determined. Each litter was examined as soon as possible after delivery to establish the number of stillbirths, live births, runts (pups were considered as runts if their weight was less than 70% of the mean litter weight) and the presence of gross abnormalities.

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, treatment-related

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
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 Aromox B-W 500/kg b.w./day. Starting at 100 mg Aromox B-W 500/kg b.w./day increased salivation was noted for a very few male and female animals at 100 mg Aromox B-W 500/kg b.w./day and most animals at 200 mg Aromox B-W 500/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 Aromox B-W 500/kg b.w./day laboured breathing was ob-served for one male and rough fur was noted several females during the pre-mating, mating, gesta-tion 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 Aromox B-W 500/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 Aromox B-W 500/kg b.w./day, though no dose-response relationship was noted. A slight but not significant re-duction was also observed for the males at 100 and 200 mg Aromox B-W 500/kg b.w./day.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
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 Aromox B-W 500/kg b.w./day.
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 Aromox B-W 500/kg b.w./day during the pre-mating, gestation and/or lactation period, respectively.
Treatment with 200 mg Aromox B-W 500/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 Aromox B-W 500/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.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No test item-related influence was noted on the female fertility index at any of the tested dose levels.
Evaluation of the pre-coital time: No test item-related influence was noted.
Evaluation of reproduction parameters of the dams: There were no test item-related differences in the number of corpora lutea, implantation sites, in the number and sex of pups, runts or malformed pups. No test item-related influence was noted in the values calculated for the gestation length, the gestation index, the birth index and the live birth index between the control group and the animals treated with 40, 100 or 200 mg Aromox B-W 500/kg b.w./day. No test item-related increase in pre-implantation loss was noted in the dams after treatment with 40 or 100 mg Aromox B-W 500/kg b.w./day throughout the study up to day 3 post-partum. Treatment with 200 mg Aromox B-W 500/kg b.w./day resulted in a statistically significant (at p  0.05) increase of the pre-implantation loss by 20.7% compared to the control (10.5%). The post-implantation loss was not influenced at any dose level.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

ORGAN WEIGHTS (PARENTAL ANIMALS)
Main study animals: No test item-related influence was noted.

GROSS PATHOLOGY (PARENTAL ANIMALS)
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 Aromox B-W 500/kg b.w./day.
Recovery period: Satellite animals: An increased salivation was still noted for 2 of 5 males previously treated with 200 mg Aromox B-W 500/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 (PARENTAL ANIMALS)
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 Aromox B-W 500/kg b.w./day. These lesions were completely reversible within the 16-days re-covery 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 Aromox B-W 500/kg b.w./day. The effect was still noted at the end of the recovery period.

OTHER FINDINGS (PARENTAL ANIMALS)
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 Aromox B-W 500/kg b.w./day compared to the control. Changes in haematological parameters were noted for the animals treated with 200 mg Aromox B-W 500/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 Aromox B-W 500/kg b.w./day compared to the control.
For the male and/or female animals treated with 40, 100 or 200 mg Aromox B-W 500/kg b.w./day the following changes in biochemical parameters were observed: Increased ALAT statistically significant (p ≤ 0.01) for both males and females.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Pre-implantation loss was noted at the high dose of 200 mg/kg b.w./day.

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:

not examined

VIABILITY (OFFSPRING)
No test item-related mortality occurred.

BODY WEIGHT (OFFSPRING)
No test item-related influence was noted on the mean and total litter weight at any of the tested dose levels.

GROSS PATHOLOGY (OFFSPRING)
External examinations at dissection revealed no external abnormalities in any of the pups exam-ined.

OTHER FINDINGS (OFFSPRING)
Behaviour: No abnormal behaviour was noted.

