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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

A two-generation reproductive toxicity study is available on C12 AO. It was conducted according to Japan Ministry of Health and Welfare guideline and is comparable to the OECD 416 study design [Tesh JM (1983)]. The study was fully quality assured by the GLP certified testing facility where it was conducted.

Administration of the test substance continuously in the diet over two generations, at levels of up to 750 ppm, had no adverse effect on the general condition of male and female rats, but was associated with slight reductions in rate of bodyweight gain during maturation and gestation at levels of 375 ppm and above.

Mating performance, fertility, gestation and parturition were unaffected by treatment. Litter size at birth and viability of offspring up to weaning showed no consistent treatment-related effects.

At terminal necropsy of parents and offspring of both generations some variations in organ weights were found, but these were largely related to the deficits in bodyweight and no macroscopic or microscopic abnormalities were detected that could be related to ingestion of the substance.

The study report concludes no effects on fertility with a high dose NOAEL of 750 pmm in diet. Based on chemical intake data provided for the study, 750 ppm in diet translates to a dose range of ≥ 37 -128 mg/kg/day in male and female animals.

Link to relevant study records

Referenceopen allclose all

Endpoint:
two-generation reproductive toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
15-11-1979 to 08-03-1983
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Comparable to an OECD 416 study - Two-Generation Reproduction Toxicity Study
Justification for type of information:
1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
Please refer to the Amine Oxide Category justification attached in Section 13

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL
Please refer to the Amine Oxide Category justification attached in Section 13
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
Pre-mating dose period was 14 weeks (longer than the requirement in current OECD test guideline). Measurements of semen quality were not performed but there were no effects on fertility or observed in male reproductive organ histopathology.
Qualifier:
according to
Guideline:
other: Japan Ministry of Health and Welfare
GLP compliance:
not specified
Remarks:
Study was conducted in a GLP certified laboratory and was subject to extensive quality assurance inspections, but no GLP certificate was included in the study report.
Limit test:
no
Justification for study design:
Comparable to an OECD 416 study - Two-Generation Reproduction Toxicity Study.
Specific details on test material used for the study:
Surfactant A
N,N-dimethyl-dodecylamine oxide, CAS RN 1643-20-5; EC 216-700-6
Batch No. L-9146S
30% active ingredient in aqueous solution.
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat was selected because it satisfies the requirements of regulatory authorities; the Charles River CD rat (of Sprague-Dawley origin) in particular was chosen because of the background data available for this strain in the testing laboratories.
Sex:
male/female
Details on test animals and environmental conditions:
Weanling rats of the CD strain (of Sprague-Dawley origin) were obtained from Charles River U.K. Limited, Margate, Kent, England to form the F0 generation. They were approximately 30 days old on arrival and were allowed eight days of acclimatisation before commencement of treatment, during which time they were maintained on basal laboratory diet (Spratt's Laboratory Diet No. 2) and examined daily to check their physical condition. At commencement of the study, males were in the weight range of 158 to 187 g and females in the weight range of 116 to 144 g.

The animals were housed inside a barriered limited access rodent facility. Each animal room had its own supply of filtered air which was passed to atmosphere without re-circulation; there were approximately 15 room air changes per hour. The temperature and relative humidity in the animal room were recorded daily – 18-23°C, RH 40%-72%. The animals were subjected to a 12-hour light: 12-hour dark cycle.

Tap water was supplied to the cages via polythene bottles and chromium plated sipper tubes. A commercially available laboratory animal diet (Spratt's Laboratory Diet No. 2, ground) was fed ad libitum throughout the study.

Rats were housed in RC1 or RB3 cages; the cages consisted of high density polypropylene bodies with stainless steel lids and mesh floors (if present). Cages with mesh floors were suspended in batteries over trays covered with crepe absorbent paper; the latter was changed on alternate weekdays. Autoclaved wood shavings were provided for bedding during the littering phase. Cages were cleaned at least fortnightly.
Route of administration:
oral: feed
Vehicle:
other: Basal diet.
Remarks:
Spratt's Laboratory Diet No. 2, ground.
Details on exposure:
Continuously via the diet throughout the study.
Details on mating procedure:
After 101 days of treatment, the rats were mated by caging together one male with two females from the same treatment group. Each morning following pairing, the trays beneath the cages were checked for ejected copulation plugs and a vaginal smear was prepared from each female and examined for the presence of sspermatozoa. The day on which evidence of mating was found was designated Day 1 of gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the test diet mixtures (all concentrations) were taken at intervals throughout the study and a representative number of these were analysed for test chemical content and homogeneity of mixing. The analytical results showed the mean recovery was 92.7% ±11.1 for all diets analysed.
Duration of treatment / exposure:
Continuously via the diet throughout the study through out two generations until termination of study.
Frequency of treatment:
Continuously via the diet throughout the study.
Details on study schedule:
See attached background material section for details on the overall study design/schedule.
Dose / conc.:
0 ppm
Remarks:
Control
Dose / conc.:
750 ppm
Remarks:
See conversion to mg/kg bw/day below in "Any other information on materials and methods".
Dose / conc.:
1 500 ppm
Remarks:
Reduced after 6.5 weeks to lower dosage of 375 ppm due to marked inhibition of first parental bodyweight gain. See conversion to mg/kg bw/day below in "Any other information on materials and methods"
Dose / conc.:
3 000 ppm
Remarks:
Reduced after 6.5 weeks to lower dosage of 188 ppm due to marked inhibition of first parental bodyweight gain. See conversion to mg/kg bw/day below in "Any other information on materials and methods"
Dose / conc.:
375 ppm
Remarks:
Original 1500 ppm dosage reduced to 375 ppm after 6.5 weeks. See conversion to mg/kg bw/day below in "Any other information on materials and methods"
Dose / conc.:
188 ppm
Remarks:
Original 3000 ppm dosage reduced to 188 ppm after 6.5 weeks.. See conversion to mg/kg bw/day below in "Any other information on materials and methods"
No. of animals per sex per dose:
15 males and 30 females were treated for first parental (F0) generation. Following weaning, 15 males and 30 females from F1 generation were selected for continued treatment and were mated 120 days after selection.
Control animals:
yes, concurrent vehicle
Details on study design:
See attached background material section for details on the overall study design/schedule.
Positive control:
No
Parental animals: Observations and examinations:
All animals were examined daily throughout the study, and any visible signs of reaction to treatment were recorded, with details of type, severity, time of onset and duration.
Any animals found dead or killed in extremis were subjected to a thorough macroscopic examination of the visceral organs with the object of identifying the cause of death. Specimens of abnormal tissues were retained.

Bodyweights
Males were weighed weekly until termination. Females were weighed weekly until mating was detected, on Days 1, 3, 7,
14 and 21 post coitum and on Days 1, 4, 11, 18 and 25 post-partum. Weekly weighing was resumed thereafter until termination, but were not reported.' In addition males and females were weighed at Week 6| on the day that the dosages were lowered.

Food consumption
Food consumption was recorded weekly for each generation until the animals were paired for mating, with the exception of half weekly recordings in the week that dose levels were lowered.
Food conversion ratio and chemical intake
Food conversion ratio and chemical intake were calculated for the periods that food consumption was measured.

Once mating had been confirmed the males and females were separated and smearing discontinued.
If mating had not been detected within seven days of pairing, the female was removed and placed, where possible, with another male from the same treatment group. This could be repeated on one further occasion, thus allowing each animal a maximum of 21 days to achieve mating.
The same mating procedure was adopted for the Fi animals after the appropriate dosing period (i.e. 120 days after selection).
Fi males and females were paired within treatment groups avoiding matings between siblings.

Water consumption
Water intake was recorded daily for each generation until the animals were paired for mating.

Vaginal smears
For ten days before the scheduled day of pairing, daily vaginal- smears were taken from all groups to establish the duration and characteristics of the oestrous cycle. Smearing was continued after pairing with the male until evidence of mating was observed.

Pre-coital interval
The time elapsing between initial pairing and detection of mating was noted.

Duration of gestation
The time elapsing between the detection of mating and- commencement of parturition was recorded.

Duration of parturition
When labour difficulties were observed, an attempt was made to record duration of the parturition process.

Oestrous cyclicity (parental animals):
For ten days before the scheduled day of pairing, daily vaginal- smears were taken from all groups to establish the duration and characteristics of the oestrous cycle. Smearing was continued after pairing with the male until evidence of mating was observed.
Sperm parameters (parental animals):
Histopathological examinations of the testes and epididymides was performed.
Litter observations:
Observations on Day 1 post partum
All litters were observed and individually toe-marked at approximately 24 hours after birth and the following recorded for each litter:
a) number born (number alive and number dead)
b) individual birth weight
c) individual sexes
d) observations on individual offspring.

Signs
Litters were observed daily for evidence of abnormal behaviour.

Mortality and litter size
Daily records were maintained of mortality and consequent changes in litter size. Wherever possible, any offspring found dead were examined externally and internally in an attempt to determine the cause of death.

Bodyweight
Individual bodyweights of offspring were recorded on Days 1, 4, 11, 18 and 25 post-partum. Animals selected to establish the Fi generation were weighed weekly commencing with the day of selection.

Sex ratio
The offspring were sexed on Days T, 11 and 25 post-partum.

Physical development
The speed of physical development of the offspring was assessed on a total litter'basis by maintaining records of the days on which the onset of and completion of the following parameters occurred:
a) Pinna unfolding - detachment of the edge of the pinna
b) Hair growth - macroscopic observation of generalised growth of body hair
c) Tooth eruption - eruption of upper incisors through'.' the-gums
d) Eye opening - separation of the upper and lower eyelids.

Auditory and visual functions
After 25 days post-partum, auditory and visual responses- of the progeny were examined in a qualitative manner by means of the following techniques:
a) Auditory function
Auditory function was assessed using the startle response to a sudden sharp noise.
b) Visual function
Visual function was assessed by:
i) examination of the pupil closure response to a bright point source of 1ight ii) assessment of the visual placing response.

Assessment of development and reproductive performance (Fi generation)
Selection
Following weaning, 15 male and 30 female offspring, were selected from each group using random number tables, to form the Fi generation (Addendum 4). Excessively heavy or light offspring were excluded from the selection.
Each animal was uniquely identified by an ear-notch.
The design conditions and serial observations were as described for the first generation.

Postmortem examinations (parental animals):
Males
Those males not selected for complete gross necropsy and histopathology were killed by inhaled carbon, dioxide and examined internally and externally for macroscopic abnormalities. Specimens of abnormal tissues were retained.

Females
Those F0 and F1 females not selected for complete gross necropsy and. histopathology or whose litter died were killed by inhaled carbon dioxide and examined externally and internally for macroscopic abnormalities.
When a litter died before weaning the mammary tissue of the female was examined and a specimen retained.
If any female failed to produce a viable litter by Day 26 post coitum, it was killed by inhaled carbon dioxide and examined externally and internally for macroscopic abnormalities and for the presence of implantation sites.
Females failing to mate within the 21-day pairing period were killed approximately 26 days after the last day of pairing by inhaled carbon dioxide and examined externally and internally for macroscopic abnormalities and for the presence of implantation sites.

Specimens of abnormal tissues were retained.

Histopathology (see Tissues list below)
From, the F0 and F1 generations, 10 males and 25 females (adults) from each group were killed by inhaled carbon dioxide and subjected to a complete gross necropsy and histopathological examination. Testes from the remaining F0 males were also retained for histopathological examination. Histopthological examinations included the eye.
In the case of the Fi and F2 generation weanlings, five randomly selected males and females from each group were killed by inhaled carbon dioxide and subjected to a complete gross necropsy and histopathological examination. Histopathological examinations included the eye.

Gross pathology
Any evidence of adhesion, deformation, invasion or other interaction between presumptive tumours and neighbouring structures was carefully recorded. Abnormalities not suggestive of neoplasia were also noted. The following sequence for locating macroscopically detectable abnormalities was adopted:
a) external openings
b) cranial cavity (including cranial nerves, pituitary and olfactory lobes)
c) subcutaneous structures (mammary glands, salivary glands, regional lymph nodes)
d) thoracic cavity (including openings of all heart chambers and inflation of the lungs)
e) abdominal cavity (including brief fixation of the bladder and examination of the mucosa; liver and kidneys were sectioned, and the stomach and caecum were opened, irrigated and examined on both surfaces).

Organ weight analysis
The following organs were weighed and expressed as percentages of bodyweight:
Pituitary
Prostate
Seminal vesicles
Spleen
Testes
Thyroid
Uterus.

Microscopic evaluation
Buffered 4% formaldehyde was used as fixative. Sections were stained with haematoxylin and eosin; other stains were used as required or indicated.
Additionally, all tissues displaying evidence of neoplastic change, from animals in all groups, were similarly evaluated.

Tissues examined by histopathology:
Adrenals
All tumours
Brain - cerebral cortex
- medulla
- cerebellum
Bone marrow smear (after air-drying fixation in anhydrous methanol)
Caecum
Duodenum
Epididymides
Eye and optic nerve (in Davidson's fluid)
Heart
Ileum
Kidneys
Liver
Lungs
Lymph nodes - cervical
- mesenteric
Mammary glands - posterior
Oesophagus
Ovaries
Pancreas
Pituitary
Prostate
Seminal vesicles
Spleen
Stomach
Testes
Thymus {where present)
Thyroid
Urinary bladder
Uterus





Postmortem examinations (offspring):
Unselected offspring
Those F1 and F2 offspring not selected for continuation of the study or for complete gross necropsy and histopathology were killed by inhaled carbon dioxide and examined externally and internally for macroscopic abnormalities. Specimens of abnormal tissues were retained.

Histopathology (see Tissue list under Postmortem examinations (parental animals))
In the case of the Fi and F2 generation weanlings, five randomly selected males and females from each group were killed by inhaled carbon dioxide and subjected to a complete gross necropsy and histopathological examination. Histopathological examinations included the eye.
Statistics:
The significance of inter-group differences was tested using appropriate statistical tests. The tests used were multiple 't'-test, Mann-Whitney U-test, Chi-squared test and Fisher's exact probability test (Armitage modification)
Reproductive indices:
Oestrous cycles
Pre-coital interval
Mating performance and fertility: % mating, conception rate, fertility index
Gestation length
Gestation index
Offspring viability indices:
Litter size
Offspring body weights
Live birth index
Vialbility index
Percentage mortality
Lacation index
Sex ratio
Offspring development timing: pinna unfolding, hair growth, tooth eruption, eye opening
Auditory and visual function tests
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The general condition of the P0 males and females showed no effects that could be attributed to treatment with Surfactant A.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female from Group 2 (750 ppm) was killed in extremis in the fifth week of treatments having shown a nasal discharge, pallor, hypoactivity, hunched back and weight loss. At necropsy the animal presented pallor of all internal tissues and an enlarged spleen and pancreatic lymph nodes. Microscopic examination revealed the presence of malignant lymphomas in the spleen and thymus.

A second Group 2 female (750 ppm) was killed in extremis on Day 25 post coitum following parturition difficulties. Necropsy revealed a torsion in the uterus adjacent to the cervix. The isolated nature of these findings suggested no involvement of Surfactant A.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was a marked, dose-related reduction in bodyweight gain of both sexes in all treated groups, commencing from initiation of treatment.
At six and a half weeks after commencement of treatment, the levels of Surfactant A were altered, such that Group 3 was reduced from 1500 to 375 ppm and Group 4 from 3000 to 188 ppm; Group 2 remained unaltered at 750 ppm. A period of recovery of bodyweight then occurred in Groups 3 and 4, with animals in these groups eventually attaining absolute values similar to those of Group 2; the rate of weight gain in all treated groups stabilised at a rate comparable with that of the controls. However, absolute bodyweight in all treated groups remained lower than that of controls until termination in the males, and until pairing in the females.

The rate of weight gain of females in Groups 3 and 4 (375 and 188 ppm) during gestation was slightly superior to that of the controls.

There was no effect on weight change during lactation.

See - Any other information on results section for male P0 body weights. See attached key data tables from the original study report.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
During the first six weeks, food intake showed a dosage-related reduction, the effect being slight in Group 2 (750 ppm) but more marked in Groups 3 and 4 (1500 and 3000 ppm). After the reduction in dose levels, food intakes of all groups became similar to those of controls, and thereafter no treatment-related effects were apparent.


Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Food conversion efficiency was reduced during the first week of treatment in Group 4 (3000 ppm) and was considerably increased in the same Group 4 during the first week after the levels had been reduced (188 ppm). At all other times, and in other treatment groups, consistent treatment-related effects on food conversion efficiency were apparent.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
During the first six weeks, water intake showed a dosage-related reduction, the effect being slight in Group 2 (750 ppm) but more marked in Groups 3 and 4 (1500 and 3000 ppm). After the reduction in dose levels, water intakes of all groups became similar to those of controls, and thereafter no treatment-related effects were apparent.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes associated with treatment.

There was a wide range of banal degenerative and inflammatory changes present, similar in type and incidence to those considered usual in CD rats at the testing laboratory. These included cortical fatty vacuolation and congestion in the adrenals; chronic myocarditis and congestion in the heart; slight nephrocalcinosis, congestion, lymphocytic infiltration and the presence of basophilic tubules and tubules dilated with eosinophilic proteinaceous material in the kidneys; hepatocytic (glycogen) pallor, anoxic vacuolation, congestion and pleomorphic cell and lymphocytic infiltration in the liver; slight peribronchiolar lymphoid hyperplasia, slight perivascular lymphocytic infiltration, accumulations of alveolar macrophages and focal vascular metastatic mineralisation in the lungs; paracortical plasmacytosis in the cervical lymph nodes; hypercellularity in the mesenteric lymph nodes; siight haemosiderosis and erythrocytes and erythrophagocytosis in sinuses in thymic lymph nodes (females); fibrosis, acinar degeneration and slight haemosiderosis in the pancreas; haemosiderosis in the spleen; focal pooling of eosinophilic proteinaceous material in the interstitium in testes and hydrometra in the uterus.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Majority of females in all groups had normal regular oesrous cycles.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no histopathological findings to suggest an effect on spermatogenesis.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Some slight inter-group differences were recorded in mating performance, conception rate, and overall fertility index but they were not consistent or treatment-related. Gestation length was similar in all groups. Gestation index was marginally reduced in Group 3 (375 ppm) due to three females that failed to litter but showed no evidence of implantations and there were no effects at 750 ppm.
The only adverse effects were a slight reduction in the rate of body weight gain in males at 375 ppm (from initial 6 weeks of treatment at the higher dose level) and both males and females at 750 ppm, but there were no treatment-related effects on reproductive performance.
Key result
Dose descriptor:
NOAEL
Remarks:
general
Effect level:
375 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
LOAEL
Remarks:
general
Effect level:
750 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive
Effect level:
750 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive
Effect level:
>= 37 - <= 128 mg/kg bw/day
Based on:
act. ingr.
Sex:
male
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive
Effect level:
>= 47 - <= 119 mg/kg bw/day
Based on:
act. ingr.
Sex:
female
Basis for effect level:
reproductive performance
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
The general condition of P1 animals was similar in all groups.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One Group 2 (750 ppm) female died in the sixth week of treatment. The animal had shown no previous adverse symptoms and, at necropsy the cause of death could not be established.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The rate of bodyweight gain of P1 males showed a slight dosage-related reduction at 750 and 375 ppm, but was similar to that of controls at 188 ppm. In females, the rate of bodyweight gain was also slightly lower at 750 ppm before pairing and during the later stages of gestation. Group 3 and 4 (375 and 188 ppm) females were unaffected and, during lactation, weight change was similar in all groups.

See attached key data tables from the original study report.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Although some inter-group variations were observed, food intake was essentially similar in all groups.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
Food conversion efficiency was essentially similar in all groups.
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Although some inter-group variations were observed, water intake was essentially similar in all groups.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The reduced bodyweight of treated males was accompanied by a reduction in absolute weight of several organs, a small number of which achieved statistical significance. However, when organ weights were expressed relative to bodyweight, no significant inter-group differences were found.

The majority of absolute and relative organ weights of females were similar in all groups, and no consistent treatment-related effects were apparent.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic changes were noted at terminal necropsy of the P1 animals that could be attributed to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no changes associated with treatment.

There was a wide range of banal degenerative and inflammatory changes present, similar in type and incidence to those considered usual in CD rats at this laboratory. These included slight diffuse cortical fatty changes in the adrenals; myocarditis slight geriatric nephropathy and nephrocalcinosis in the kidneys; lymphocytic infiltration, hepatocytic (glycogen) pallor, periacinar anoxic vacuolation and slight centriacinar hepatocytic fine vacuolar fatty change in the liver; peribronchiolar lymphoid hyperplasia, perivascular lymphocytic infiltration and focal vascular mineralisation in the lungs; hypercellularity in the cervical lymph nodes; acinar hyperplasia in the mammary glands; chronic inflammation of islet tissue and peri-islet haemosiderosis in the pancreas; haemosiderosis in the spleen, dilatation of glands in the stomach and dilatation of the lumen of the uterus.

One observation of a cataract was noted in a low-dose animal in this generation by histopathology. This single instance was not dose-related and therefore not deemed treatment-related.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The majority of females in all groups had normal regular oestrous cycles of four to five days duration.

See attached key data tables from the original study report.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No effects on the reproductive organs were evident after histopathological examination.
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating performance, conception rate and fertility of P1 males and females showed no adverse effects that could be related to treatment.
Gestation lentth and gestation index were similar in all dose groups.

See attached key data tables from the original study report.
The only adverse effects were a very slight reduction in body weight gain in males at 375 and a slight reduction in males at 750 ppm. In females, the rate of bodyweight gain was slightly lower at 750 ppm only before pairing and during the later stages of gestation. There were no treatment-related effects on reproductive performance.
Key result
Dose descriptor:
NOAEL
Remarks:
general
Effect level:
375 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
LOAEL
Remarks:
general
Effect level:
750 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive
Effect level:
750 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive
Effect level:
>= 37 - <= 128 mg/kg bw/day
Based on:
act. ingr.
Sex:
male
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
NOAEL
Effect level:
>= 47 - <= 119 mg/kg bw/day
Based on:
act. ingr.
Sex:
female
Basis for effect level:
reproductive performance
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
The general condition of F1 animals was similar in all groups.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
One Group 2 female (750 ppm) died in the sixth week of treatment. The animal had shown no previous adverse symptoms and, at necropsy, the cause of death could not be established.