Reproductive effects observed:

not specified

Conclusions:

Aromox B-W 500 (C12-18 alkyldimethylamine oxide) was assessed in a screening test for reproductive/developmental toxicity according to OECD guideline 422. No test item-related mortality was noted. No test item-related influence was noted on the female fertility index at any of the tested dose levels. No test item-related influence was noted on the pre-coital time. There were no test item-related differences in the number of corpora lutea, implantation sites, in the number and sex of pups, runts or malformed pups. No test item-related influence was noted in the values calculated for the gestation length, the gestation index, the birth index and the live birth index between the control group and the animals treated with 40, 100 or 200 mg Aromox B-W 500/kg b.w./day. No test item-related increase in pre-implantation loss was noted in the dams after treatment with 40 or 100 mg Aromox B-W 500/kg b.w./day throughout the study up to day 3 post-partum. Treatment with 200 mg Aromox B-W 500/kg b.w./day resulted in a statistically significant (at p  0.05) increase of the pre-implantation loss by 20.7% compared to the control (10.5%) The post-implantation loss was not influenced at any dose level. No test item-related mortality occurred In the F1 generation. External examinations at dissection revealed no external abnormalities in any of the pups examined.
Due to the pre-implantation loss noted at the high dose of 200 mg/kg b.w./day, p.o. the no-observed-effect level (NOEL) for reproductive toxicity was 100 mg/kg b.w./day, p.o. via gavage.

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:

Two screening studies are available, both of which are Klimisch 1. Quality is therefore high.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No reproductive toxicity studies are available for C12 -16 amine oxide. Data are read across from C12-14 and C12-18 AO on the basis of the equivalence in chemical structure, large overlap in chain length distributions between the substances and expected similarities in physico-chemical properties.

A combined repeat dose toxicity study with the reproduction/developmental toxicity screening test conducted under GLP according to OECD TG 422 is available for C12 -18 AO [Hansen B (2010)]. In this study rats (CD/Crl:CD(SD); 10 animals/sex/group) were dosed by oral gavage with C12 -18 AO at levels of 0, 40, 100 or 200 mg AO/kg bw/day for two weeks prior to mating, during mating and approximately two weeks after mating (males) and for two weeks prior to mating and continuing up to and including day 3 post-partum or the day prior to sacrifice for females. General toxicity effects in parent animals are discussed in the repeated dose toxicity section. No test item related influence was noted on the female fertility index or pre-coital time at any of the tested dose levels. There were no test item related differences in the number of corpora lutea, implantation sites, in the number and sex of pups, runts or malformed pups. No test item related influence was noted in the values calculated for the gestation length, the gestation index, the birth index and the live birth index between the control group and animals in any test group. No test item related increase in pre-implantation loss was noted in the dams after treatment with 40 or 100 mg AO/kg bw/day throughout the study up to day 3 post-partum. Treatment with 200 mg AO/kg bw/day resulted in a statistically significant (p<= 0.05) increase of the pre-implantation loss by 20.7 % compared to the control (10.5 %). The post implantation loss was not influenced at any dose level. In pups no test item related effects were noted for mortality, abnormal behaviour, mean and total litter weight or external abnormalities at any of the doses tested. Due to the pre-implantation loss noted at the high dose of 200 mg AO/kg bw/day the NOAEL for reproduction/developmental toxicity was 100 mg AO/kg bw/day.

In a combined repeat dose toxicity study with the reproduction/developmental toxicity screening test conducted under GLP according to OECD TG 422 C12 -14 AO 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. General toxicity effects on parent animals are discussed in the section on repeat dose toxicity. Microscopic examination of the reproductive organs of males and females treated at 250 mg AO/kg bw/day failed to reveal any abnormalities.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 NOAEL for reproduction/developmental toxicity was considered to be 100 mg/kg bw/day.

Short description of key information:
No reproductive toxicity studies are available for C12 -16 AO. Data are read across from C12-14 and C12-18 AO on the basis of the equivalence in chemical structure, large overlap in chain length distributions between the substances and expected similarities in physico-chemical properties.
In a combined repeat dose toxicity study with the reproduction/developmental toxicity screening test conducted under GLP according to OECD TG 422 C12-18 AO was administered via gavage to rats. The NOAEL for reproduction/developmental toxicity was considered to be 100 mg/kg bw/day.
In a similar study C12-14 AO was administered via gavage to 10 animals/sex/group at 0, 40, 100 or 250 mg/kg bw/day. The NOAEL for reproduction/developmental toxicity was considered to be 100 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

No developmental toxicity studies are available for C12 -16 amine oxide. Data are read across from C12-14 and C12-18 AO on the basis of the equivalence in chemical structure, large overlap in chain length distributions between the substances and expected similarities in physico-chemical properties.
In a prenatal developmental toxicity study in which pregnant female rats were dosed by gavage with C12-14 AO at 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:

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

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 for cross-reference:

reference to same study

Qualifier:

according to guideline

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 or test system 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

Type of inhalation exposure (if applicable):

other: N/A

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 (Klimisch score = 1) and two screening studies (Klimisch score = 1) available. Quality is therefore high.