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At Day 1 post partum, bodyweights of offspring were slighltly lower than those of concurrent controls but the values were within historical control range. During lacation a dose-related reduction in weight gain was recorded for all treatment groups.

See attached key data tables from the original study report.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
There was some inter-group variation in sex ratio, but no treatment-related trends were apparent. Offspring development as assessed by the onset of pinna unfolding, hair growth, tooth eruption, and eye opening, were similar in control and treated groups.

See attached key data tables from the original study report.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Animals not selected for the mating phase:
Absolute and relative weights of the spleen were slightly reduced in male offspring in Groups 2 and 3 (750 and 375 ppm) and in female offspring of all treated groups. A number of other inter-group differences in absolute and/or relative organ weights were recorded, but none showed any consistent association with treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic changes were noted at terminal necropsy of the F1 animals that could be attributed to treatment.
Histopathological findings:
no effects observed
Description (incidence and severity):
Animals not selected for the mating phase:
There were no changes associated with treatment in either the testes or other organs.

There was a range of banal degenerative and inflammatory changes present, similar in type and incidence to those considered usual in CD rats at this laboratory. These included hepatocytic (glycogen) pallor, hepatocytic fine fatty vacuolation and slight extramedullary haemopoiesis in the liver, slight peribronchiolar lymphoid hyperplasia and splenic extramedullary haemopoiesis.
Other effects:
no effects observed
Description (incidence and severity):
Auditory and visual responses were normal in all offspring of control and treated groups.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
At Day 1 post partum, bodyweights were slighltly lower than those of concurrent controls but the values were within historical control range.

Litter size at birth and live birth index were unaffected by treatment. Slight inter-group differences in viability indices were recorded, but there were no patterns of any adverse effects of treatment with Surfactant A.

Live birth and viability indices were unaffected in all groups.
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
750 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
Key result
Dose descriptor:
NOAEL
Remarks:
developmetal
Generation:
F1
Effect level:
>= 37 - <= 128 mg/kg bw/day
Based on:
act. ingr.
Sex:
male
Basis for effect level:
viability
sexual maturation
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
>= 47 - <= 119 mg/kg bw/day
Based on:
act. ingr.
Sex:
female
Basis for effect level:
viability
sexual maturation
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The number of offspring born in all groups was low in the 750 ppm group when compared to other groups.

Live birth index showed no treatment-related effect, but subsequent offspring viability was slightly reduced in Groups 2 and 4 (750 and 188 ppm) and mortality index correspondingly slightly elevated. Majority of offspring tht died did so during first four days post-partum and had no milk in their stomachs at necropsy. Offspring viability in Group 3 (375 ppm) was similar to controls.

The majority of offspring that died did so during the first four days post partum; at necropsy several were found to have no milk in their stomachs. Offspring viability in Group 3 (375 ppm) was similar to that of the controls.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At Day 1 post partum, bodyweights of offspring were all within the normal range. At Day 25 post-partum only was a dosage-related reduction in rate of weight gain at 375 and 750 ppm seen.

See attached key data tables from the original study report.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
The ratio of male to female offspring showed no treatment-related effects. Offspring development as assessed by the onset of pinna unfolding, hair growth, tooth eruption, and eye opening, were similar in control and treated groups.

See attached key data tables from the original study report.

See attached key data tables from the original study report.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were a small number of inter-group differences in absolute and relative organ weights, the majority of which did not appear to be related to treatment. Slight reductions in the absolute and relative spleen weights of male offspring in Groups 2 and 4 (750 and 188 ppm) and of female offspring in all treated groups was observed.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A small number of abnormalities were recorded in all groups at terminal necropsy. Two Group 2 offspring (750 ppm), not selected for full histopathology, had cerebral anomalies, one with hydrocephaly and the other with a domed skull and congested area in one cerebral hemisphere. The isolated nature of these observations did not suggest they were treatment-related.
Histopathological findings:
no effects observed
Description (incidence and severity):
There were no changes associated with treatment.

There was a range of banal degenerative and inflammatory changes present, similar in type and incidence to those considered usual in CD rats at this laboratory. These included lymphocytic infiltration and hepatocytic (glycogen) pallor in the liver; slight peribronchiolar lymphoid hyperplasia in the lungs and extramedullary haemopoiesis in the spleen
Other effects:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on the auditory and visual responses of the offspring.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F2
Effect level:
750 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F2
Effect level:
>= 37 - <= 128 mg/kg bw/day
Based on:
act. ingr.
Sex:
male
Basis for effect level:
viability
sexual maturation
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F2
Effect level:
>= 47 - <= 119 mg/kg bw/day
Based on:
act. ingr.
Sex:
female
Basis for effect level:
viability
sexual maturation
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
750 ppm
Treatment related:
no

Group mean bodyweights (g ± S.D.) of males - Weeks 0 - 6 – F0

Group

1

2

 3               4

 

Compound

Control

Surfactant A    

 

Level (ppm)

0

750

 1500       3000

 

 

 

Group

Number

of

animals

 

Week of treatment

0

1

2

3

4

5

6

6 BWC

1

15

Mean

S.D.

175

8

235

12

285

17

331

22

368

26

398

30

426

34

434'

35

2

15

Mean

S.D.

176

9

233

12

281

16

323

18

357

22

386

22

410

29

419

30

3

15

Mean

S.D.

174

8

226

10

270

13

309

18

343

21

372

23

396

27

404*

28

4

15

Mean

S.D.

175

7

210

13

250

16

286

20

315

24

338

26

358

30

365***

31

* Bodyweight change Weeks 0 – 6 significantly different from Controls, p < 0.05 (Multiple t-test). 
*** Bodyweight change Weeks 0 - 6| significantly different from Controls, p < 0.001 (Multiple test).

Group mean bodyweights (g ± S.D.) of males - Weeks 6- 14 – F0

 

Group

Number

of

animals

 

Week of treatment

6|

7

i

8

9

10

n

12

13

14

1

15

Mean

S.D.

434

35

443

38

460

44

476

47

487

50

500

52

509

55

520

58

529

60

2

15

Mean

S.D.

419

30

427

32

443

35

458

37

470

38

478

42

487

44

496

43

509

44

    3

15

Mean

S.D.

404

28

415

32

432

34

445

36

460

35

466

38

477

40

490

43

502

47

4

15

Mean

S.D.

365

31

382

34

407

38

424

41

441

41

454

42

467

46

485

49

499

49

Conclusions:
This two-generation study in rats was conducted using a test material that was 30% linear C12 amine oxide in water. Linear C12 amine oxide is within the category established for read across. The study was conducted according to Japanese MHW guidelines in place at the time. It is considered to be of high quality (K1). The study protocol was comparable to two-generation protocols used around the world at the time the study was performed (1979-80) and is very similar in design to OECD TG 416.

The study used CD rats. Both males and females were dosed. There were 15 males and 30 females per group. The test material was administered in the diet at three concentrations: 750, 1500 and 3000 ppm. After six-and-a-half weeks, the two highest concentrations were producing an excessive amount of toxicity and therefore these doses were reduced from 3000 ppm to 188 ppm, and from 1500 ppm to 375 ppm, for the remainder of the study. Because animals decrease food consumption as they age, the dosages (adjusted for active material) in each group were calculated based on food/chemical intake and are given as ranges (see table below). After dosing for 101 days (approximately 14 weeks), the animals were mated, two females per male, within treatment groups. The pre-mating dose period of 14 weeks is longer than the requirement in current guidelines and is sufficient to cover more than one spermatogenic cycle in males, and many estrous cycles in females. Dosing was continued through mating, gestation, and rearing of the F1 generation. At weaning, F1 animals were selected at random to constitute groups of 15 male and 30 female rats per group. Dosing continued in these groups. After 120 days these animals were bred to produce an F2 generation.

Concentration in diet (ppm) Chemical intake range (F0), mg/kg/day (based on active) Chemical intake range (F1), mg/kg/day (based on active)
750 (males) 39-87 37-128
750 (females) 49-94 47-119
1500 (males; first 6.5 weeks) 97-168 NA
1500 (female; first 6.5 weeks) 120-192 NA
3000 (male; first 6.5 weeks) 194-308 NA
3000 (female; first 6.5 weeks) 259-353 NA
375 (males) 19-25 19-58
375 (females) 25-30 24-62
188 (males) 10-13 9-31
188 (females) 12-17 11-29

Observations included measurements of body weight, food and water consumption and cage-side observations at regular intervals in both generations. Estrous cyclicity was evaluated in females prior to mating. Fertility was assessed as a measure of reproductive health, as was gestation length, and viability and growth of offspring. In addition, F1 animals were evaluated for the time of achieving various developmental milestones (tooth eruption, eye opening, pinna detachment, appearance of fur), as well as neurobehavioral development (auditory startle and visual reflexes, motor activity, and development of strength and coordination. Animals in each generation were evaluated histopathologically. The only measurements not included in this study that are part of a more modern two-generation study (or extended one-generation study) are measurements of semen quality. However, that there were no effects on fertility or on histology of the reproductive organs, and by inference then sperm quality was unaffected.

The 1500 and 3000 ppm concentrations had to be reduced (as described above) in order to continue the study because they produced an unacceptably severe effect on body weight. Subsequently, the only adverse effects were a slight reduction in the rate of body weight gain in males in the 375 and 750 ppm groups and females in the 750 ppm group. There were no effects on estrous cyclicity or on fertility in either generation. There was a slight reduction in number of F2 offspring in the 750 ppm group, but otherwise there were no effects on any of the developmental parameters evaluated in this study. The study report concludes that there were no effects on fertility or development, with a NOAEL of 750 ppm or greater. Using the chemical intake data from the study report, this translates to a dose range of > 37-128 mg/kg/day for males and 47-119 mg/kg/day for females.

While this study does not include all of the endpoints present in the current extended one-generation study, it is sufficient to establish a lack of effect on reproduction and fertility. There were no effects on estrus cyclicity in any of the groups. The time required for successful mating was similar across all groups, as was the pregnancy rate. Histopathological evaluation of reproductive organs indicated no effects of the test agent. The current extended one-generation protocol calls for neurobehavioral or immunotoxicity measurements to be added to the study if there are any signals, such as neurotoxicity or immunotoxicity of analogs, or morphological/ histological effects on the nervous system or immune system. There was no evidence of either histologically. The study design used in this study evaluated a number of neurobehavioral parameters, none of which was affected by treatment. It can be concluded that neurobehavioral or immunological evaluations would not have been triggered. The study report concludes that there were no effects on fertility or development, with a NOAEL of 750 ppm or greater. Using the chemical intake data from the study report, this translates to a dose range of > 37-128 mg/kg/day.

It should be noted that in repeated dose studies with this substance, treatment-related ocular changes were noted. In this study however, there were no treatment-related observations of ocular problems noted in any of the three generations (no effects noted clinically or in histopathology). One observation of a cataract was noted in a low-dose animal in the F1 generation by histopathology. This single instance was not dose-related. Given the high spontaneous rate of cataracts in albino rats (Durand et al. 2001), it is not surprising to see a single observation of a cataract in a study in which a large number of animals were evaluated. It is also worth noting that the observation of a cataract provides evidence of the laboratory’s ability to detect cataract by histopathology.
1) Durand, G et al. Spontaneous polar anterior subcapsular lenticular opacity in Sprague-Dawley rats. Comp Med. 2001 Apr;51(2):176-179.
Executive summary:

The study design used was comparable to the OECD 416 two-generation study. Surfactant A was administered in the diet initially, at levels of 750, 1500 and 3000 ppm, to male and female rats of the P0 generation. However, following a marked inhibition of bodyweight gain at the two highest levels, these were reduced respectively to 375 and 188 ppm, after six and a half weeks of treatment. Treatment of the P0 generation continued at these levels for the remainder of the maturation period and throughout mating, gestation and lactation. Using selected animals from the F1 offspring, treatment continued at dietary levels of 188, 375 and 750 ppm throughout maturation, mating, gestation and lactation of a second generation. Throughout the study, a fourth group serving as controls, received untreated diet.

The general condition of animals throughout the study was unaffected by Surfactant A treatment. After the reduction of treatment level's, the rate of bodyweight gain increased in animals that had been receiving 1500 and 3000 ppm, but, at all treatment levels, absolute bodyweight of both sexes remained slightly below that of controls. The rate of bodyweight gain was slightly reduced in parental males at levels of 375 ppm and 750 ppm (not significant), and in parental females at 750 ppm.

Mating performance, fertility and conception rate presented no effects that could be related to treatment in either generation.

Gestation and parturition proceeded normally and, except for a slight (not significant) reduction in the number of F2 offspring born at the 750 ppm level. However, there were no adverse effects of treatment on litter size at birth, live birth index and birth weight in either generation.

Viability of offspring was unaffected in the first generation, but there were slight reductions (not significant) in viability of F2 offspring at the 138 and 750 ppm levels. At all treatment levels the rate of bodyweight gains of F1 and F2 offspring was reduced (but only on one occasion, Day 25 post-partum) during the lactation period.

At terminal necropsy of P0 and P1 adults and F1 and F2 offspring, no macroscopic abnormalities attributable to treatment with Surfactant A were observed. There were some slight inter-group differences in organ weights, but the majority were related to bodyweight deficits, and no treatment-related histopathological changes were shown.

It was concluded from this investigation that dietary administration of Surfactant A to male and female rats for two generations, at concentrations of 188, 375 and 750 ppm, was associated with slight reductions in weight gain of both parents and offspring, but was without adverse effect on their mating performance and fertility or on development of the offspring.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
07-11-2007 to 28-05-2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
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.
Dose / conc.:
0 other: mg AO/kg bw/day (actual dose received)
Dose / conc.:
40 other: mg AO/kg bw/day (actual dose received)
Dose / conc.:
100 other: mg AO/kg bw/day (actual dose received)
Dose / conc.:
200 other: mg AO/kg bw/day (actual dose received)
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous dose range finding toxicity study in Han Wistar rats, RCC study No. B51592 in which dose levels of 30, 60, 120, 500 and 1000 mg/kg/day corrected for purity were tested. Both the 1000 and 500 mg/kg/day resulted in lethality after a single or two doses, respectively. The overall NOEL was 120 mg/kg/day (corrected for purity).
- Rationale for animal assignment: Computer-generated random algorithm was used, with body weights taken into consideration in order to ensure similar mean body weights in all groups.
10 mL/kg bw was administered.
Positive control:
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
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:
act. ingr.
Sex:
male/female
Basis for effect level:
other: At doses of ≥100 mg AO/kg bw/day: reduced activity, body weight gain and food consumption noted; forestomach lesions observed. At 250 mg AO/kg bw/day: increased liver weights and microscopic changes in liver and kidney.
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.
Dose descriptor:
NOEL
Generation:
F1
Effect level:
100 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Post-natal day 0-4 pup loss and reduced mean pup weight at 250 mg AO/kg bw/day
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.
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
23-03-2010 to 19-05-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
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. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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.
Dose / conc.:
40 other: mg AO/kg bw/day
Dose / conc.:
100 other: mg AO/kg bw/day
Dose / conc.:
200 other: mg AO/kg bw/day
No. of animals per sex per dose:
Main Study:
80 animals (40 males and 40 females)

Recovery period: 20 animals (10 males and 10 females) were allocated to groups 1 and 4 for a 14-day recovery period. These animals were not mated.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on available toxicological data and a preliminary dose-range finding study (LPT study no. 25122). No mortality was noted in this dose-range-finding study no. 25122. Signs of systemic intolerance were noted in the animals of both sexes starting at 40 mg AO/kg b.w./day. A slightly to severely reduced mean food consumption and mean body weight were noted in the male rats starting at 100 mg AO/kg b.w./day or at 250 mg AO/kg b.w./day, respectively. The females were not affected.
Necropsy revealed a thickened cardiac region of the stomach in one male rat treated with 250 mg AO/kg b.w./day as well as in all high dosed male and female rats treated with 400 mg AO/kg b.w./day.

- Rationale for animal assignment: Random. At commencement of the study, the weight variation of animals used was minimal and did not exceed 20% of the mean weight of each sex.
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:
40 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Histopathological changes noted in the forestomach and the mesenteric lymph nodes and signs of systemic toxicity (salivation) observed at dose levels of 100 and/or 200 mg AO/kg bw/day
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.
Dose descriptor:
NOAEL
Generation:
F1
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 AO/kg b.w./day.
Reproductive effects observed:
not specified
Conclusions:
C12-18 AO 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 AO/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 AO/kg b.w./day throughout the study up to day 3 post-partum. Treatment with 200 mg AO/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 AO/kg b.w./day.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
37 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Reliability 1 for the 2-generation study.
Reliability 1 for the entire database.
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 addition to the key study (i.e. the two-generation reproductive toxicity study) on C12 AO, there are combined repeat dose toxicity and reproductive developmental toxicity screening studies, conducted under GLP according to OECD TG 422, available on the registered substance, C12-14 AO and on a category member, C12-18 AO.

In the C12-14 AO study the substance was administered via gavage to 10 animals/sex/group at 0, 40, 100 or 250 mg AO/kg bw/day [Ceccatelli et al (2008)]. 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. No adverse effects on fertility were noted at any dose level. Litter size and mean number of pups at first litter check and sex ratio were not affected by treatment. No abnormal pups were noted at any dose level. However, a statistically significant increase in pup deaths was observed on postnatal days 0 -4 at the high dose of 250 mg AO/kg/day. Although the higher postnatal loss was within the range of historical control data, this resulted in a reduced number of pups and mean pup weight during development was reduced at this dose. The NOEL for reproductive/developmental toxicity in this study was 100 mg AO/kg bw/day.

In the C12-18 AO study the substance was administered via gavage to 10 rats/sex/group at 0, 40, 100 or 200 mg AO/kg bw/day [Hansen B (2010)]. Males were dosed for two weeks prior to mating, during mating and approximately two weeks after mating. Females were dosed for two weeks prior to mating and continuing up to and including day 3 post-partum or the day prior to sacrifice. No adverse effects on fertility were noted at any dose level. Litter size and mean number of pups at first litter check and sex ratio were not affected by treatment. No abnormal pups were noted at any dose level. However, treatment with 200 mg AO/kg bw/day resulted in a statistically significant increase in pre-implantation loss compared to controls. Post implantation loss was not influenced at any dose level. The NOAEL for reproductive/developmental toxicity in this study was 100 mg AO/kg bw/day.

Effects on developmental toxicity

Description of key information

A prenatal developmental toxicity study is available on C12 -14 AO. The study was performed under GLP according to EPA OTS 798.4900 test guideline [York RG (1999)]. In the 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/day on days 6 through 19 of presumed gestation. 

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 was occurred in 100 mg AO/kg bw/day dose groups. All necropsy observations were considered unrelated to treatment with the test substance.

Rats in the 100 mg AO/kg bw/day dose 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 dose group for the entire gestation period (DGs 0 to 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 and relative feed consumption values were significantly reduced in the 200 mg AO/kg bw/day dose group. Relative feed consumption values were also significantly reduced in the 100 mg AO/kg bw/day dose group for the entire doseage period, while values for the gestation period were significantly reduced in all treatment groups.

Fetal 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 primarily as the result of one dam in this dose group that had a litter consisting of 16 early resorptions.

The percentages of fetuses 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 fetal body weights in this group. Similar but lower incidence and less severe delays in ossification also occurred in the 100 mg AO/kg bw/day dose group. The, the NOAELs for both maternal toxicity and developmental toxicity in this study are 25 mg AO/kg bw/day.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Remarks:
Rabbit - dose range finding study.
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
04-07-1979 to 29-11-1979
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
Rabbit - dose range finding study.
Qualifier:
no guideline required
Principles of method if other than guideline:
Study was a dose range finding study for a main developmental toxicity study.
GLP compliance:
not specified
Remarks:
Study conducted in a GLP certified laboratory to GLP standards.
Limit test:
no
Specific details on test material used for the study:
Surfactant A
N,N-dimethyl-dodecylamine oxide, CAS RN 1643-20-5; EC 216-700-6
Batch No. L-9264S
30% active ingredient in aqueous solution.
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Sexually mature virgin female New Zealand White rabbits of the Morton Line, Morton Rabbi tries, Stansted, Essex, England, were used in the investigation. They were approximately 24 weeks old on arrival and were allowed a minimum of one week's acclimatisation before insemination. At commencement of the study the animals were in the bodyweight range 3.0 - 3.8 kg.
All animals were housed singly in galvanised steel caging. They were allowed free access to Beta Rabbit Standard Diet (803 181W) and to tap water.
Route of administration:
oral: gavage
Details on exposure:
Days 6 to 18 of gestation.
Analytical verification of doses or concentrations:
no
Details on mating procedure:
Females were artificially inseminated using pooled semen from fertile males of the same strain. Following insemination each female was injected intravenously with 25 i.u. luteinising hormone (Pregnyl, Organon) to ensure successful ovulation. The day of insemination was designated Day 0 of gestation.
Duration of treatment / exposure:
Days 6 to 18 of gestation.
Frequency of treatment:
Daily Days 6 to 18 of gestation.
Duration of test:
Day 0 to Day 29 of gestation.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
75 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 or 6
Control animals:
yes, concurrent vehicle
Details on study design:
Dose range finding study design was essentially that of a developmental toxicity study but without detailed skeletal or visceral examinations.
Maternal examinations:
All animals were examined daily throughout the study and any visible signs of reaction to treatment were recorded with details of type, severity, duration and time of onset.
Any animals found dead or killed in extremis were subjected to a thorough macroscopic examination of the visceral organs with the object of identifying the cause of death.
Animals were weighed daily and means calculated and reported on Days 0, 6, 8, 10, 12, 14, 16, 18, 23 and 28 after insemination.
The food and water consumption of each animal was recorded over five phases during pregnancy:
Phase 1 Days 1-5 after insemination
Phase 2 Days 6-11 after insemination
Phase 3 Days 12-17 after insemination
Phase 4 Days 18-23 after insemination
Phase 5 Days 24-28 after insemination.
Ovaries and uterine content:
On Day 29 after insemination females were killed by cervical dislocation for examination of their uterine contents. Each animal was first examined macroscopically for evidence of disease or adverse reaction to treatment. The reproductive tract, complete with ovaries, was dissected out and the following recorded:
a) number of corpora lutea in each ovary
b) number of implantation sites
c) number of resorption sites (classified as early or late)
d) number and distribution of live and dead foetuses in each uterine horn (including an estimation of the time of death of non-viable foetuses)