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

No developmental toxicity studies are available for C12 -16 amine oxide. Data are read across from C12-14 and C12-18 AO on the basis of the equivalence in chemical structure, large overlap in chain length distributions between the substances and expected similarities in physico-chemical properties.

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 C12 -14 AO at 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 foetal 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 foetuses and early and late resorptions. The gravid uterus was weighed. Each foetus was removed from the uterus and subsequently weighed and examined for sex and gross external alterations. Approximately one-half of the foetuses in each litter were examined for soft tissue alterations using a variation of the microdissection techniques. The remaining foetuses in each litter were examined for skeletal alterations.

Two rats in the 200 mg AO/kg bw/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 bw/day dosage group rats and in significantly increased numbers of 200 mg AO/kg bw/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 faeces occurred in one or two rats in the 200 mg AO/kg bw/day dosage group. All necropsy observations were considered unrelated to the test substance.

Rats in the 100 and 200 mg AO/kg bw/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 bw/day dosage group for the entire gestation period (DGs 0 to 20). Body weights were significantly reduced in the 200 mg AO/kg bw/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 bw/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 bw/day dosage group. Relative feed consumption values for the entire dosage period were also significantly reduced in the 100 mg AO/kg bw/day dosage group, while values for the gestation period were significantly reduced in the 25, 100 and 200 mg AO/kg bw/day dosage groups.

Male and female foetal body weights were significantly reduced in the 200 mg AO/kg bw/day dosage group. Live litter size was decreased and the number of early resorptions was increased in the 200 mg AO/kg bw/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 foetuses and litters with alterations in the 200 mg AO/kg bw/day dosage group were significantly increased and reflected delays in skeletal ossification related to the significantly reduced foetal body weights in this group. These delays in ossification included significant increases in the foetal 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 bw/day.

A combined repeat dose toxicity study with the reproduction/developmental toxicity screening test conducted under GLP according to OECD TG 422 is available for C12 -18 AO [Hansen B (2010)]. In this study rats (CD/Crl:CD(SD); 10 animals/sex/group) were dosed by oral gavage with C12 -18 AO at levels of 0, 40, 100 or 200 mg AO/kg bw/day for two weeks prior to mating, during mating and approximately two weeks after mating (males) and for two weeks prior to mating and continuing up to and including day 3 post-partum or the day prior to sacrifice for females. General toxicity effects in parent animals are discussed in the repeated dose toxicity section. No test item related influence was noted on the female fertility index or pre-coital time at any of the tested dose levels. There were no test item related differences in the number of corpora lutea, implantation sites, in the number and sex of pups, runts or malformed pups. No test item related influence was noted in the values calculated for the gestation length, the gestation index, the birth index and the live birth index between the control group and animals in any test group. No test item related increase in pre-implantation loss was noted in the dams after treatment with 40 or 100 mg AO/kg bw/day throughout the study up to day 3 post-partum. Treatment with 200 mg AO/kg bw/day resulted in a statistically significant (p<= 0.05) increase of the pre-implantation loss by 20.7 % compared to the control (10.5 %). The post implantation loss was not influenced at any dose level. In pups no test item related effects were noted for mortality, abnormal behaviour, mean and total litter weight or external abnormalities at any of the doses tested. Due to the pre-implantation loss noted at the high dose of 200 mg AO/kg bw/day the NOAEL for reproduction/developmental toxicity was 100 mg AO/kg bw/day.

In a combined repeat dose toxicity study with the reproduction/developmental toxicity screening test conducted under GLP according to OECD TG 422 C12 -14 AO 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 NOAEL 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 CO2 and 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 oil via gavage 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 C12-18 AO and 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

No reproductive/developmental toxicity studies are available for C12 -16 amine oxide. Data are read across from C12-14 and C12-18 AO on the basis of the equivalence in chemical structure, large overlap in chain length distributions between the substances and expected similarities in physico-chemical properties.

In the prenatal developmental toxicity study performed with C12-14 AO 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, laboured 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 a secondary non-specific consequence of maternal toxicity.

In the OECD TG 422 study performed using C12-14 AO 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 NOAEL 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 a secondary non-specific consequence of maternal toxicity.

Based on the findings in these 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.