Fetal examinations:
a) weight and sex of individual foetuses
b) individual placental weights
c) external abnormalities of individual foetuses
d) internal examinations: the thoracic and abdominal cavities of all foetuses of each litter were dissected out and examined. Low-power magnification was used if necessary.
After internal examination, all foetuses were eviscerated and placed in industrial methylated spirit (74 o.p.) and stored.
Statistics:
The significance of inter-group differences was tested using appropriate statistical tests: Multiple t-test, t-test or Mann-Whitney U-test.
Indices:
Foetal and placental weights.
Resorptions: pre- and post-implantation loss.
Historical control data:
Yes, the test facility background control range.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The general condition of animals in the treated groups was inferior to that of the controls, with increased incidences of scouring, emaciation, nasal discharge, dyspnoea and respiratory noise. Two Group 4 females (300 mg/kg/day) developed sores around the mouth.
Mortality:
mortality observed, treatment-related
Description (incidence):
A total of ten animals died or were killed in extremis during the study, with the mortality rate greatest in the higher dosage groups. At necropsy, increased incidences of enteric disorder and respiratory tract lesions were recorded in all treated groups, but especially at the highest dosage level.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The overall maternal weight gain was similar in all groups. The weight loss recorded in Group 4 (300 mg/kg/day), between Days 16 and 23 of gestation was due to a single female (animal No. 3TA010).
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Despite some inter-group variation in food and water consumption, no significant trends were observed.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The majority of females killed on Day 29 of gestation showed no treatment-related anomalies. However, one female from Group 4 (300 mg/kg/day; animal No. 3TA010) had the right cranial lung lobe hard, necrotic and containing pus, with the other lung lobes slightly necrotic. This condition resembled that seen in several of the treated females that died during the study.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
See data table: Any other information on results.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
See data table: Any other information on results.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
See data table: Any other information on results.
One control female exhibited total litter resorption, but all other pregnant females carried their litters successfully through to Day 29 of gestation
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
See data table: Any other information on results.
The numbers of implantations and viable young showed no adverse effects of treatment, but in Group 4 (300 mg/kg/day) there was a slight increase in early resorptions resulting in an elevated post-implantation loss.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
The general condition of animals in the treated groups was inferior to that of the controls, with increased incidences of scouring, emaciation, nasal discharge, dyspnoea and respiratory noise. Two Group 4 females (300 mg/kg/day) developed sores around the mouth. A total of ten animals died or were killed in extremis during the study, with the mortality rate greatest in the higher dosage groups - two rabbits were killed in extremis at 300 mg/kg/day. At necropsy, increased incidences of enteric disorder and respiratory tract lesions were recorded in all treated groups, but especially at the highest dosage level.
Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
mortality
Abnormalities:
effects observed, treatment-related
Description (incidence and severity):
Mortality at 300 and 150 mg/kg/day.
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
See data table: Any other information on results.
Mean foetal weight was slightly lower than that of the controls in all treated groups, although the difference was not statistically significant, and the concurrent control value was higher than the background mean.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
See data table: Any other information on results.
Examination of foetuses revealed an apparent treatment-related increase in the incidence of small foetuses (less than 32.0 g). Values were mostly within the laboratory background control range and in view of the small group size and intragroup variation it was considered that the biological significance of these observations was equivocal.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
See data table: Any other information on results.
Examination of foetuses revealed an apparent treatment-related increase in the incidence of small foetuses (less than 32.0 g) and of foetuses with gall bladder variants. However, in all but one instance the values were within the laboratory background control range and in view of the small group size and intragroup variation it was considered that the biological significance of these observations was equivocal.
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
See data table: Any other information on results.
Examination of foetuses revealed an apparent treatment-related increase in the incidence of small foetuses (less than 32.0 g). Values were mostly within the laboratory background control range and in view of the small group size and intragroup variation it was considered that the biological significance of these observations was equivocal.
Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Small foetus (less than 32.0 g weight)
Remarks on result:
other: Within historical background control data.
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No foetal effect.
Developmental effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
no

Group mean litter data (± S.D.) - females killed on Day 29 of gestation:

Group

1

2

3

4

Treat.

Control

Surfactant A

Dosage

0

75

150

300

Mean

Group

 

Number of pregnant animals

%

Abortion and total litter loss

Corpora

lutea

count

Implantations

Viable young

Resorptions

Implantation

loss (%)

Foetal

weight

(g)

Placental

weight

(g)

M

F

Total

Early

Late

Total

Pre-

Post-

A

1

Mean

S.D.

3y

33.3

7.3

4.0

4.3

2.5

2.0

2.0

1.7

1.5

3.7

3.5

0.7

0.8

0.0

0.0

0.7

0.8

40.9

15.4

 

 

A

2

Mean

S.D.

2

0.0

12.5

3.5

8.0

1.4

3.0

2.8

3.0

2.8

6.0

0.0

2.0

1.4

0.0

0.0

2.0

1.4

36.0

25.0

 

 

A

3

Mean

S.D.

2

0.0

12.0

1.4

7.5

0.7

4.5

0.7

3.0

1.4

7.5

0.7

0.0

0.0

0.0

0.0

0.0

0.0

37.5

0.0

 

 

A

4

Mean

S.D.

2

0.0

10.5

2.1

9.5

3.5

2.0

1.4

4.5

0.7

6.5

0.7

3.0

1.7

0.0

0.0

3.0

1.7

9.5

3T.6

 

 

B

1

Mean

S.D.

2

-

9.5

2.1

5.5

2.1

3.0

1.4

2.5

0.7

5.5

2.1

0.0

0.0

0.0

0.0

0.0

0.0

42.1

0.0

46.1

2.4

5.9

0.4

B

2

Mean

S.D.

2

-

12.5

3.5

8.0

1.4

3.0

2.8

3.0

2.8

6.0

0.0

2.0

1.4

0.0

0.0

2.0

1.4

36.0

25.0

42.4

1.5

6.3

0.2

B

3

Mean

S.D.

2

-

12.0

1.4

7.5

0.7

4.5

0.7

3.0

1.4

7.5

0.7

0.0

0.0

0.0

0.0

0.0

0.0

37.5

0.0

36.9

1.6

4.8

0.3

B

4

Mean

S.D.

2

-

10.5

2.1

9.5

3.5

2.0

1.4

4.5

0.7

6.5

0.7

3.0

1.7

0.0

0.0

3.0

1.7

9.5

31.6

38.0

1.2

5.4

0.4

Background

control

values

 

104

Mean 7.2

95% 0.0

37.8

 

10.7

7.5

13.9

9.0

4.8

13.2

4.1

1.3

6.9

4.1

1.1

7.1

8.2

4.4

12.0

0.3

0.0

1.5

0.5

 0.0

 1.9

0.8

0.0

2.6

16.2

0.0

45.0

8.4

0.0

22.4

5.5

32.7

43.7

40.7

4.1

6.9

Mean A is derived from ail animals that survived to term and bore evidence of implantation. Mean B is derived only from animals that survived to term and bore viable young. y Female No. 120 - total litter loss.

Summary of foetal observations at examination post mortem:

Group:

1

2

3

4

Control

incidence

(%)

Study

control

ranges

Number of foetuses examined.

11

12

15

13

3819

%incidence:

 

 

 

 

 

 

Head shortened; vibrissae underdeveloped

0.0

0.0

6.7

0.0

0.00

-

Ductus arteriosus 75% closed

0.0

0.0

6.7

0.0

0.08

0.0 - 3.7

Gall bladder variants

Agenesis of one kidney, ureter and epididymis

Testicular vascular supply reduced; one testis misshapen

9.1

25.0

33.3

38.5

11.57

0.0 - 37.0

Agenesis of one kidney, ureter and epididymis

0.0

16.7

0.0

0.0

0.00

-

Testicular vascular supply reduced; one testis misshapen

0.0

0.0

11.1

0.0

0.00

-

Subcutaneous haemorrhage

0.0

0.0

6.7

7.7

0.08

0.0 - 2.6

Small foetus (less than 32.0 g)

9.1

16.7

33.3

38.5

14.31

0.0-55.6


Conclusions:
It was concluded from this preliminary study that dosages of Surfactant A of up to 150 mg/kg/day should be suitable for use in a main rabbit teratology study.
Executive summary:

In this dose range finding study, mature virgin female New Zealand White rabbits were treated with Surfactant A, by oral gavage, from Day 6 to Day 18 of gestation inclusive, at dose levels of 75, 150 and 300 mg/kg/day. A fourth group, serving as controls, received the vehicle, distilled water, during the same treatment period. On Day 29 of gestation all animals were sacrificed to allow examination of their uterine contents.

Increased incidences of gastro-intestinal and respiratory tract disorders were observed in all treated groups, both during the study and at necropsy. A total of ten animals died or were killed in extremisduring the study, with the mortality rate greatest in the higher dosage groups. At necropsy, increased incidences of enteric disorder and respiratory tract lesions were recorded in all treated groups, but especially at the highest dosage level. Maternal weight gain and food and water consumption were unaffected by treatment with Surfactant A. Animals receiving 300 mg/kg/day exhibited an elevated post implantation loss, but litter size of all treated groups was superior to that of the controls. Mean foetal weight of treated animals was slightly lower than that of the concurrent controls, although the value recorded for the latter was higher than the background mean. Examination of foetuses indicated a treatment-related incidence of small foetuses and of foetuses with gall bladder variants. However, these data were only of a very limited nature and should be interpreted with caution. It was concluded from this preliminary study that dosages of Surfactant A of up to 150 mg/kg/day should be suitable for use in a main rabbit teratology study.

Endpoint:
developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
29-05-1980 to 08-01-1981
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Comparable to an OECD 414 study - Prenatal Developmental Toxicity Study
Justification for type of information:
1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
Please refer to the Amine Oxide Category justification attached in Section 13

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL
Please refer to the Amine Oxide Category justification attached in Section 13
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Dosing period shorter than in the current OECD 414 guideline.
Qualifier:
according to
Guideline:
other: Japan Ministry of Health and Welfare
GLP compliance:
yes
Remarks:
Study was conducted in a GLP certified laboratory and was subject to extensive quality assurance inspections.
Limit test:
no
Specific details on test material used for the study:
Surfactant A
N,N-dimethyl-dodecylamine oxide, CAS RN 1643-20-5; EC 216-700-6
Batch No. L-9146S
30% active ingredient in aqueous solution.
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Sexually mature virgin female New Zealand White rabbits, approximately 24 weeks old on arrival, were obtained from Ranch Rabbits Ltd., Crawley Down, Sussex, England. Shortly after arrival oestrus was synchronised by intravenous injection of 25 i.u. luteinising hormone ("Pregnyl", Organon). The animals were allowed a minimum of three weeks acclimatisation during which time they were inspected daily to check their physical condition. At commencement of the study the animals were in the bodyweight range of 3.48 to 4.46 kg.
All animals were housed singly in galvanised steel caging from Cope and Cope Limited, Reading, Berkshire, England. They were allowed free access to BETA Rabbit Standard Diet (813 181W) supplied by BP Nutrition (U.K.) Limited, Witham, Essex, England, and to tap water.

The animals were housed inside a limited access rabbit facility with its own supply of filtered air which was passed to atmosphere without re-circulation; there were approximately 17-20 room air changes per hour. The temperature and relative humidity in the rabbitry were recorded daily and were in the range 16 to 18°C with a relative humidity of 75% ± 15%. The animals were subjected to a 10-hour dark : 14-hour light cycle.
Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
Females were artificially inseminated using pooled semen from New Zealand White bucks of established fertility. Following insemination, each female was injected intravenously with 25 i.u. luteinising hormone ("Pregnyl", Organon) to ensure successful ovulation. The day of insemination was designated Day 0 of gestation.
Duration of treatment / exposure:
Animals were dosed daily by the oral route from Day 6 to Day 18 inclusive of gestation at a dose volume of 5 ml/kg bodyweight.
Frequency of treatment:
Animals were dosed daily by the oral route from gestation Day 6 to 18.
Duration of test:
Day 0 to 29 of gestation.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 = Control
Dose / conc.:
40 mg/kg bw/day (nominal)
Remarks:
Group 2 = 40 mg/kg/day nominal, actual dose is 12 mg/kg/day after correction for 30% active ingredient
Dose / conc.:
80 mg/kg bw/day (nominal)
Remarks:
Group 3 = 80 mg/kg/day nominal, actual dose is 24 mg/kg/day after correction for 30% active ingredient
Dose / conc.:
160 mg/kg bw/day (nominal)
Remarks:
Group 4 = 160 mg/kg/day nominal, actual dose is 48 mg/kg/day after correction for 30% active ingredient
No. of animals per sex per dose:
14 or 15 (includes replacements for early decedent animals)
Control animals:
yes, concurrent vehicle
Details on study design:
This study was designed to examine the effects of repeated oral administration of Surfactant A upon the progress and outcome of pregnancy in the rabbit. The New Zealand White rabbit was selected because of the requirements for its use by regulatory authorites. Surfactant A was administered orally, to simulate the conditions of human exposure, at dosages based on the results of a preliminary investigation (LSR Report No. 79/LIF049/542). Dosing was through the organogenesis phase of pregnancy in accordance with Japanese Ministry of Health and Welfare Guidelines at the time of the study. The animals were killed on Day 29 of gestation for examination of their uterine contents.
Maternal examinations:
Clinical signs of reaction to treatment were recorded with details of type, severity, duration and time of onset.
Any animals found dead or killed in extremis were subjected to a thorough macroscopic examination of the visceral organs with the object of identifying the cause of death.
Animals were weighed daily and means calculated and reported on Days 0, 6, 8, 10, 12, 14, 15, 18, 23 and 28 after insemination. Weight changes were calculated with respect to Day 6 after insemination.
Food and water consumption of each animal was recorded over five phases during the study on Days 1-5, 6-11, 12-17, 18-23, and 24-28, inclusive.
Ovaries and uterine content:
On Day 29 after insemination the females were killed by intravenous injection of Pentobarbitone Sodium for examination of their uterine contents. Each animal was first examined macroscopically for evidence of disease or adverse reaction to treatment. The reproductive tract, complete with ovaries, was dissected out and examined, and the following recorded:
a) number of corpora lutea in each ovary
b) number of implantation sites
c) number of resorption sites (classified as early or late).
d) number and distribution of live and dead foetuses in each uterine horn, including an estimation of the time of death of non-viable foetuses.
Fetal examinations:
For each animal, the following were recorded:
a) Weight and sex of individual foetuses
b) individual placental weights
c) external abnormalities of individual foetuses.
d) skeletal examinations: the neck and thoracic and abdominal cavities of all the foetuses from each litter were dissected and the contents examined. Following examination and evisceration, the foetuses were placed in industrial methylated spirit (74 o.p.) before processing, which utilised a modification of the Dawson staining technique and subsequent skeletal examination.
Statistics:
The significance of inter-group differences was tested using appropriate statistical tests. The tests used were multiple 't'-test, Mann-Whitney U-test, Chi-squared test and Fisher's exact probability test (Armitage modification)
Indices:
Group mean foetal and placental weights
Resorptions: Pre-implantation loss
Resorptions: Post-implantation loss
Historical control data:
Not specified
Clinical signs:
no effects observed
Description (incidence and severity):
The general condition of treated animals was similar to that of controls throughout the investigation.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Three animals in each of Groups 3 and 4 (80 and 160 mg/kg/ day) died or were killed in extremis during the study. Necropsy revealed no evidence to suggest any direct involvement of Surfactant A.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
During the first two days of treatment, a slight loss of bodyweight was recorded in Groups 2 and 4 (40 and 160 mg/kg/day), but not in Group 3 (80 mg/kg/day). During the remainder of the treatment period, the rate of weight gain improved to control values for these groups. Following completion of treatment, Groups 3 (80 mg/kg/day) mean body weight was slightly depressed.

See data tables in 'Any other information on results incl. tables' section.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food intake, when compared with pre-treatment values, was reduced during the second half of the treatment period until termination in Groups 2 and 3 (40 and 80 mg/kg/day), and from the commencement of treatment until termination in Group 4 (160 mg/kg/day).

See data tables in 'Any other information on results incl. tables' section.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Water intake was decreased in all Surfactant A dosed groups during the early part of the treatment period, with some recovery during the latter-half of treatment (Days 12 to 12). Following cessation of treatment, water intake was similar in all groups.

See data tables in 'Any other information on results incl. tables' section.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
At terminal necropsy on Day 29 of gestation, one female in Group 2 (40 mg/kg/day) was found to have suffered total litter resorption.
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
At terminal necropsy on Day 29 of gestation, one female in Group 2 (40 mg/kg/day) was found to have suffered total litter resorption (see above).
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not specified
Details on maternal toxic effects:
Three animals in each of Groups 3 and 4 (80 and 160 mg/kg/ day) died or were killed in extremis during the study. Necropsy revealed no evidence to suggest any direct involvement of Surfactant A.
Key result
Dose descriptor:
NOAEL
Effect level:
> 160 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
other: No adverse effects seen.
Dose descriptor:
NOAEL
Effect level:
> 48 mg/kg bw/day
Based on:
act. ingr.
Remarks on result:
other: No adverse effects seen
Fetal body weight changes:
no effects observed
Description (incidence and severity):
See data tables in 'Any other information on results incl. tables' section.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
See data tables in 'Any other information on results incl. tables' section.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
See data tables in 'Any other information on results incl. tables' section.
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Examination of foetuses at necropsy on Day 29 of gestation revealed a small number of anomalies in all groups, the incidence and distribution of which did not indicate any adverse effect of Surfactant A upon morphogenesis.

See data tables in 'Any other information on results incl. tables' section.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Examination of foetuses following skeletal processing revealed a small number of abnormalities in all groups, the incidence and distribution of which did not indicate any treatment-related aetiology. The degree of foetal ossification was similar in all groups.

See data tables in 'Any other information on results incl. tables' section.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Examination of foetuses at necropsy on Day 29 of gestation revealed a small number of anomalies in all groups, the incidence and distribution of which did not indicate any adverse effect of- Surfactant A upon morphogenesis.

See data tables in 'Any other information on results incl. tables' section.
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
Not embryotoxic or teratogenic.
Key result
Dose descriptor:
NOAEL
Effect level:
> 160 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No adverse effects evident.
Dose descriptor:
NOAEL
Effect level:
> 48 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Remarks on result:
other: No adverse effects evident
Key result
Abnormalities:
no effects observed
Description (incidence and severity):
.
Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
160 mg/kg bw/day (nominal)
Treatment related:
no

Group mean bodyweights ( ± S.D.) of females during gestation

Group

Number of pregnant animals

 

Day of gestation

0

6

8

10

12

. 14

16

18

23

28

1

Control

14

Mean

S.D.

3.81

0.18

3.93

0.23

3.96

0.21

4.02

0.22

4.08

0.23

4.12

0.22

4.19

0.26

4.16

0.25

4.29

0.23

4.40

0.27

2

40 mg/kg bw/day

13

Mean

S.D.

4.02

0.20

4.20

0.25

4.19

0.27

4.23

0.27

4.29

0.29

4.35

0.28

4.39

0.28

4.38

0.27

4.53

0.29

4.65

0.29

3

80 mg/kg bw/day

12

Mean

S.D.

3.98

0.28

4.06

0.27

4.10

0.27

4.16

0.27

4.16

0.27

4.20

0.30

4.25

0.32

4.26

0.33

4.34

0.32

4.36*

0.33

4

160 mg/kg bw/day

10

Mean

S.D.

3.98

0.26

4.11

0.26

4.05*

0.29

4.13

0.33

4.12**

0.34

4.21

0.33

4.26

0.35

4.30

0.37

4.37

0.35

4.45

0.36

*Bodyweight change from Day 6 significantly different from Controls, P < 0.05 (Multiple t-test).

** Bodyweight change from Day 6 significantly different from Controls, P < 0.01 (Multiple t-test).

Food intake - group mean values (g/rabbit/day ± SD)

Group

Number of pregnant animals

 

Days after insemination

1-5

6-11

12-17

18-23

24-28

1

Control

14

Mean

S.D.

207

51

201

41

209

70

190

35

172

57

2

40 mg/kg bw/day

13

Mean

S.D.

242

62

232

73

206

 41

202

 48

153

26

3

80 mg/kg bw/day

12

Mean

S.D.

198

 37

199

 19

166

40

171

41

109

51

4

160 mg/kg bw/day

10

Mean

S.D.

241

 81

171

 61

188

67

183

60

134

41

Water intake – group mean values (ml/rabbit/day ± SD)

Group

Number of pregnant animals

 

Days after insemination

1-5

6-11

12-17

18-23

24-28

1

Control

14

Mean

S.D.

614

300

664

268

670

340

607

315

539

338

2

40 mg/kg bw/day

13

Mean

S.D.

706

277

571

255

649

295

758

334

563

213

3

80 mg/kg bw/day

12

Mean

S.D.

548

230

444

125

494

264

521

246

432

257

4

160 mg/kg bw/day

10

Mean

S.D.

617

314

531

320

559

320

642

317

589

 353

Group mean litter data  (± S.D.) females killed on Day 29 of gestation

Group

Number of pregnant animals

 

%

Abortion and total litter loss

Corpora

lutea

count

Implantations

Viable young

Resorptions

Implantation loss (%)

Foetal weight (g)

Placental

weight

(g)

M

F

Total

Early

Late

Total

Pre-

Post-

1

Control

14

Mean

 S.D.

-

14.6

4.5

11.4

4.6

5.0

1.9

5.1

2.8

10.1

4.3

0.5

0.7

0.9

0.9

1.4

1.2

21.6

 

11.9

41.5

1.9

5.9

0.4

2

40 mg/kg bw/day

13

Mean

 S.D.

 

-

14.7

3.9

13.2

4.1

5.9

2.7

5.4

1.6

11.3

3.6

0.5

0.7

1.4

1.2

1.9

1.4

 

9.9

 

14.5

38.3

1.6

5.6

0.3

3

80 mg/kg bw/day

12

Mean

 S.D.

 

-

12.7

2.8

10.9

3.4

4.6

2.1

5.3

2.3

9.8

3.0

0.2

0.4

0.9

1.0

1.1

.1.0

 

13.8

 

9.9

38.9

1.8

5.6

0.4

4

160 mg/kg bw/day

10

Mean

S.D.

 

-

12.8

2.5

10.2

3.1

4.4

2.3

4.8

1.9

9.2

2.9

0.1

0.3

0.9

0.9

1.0

1.0

 

20.3

 

9.8

42.2

1.5

6.2

0.3

Summary of foetal observations at examination post-mortem

Group:

1

Control

2

40 mg/kg bw/day

3

80 mg/kg bw/day

4

160 mg/kg bw/day

Number of foetuses examined:

141

147

118

92

%incidence:

 

 

 

 

Thin skin; abdomen fluid-distended;

 

 

 

 

heart and aortic arch enlarged;

0.7

 

 

 

abnormal stomach, spleen, and

 

 

 

 

liver; pale placenta

 

 

 

 

Interocular haemorrhage

0.7

-

-

-

Bilateral forelimb flexure

0.7

-

-

-

Haemorrhage on hind limbs

-

-

0.8

-

Thyroid gland enlarged.

-

0.7

0.8

-

Thyroid gland haemorrhagic

0.7

0.7

-

-

Ductus arteriosus 25% closed

-

-

0.8

-

Agenesis of median lung lobe

1.4

-

-

-

Gall bladder variants

24.1

20.4

30.5

25.0

Pale area on liver

-

-

-

1.1

Gas in stomach

7.8

6.8

6.8

4.3

Renal haemorrhage

0.7

-

-

-

Unilateral hydronephrosis

-

-

0.8

-

Haemorrhagic testis

-

-

1.8

-

Small foetus (less than 32.0 g)

25.5

24.5

19.5

10.9

Placental anomalies

0.7

0.7

-

-

Summary of foetal observations at skeletal examination

Group:

1

Control

2

40 mg/kg bw/day

3

80 mg/kg bw/day

4

160 mg/kg bw/day

Number of foetuses examined:

141

147

118

92

Head -%with:

Size of anterior fontanelle - small

17.7

10.2

9.3

16.3

medium

74.5

76.9

81.4

78.3

large

7.8

12.9

9.3

5.4

Misshapen anterior fontanelle

-

2.0

3.4

1.1

Discrete unossified area at sutural margins of one or both parietal bones

 

0.7

0.7

 

 

Interparietal bone slightly misshapen and divided into

 

 

 

 

two parts by a longitudinal fissure

-

0.7

-

-

Interparietal bone reduced in size and/or misshapen

6.4

4.8

9.3

6.5

Enlarged posterior fontanelle

29.8

34.7

31.4

21.7

Unossified area(s) in cranial bones

5.0

1.4

4.2

1.1

Enlarged cranial sutures

7.1

4.1

8.5

2.2

Irregular ossification of cranial suture(s)

38.3

38.8

37.3

45.7

Fissure(s) in cranial bone(s)

9.9

4.8

10.2

10.9

Additional cranial suture

0.7

1.4

-

1.1

Incomplete ossification of hyoid bone

14.9

17.0

7.6

2.2

Bilateral or unilateral bent hyoid cornu

4.3

2.0

3.4

8.7

Hyoid cornu reduced in length Vertebral column and rib cage -%with:

 

 

0.8

 

Number of ribs 12

73.8

68.0

67.8

64.1

12/13

7.8

14.3

9.3

12.0

13

18.4

17.7

22.9

23.9

One or-more ribs-thickened air junction with costal- cartilages

2.8

 

2.5

2.2

12th rib (right) slightly elongated

0.7

-

-

-

Incomplete ossification of 9th and 10th thoracic vertebral centra

 

 

0.7

 

 

Supernumerary 10th rib and hemivertebra (right), additional 10th hemicentrum fused to 10th thoracic centrum; 9th thoracic vertebral centrum slightly asymmetrical and misaligned

 

0.7

 

 

Bilateral or unilateral absence of 1st sacral vertebral ventral processes

 

6.4

14.3

8.5

8.7

Incomplete ossification of sternebrae5number of bones affected -

 

1

71.6

58.5

67.5

78.3

2

12.1

16.3

14.5

7.6

3

2.8

3.4

0.9

1.1

4

-

0.7

-

-

Slight variations in sternebral ossification

5.0

4.1

1.7

2.2

Omosternum ossified

1.4

-

0.8

-

Limbs -%with:

 

 

 

 

Incomplete ossification of long bones

24.8

20.4

26.3

17.4

Uncomplete ossification of phalanges

12.1

12.9

10.2

13.0

Bilateral forelimb flexure

0.7

-

-

-

Absence of tarsals

0.7

-

-

-

Incomplete ossification of pubic bones

0.7

1.4

-

-

Misaligned pelvic girdle

4,3

8.2

6.8

7.6

Others - % with:

 

 

 

 

Right dorsal hemivertebra! arch of atlas reduced in size; right hyoid cornu reduced in length and displaced

-

-

0.8

-

Conclusions:
This rabbit study was conducted by oral gavage at dose levels of 40, 80 or 160 mg/kg/day Surfactant A. Surfactant A is a 30% aqueous solution of the source substance linear C12 amine oxide. Female New Zealand White rabbits were artificially inseminated, and the time of insemination was designated as gestation day 0. The pregnant animals (14-15 per group) were dosed daily on gestation days 6-18. Maternal toxicity was evaluated by regular measurement of body weight, food and water consumption, and cageside observations. Caesarean sections were performed on gestation day 29. Fetal weights, number of resorptions, fetal deaths and fetal morphology (soft tissue and skeletal) were evaluated.

Decreased body weight and food and water consumption were observed at some point during dosing in all three treatment groups and were more persistent in the mid and high dose levels, but only food consumption was reduced by the end of the study. There were three deaths in the 80 and 160 mg/kg/day groups, but the study report indicates that these deaths were not related to the treatment. One animal in the low dose group had a fully resorbed litter, but this was not considered treatment-related since it was not dose-responsive. No developmental effects were observed in any other group. Because the test material contained 30% active, the developmental NOAEL of ≥160 mg/kg/day is equivalent to ≥48 mg/kg/day based on active.

In conclusion, based on this investigation, oral administration of Surfactant A to pregnant rabbits during organogenesis, at dosages up to 160 mg/kg/day (or 48 mg/kg/day based on active), was associated with some interference with maternal body weight gain and food and water intake. Observed changes were transient and not dose-related and not considered adverse, and the maternal toxicity NOAEL was the high dose level of 160 mg/kg/day. No adverse effects were observed upon foetal survival and development in utero, and the developmental toxicity NOAEL was the high dose level of 160 mg/kg/day. As the test material was a 30% active solution, the adjusted (based on active) NOAEL for maternal and developmental toxicity was > 48 mg/kg/day.
Executive summary:

The influence of Surfactant A (a 30% aqueous solution of linear C12 amine oxide) upon the organogenesis phase of pregnancy was assessed in rabbits of the New Zealand White strain. For this purpose, Surfactant A was administered by oral gavage to groups of female rats at levels of 40, 80 and 160 mg/kg from Day 6 to 18 inclusive of gestation. A fourth group, serving as controls, received the vehicle, distilled water, on the same days of gestation and at the same volume-dosage. Maternal condition was unaffected by treatment with Surfactant A. Three females receiving 80 mg/kg/day and three females receiving 160 mg/kg/day died or were killed in extremis, but no direct involvement of Surfactant A was apparent. Maternal bodyweight gain was slightly reduced in all Surfactant A treated groups during the study, although, at 40 mg/kg/day, terminal bodyweights like those of controls were achieved following completion of treatment and the body weight reduction at the higher doses was not significant. Food and water intakes were decreased (not statistically significant) in all treated groups. No adverse effects upon litter responses and development were recorded. No teratogenic responses were observed.

It was concluded from this investigation that oral administration of Surfactant A to pregnant rabbits during organogenesis, at dosages up to 160 mg/kg/day (48 mg/kg/day based on active), had no adverse effects upon survival and development in utero.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31-07-1978 to 01-05-1980
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Comparable to an OECD 414 study - Prenatal Developmental Toxicity Study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Dosing period was shorter that in the current OECD 414 guideline. Additionally, one third of animals allowed to litter with subsequent examinations of neonates up to and including sexual maturity.
Qualifier:
according to
Guideline:
other: Japan Ministry of Health and Welfare
GLP compliance:
not specified
Remarks:
Study was conducted in a GLP certified laboratory and was subject to extensive quality assurance inspections.
Limit test:
no
Specific details on test material used for the study:
Surfactant A
N,N-dimethyl-dodecylamine oxide, CAS RN 1643-20-5; EC 216-700-6
Batch No. L-9146S
30% active ingredient in aqueous solution.
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Adult virgin female rats of the CD strain were obtained from Charles River U.K. Limited, Margate, Kent, England, and mated with stock males from the same source. On arrival female rats were in the approximate age range 58-62 days and were allowed approximately one week's acclimation before commencement of treatment, during which time they were examined daily to check their physical condition.

The animals were housed inside a barriered limited-access rodent facility. Each animal room was provided with approximately 15 changes per hour of filtered air. The target room temperature and relative humidity were 21°± 2°C and 50% ± 10% R.H. The acheived mean values ranged from 20.2 to 21.9°C and 61% ± 16.5% R.H. The animals were subjected to a 12-hour light : 12-hour dork cycle. The rats were allowed free access to a commercially-available laboratory animal diet (Spratts Laboratory Diet No. 1, Spratts Patent Ltd., Barking, Essex, England) and to tap water. Rats were housed in RC1, modified RM2 and RB3 cages from North Kent Plastics Ltd., Dartford, Kent, England. The cages consisted of high density polypropylene bodies with stainless steel lids and mesh floors. The cages were suspended in batteries over trays covered with crepe absorbent paper; the latter was changed on alternate weekdays. Autoclaved wood shavings were provided for bedding during the littering phase.
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Days 7 to 17 of gestation.
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
Females mated with stock males from the same source 1:1.
Cages were checked for ejected copulation plugs and a vaginal smear was examined for the presence of spermatozoa.
Duration of treatment / exposure:
Days 7 to 17 of gestation.
Frequency of treatment:
Daily on Days 7 to 17 of gestation.
Duration of test:
From Day 0 of gestation until week five for the F1 weanlings derived from the treated F0 females.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 = Control
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 2 = 50 mg/kg/day nominal; actual dose is 15 mg/kg/day after correction for 30% active ingredient
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 3 = 100 mg/kg/day nominal; actual dose is 30 mg/kg/day after correction for 30% active ingredient
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Group 4 = 200 mg/kg/day nominal; actual dose is 60 mg/kg/day after correction for 30% active ingredient
No. of animals per sex per dose:
32
Control animals:
yes, concurrent vehicle
Details on study design:
Surfactant A was administered to rats by oral gavage at dosages selected based on the results of an earlier study performed at the same facility (Report No. 79/LIF045/444).

The aim of the study was to assess the influence of oral administration of Surfactant A (N,N-dimethyl-dodecylamine oxide, or lauryl dimethylamine oxide) upon the organogenesis phase of pregnancy (Segment II) in the rat, in accordance with Japanese Ministry of Health and Welfare Guidelines at the time of the study.
At day 20 of gestation approximately two-thirds of the animals in each group were killed for examination of their uterine contents The remaining one-third of the animals were allowed to deliver and raise their young naturally to 25 days post partum.
Maternal examinations:
Clinical signs: Animals were examined daily throughout the study, and any visible signs of reaction to treatment were recorded, with details of type, severity, time of onset and duration.

Bodyweight: Maternal bodyweights were recorded on Days 0, 2, 7 - 17 inclusive and Day 20 post coitum, and reported on Days 0, 2, 7, 9, 11, 13, 15, 17 and 20 post coitum.

Food consumption: Food consumption was measured and recorded twice weekly during gestation.

Water consumption: Was measured and recorded twice weekly during gestation.

See 'Any other information on materials and methods incl. tables' section below for details on the Post-natal phase of the study (i.e. females allowed to litter).
Ovaries and uterine content:
On Day 20 of gestation, approximately two thirds of the females were killed by inhaled carbon dioxide for examination of their uterine contents. Each animal was first examined macroscopically for evidence of disease or adverse reaction to treatment. The reproductive tract, complete with ovaries, was then dissected out and the following recorded:
a) number of corpora lutea in each ovary
b) number of implantation sites
c) number of resorption sites (classified as early or late)
d) number and distribution of live and dead foetuses in each uterine horn, including an estimation of the time of death of non-viable foetuses.
Fetal examinations:
Weight and sex of individual foetuses
Individual placental weights
External abnormalities
Skeletal examinations: the thoracic and abdominal cavities of approximately half of each litter were dissected and examined. Following examination and evisceration, the foetuses were placed in industrial methylated spirit (74 o.p.) prior to processing, which utilised a modification of the Dawson staining technique and then subjected to skeletal examination
Visceral examination: the remaining foetuses were placed in Bouin's fixative and then subjected to free-hand sectioning following the technique of Wilson (in Teratology: Principles and Techniques, p. 251, U. Chicago Press, 1965).
Statistics:
The significance of inter-group differences was tested using appropriate statistical tests. The tests used were multiple 't'-test, Mann-Whitney U-test, Chi-squared test and Fisher's exact probability test (Armitage modification).
Indices:
Prenatal:
Pre-implantation loss
Post-implantation loss
Group mean foetal and placental weights
Postnatal (see details in 'Any other information on materials and methods incl. tables' section below):
Gestation lengths
Mean live litter size
Mean offspring weight
Live birth index
Viability index
Percentage mortality
Offspring development timing
Sex ratios
Mean activity score
Mean swimming time
Auditory and visual function; locomotor co-ordination and quadruped muscle development
Pre-coital interval (Fi generation)
Mating performance and fertility:
Percentage mating
Conception rate
Fertility index
Pregnancy index
Historical control data:
The laboratory background control ranges are mentioned.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Bodyweight gain of Group 4 females (200 mg/kg/day) was slightly, but significantly, depressed (p < 0.001) during the first three days of treatment, was comparable with the controls for the remainder of the dosing period, but again showed a slight reduction between Days 17 and 20 post coitum. Bodyweight gain of Groups 2 and 3 (50 and 100 mg/kg/day) was unaffected by treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food intake of Group 4 females (200 mg/kg/day) was slightly depressed during the dosing period (p < 0.01; Days 7-10 post coitum) but, thereafter, was similar to controls. Food intake was similar to the controls in Groups 2 and 3 (50 and 100 mg/kg/day).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water intake of Group 4 females was elevated during the dosing period and remained higher even after completion of treatment. Water intake was similar to the controls in Groups 2 and 3 (50 and 100 mg/kg/day).
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Details on results:
Females at the high dose of 200 mg/kg/day exhibited intermittent reduced body weight gain, intermittent decreased food consumption, and increased water consumption. Durations are described in relevant sections above.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
For females allowed to litter, evaluation of the following maternal reproductive parameters did not not reveal any treatment-related changes from controls: gestation length and index, body weights during lactation, and litter size/viability/mortality.
Details on maternal toxic effects:
Females at the high dose of 200 mg/kg/day exhibited intermittent reduced body weight gain, intermittent decreased food consumption, and increased water consumption. No effects were noted in reproductive parameters such as pre- and post-implantation loss, number of resorptions, etc.
Key result
Dose descriptor:
LOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No effects on maternal condition.
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
act. ingr.
Basis for effect level:
other: No effects on maternal condition
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
A significant reduction in fetal weight, together with a slight increase in the incidence of small foetuses, was recorded in Group 4 (200 mg/kg/day).
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, treatment-related
Description (incidence and severity):
A slight increased incidence of small foetus was recorded in Group 4 (200 mg/kg/day).
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Associated with the reduction in foetal weight, in Group 4 (200 mg/kg/day) there was a slight reduction in the degree of foetal ossification, as assessed from the cranial bones, thoracic and lumbar vertebral centra and metacarpals.

See table below in Any other information on results incld. tables section.
Visceral malformations:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
For females allowed to litter, evaluation of the following parameters in the pups did not reveal any treatment-related changes from controls: body weight of offspring, sex ratio, physical development of offspring, auditory and visual functions, physical activity, learning ability, and locomotor coordination and muscle development.
Details on embryotoxic / teratogenic effects:
Other than reduced foetal body weigh, small foetus, and a reduction in the degree of ossification at the high dose where maternal toxicity was also observed, there were no embryotoxic/teratogenic effects.
Key result
Dose descriptor:
LOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reduced foetal body weight, small foetus, and slight reduction in the degree of foetal ossification,
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed.
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No effects observed.
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
no

Summary of foetal observations at skeletal examination – F0 -F1

Group: mg/kg bw/day

1

Control

2

50

3

100

4

200

Previously recorded laboratory control values

Number of foetuses examined: One foetus may have more than one observation

 

142

129

139

145

2558

Head -%with:

 

 

 

 

 

Size of anterior fontanelle - small

1.4

-

-

-

0.0 -v 10.8

- medium

95.8

97.7

93.5

97.9

82,0 + 97.3

- large

2.8

2.3

6.5

2.1

1.5 + 11.0

Incomplete ossification of cranial bones

6.3

11.6

11.5

15.9

0.0 + 13.0

Indentation in cranial bones

-

0.8

-

-

0.0 + 20.8

Discrete unossified area(s) in inferior

0.7

0.8

-

-

-

occipital bone

 

 

 

 

 

Absence of hyoid bone

8.5

14.7

7.2

4,8

0.0 + 12.2

Incomplete ossification of hyoid bone

-

0.8

1.4

2.8

0.0+ 0.6

Vertebral column and rib-cage -%with:

 

 

 

 

 

Number of ribs - 13

90.1

90.7

98.6

94.5

86.8 + 98.2

- 13/14

4.2

6.2

0.7

3.4

0.6 + 11.8

- 14

5.6

3.1

0.7

2.1

1.1 + 11.8

Incomplete ossification of one or more

47.2

45.0

61 .9

71.7

18.1 + 70.3

thoracic vertebral centra

 

 

 

 

 

Asymmetrical ossification of one thoracic

3.5

2.3

1.4

1.4

0.0+ 4.1

vertebral centrum

 

 

 

 

 

Eleventh centrum slightly misaligned

-

0.8

-

-

-

Incomplete ossification of one or more

2.1

2.3

-

13.1

0.0+ 3.0

lumbar vertebral centra

 

 

 

 

 

Asymmetrical ossification of one lumbar

-

0.8

-

0.7

-

vertebral centrum

 

 

 

 

 

Incomplete ossification of 4th lumbar

-

0.8

-

2.1

0.0+ 1.0

vertebral arch

 

 

 

 

 

Reduced 5th lumbar vertebra, no other

-

-

0.7

-

-

lumbar vertebrae, no sacral or caudal vertebrae; displacement of ischial bones towards mid-line

 

 

 

 

 

Incomplete ossification of sternebrae

100.0

100.0

100.0

100.0

96.4 +100.0

Sternebrae bipartite and offset

-

0.8

-

2.8

0.0+ 1.8

Limbs - % with:

 

 

 

 

 

Number of metacarpals/metatarsals - 3/4

59.9

55.8

60.4

75.2

31.5 + 77.6

- 4/4

40,1

43.4

38.1

23.4

22.4 + 64.9

Incomplete ossification of metacarpals/

-

0.8

1.4

1.4

0.0 + 0.8

metatarsals Others -%with:

 

 

 

 

 

Generalised reduction in ossification,

-

0.8

0.7

-

-

associated with immaturity

 

 

 

 

 

F0 Females allowed to litter

Gestation length was similar in all groups; all females littered between Days 22 and 23\ post coitum. No incidences of dystocia were observed and there was no effect upon gestation index.

Bodyweight of females during the lactation period was similar in all groups.

Litter size, viability and mortality indices: were unaffected by treatment of the F0 females.

Bodyweight of offspring Day 1 post-partum and bodyweight gain throughout the lactation period was essentially similar in all groups.

Sex ratio at Days 1 and 25 post-partum showed no changes that could be related to treatment of the F0 females.

Physical development of offspring as assessed by pinna unfolding, hair growth, tooth eruption and eye opening was similar in all groups.

Auditory and visual functions were unaffected by treatment of the F0 females.

Physical activity of offspring, no treatment-related effects were apparent.

Learning ability of offspring, all groups showed similar swimming times.

Locomotor co-ordination and muscle development were similar in all groups.

Terminal studies

At necropsy of F0 females, no macroscopic abnormalities were observed that could be related to treatment.

F1 generation

General condition and mortality, no signs were noted that could be related to treatment of the F0 females and no deaths occurred.

Bodyweight gain of unselected offspring, and of selected males and females during the maturation, mating and gestation periods until termination at Day 20 post coitum, showed no effects that could be attributed to treatment of the F0 females.

Mating performance and fertility of F-, animals was similar in all groups.

Terminal studies on F1 females killed on Day 20 of gestation

Maternal observations: at necropsy, no macroscopic abnormalities were noted that could be related to treatment of the F0 females.

Litter responses, there were no treatment-related inter-group differences in the numbers of corpora lutea, implantations or viable young or in the extent of pre- and post-implantation losses.

Mean foetal weight in treated groups was similar to, or slightly greater than that of the controls (Group 4, 200 mg/kg/day to F0 females, p < 0.05); placental weight was also slightly increased in Group 4 (200 mg/kg/day to F0 females, p < 0.05) and this was associated with an increased incidence of large placentae (more than 0.70 g). However, all values were within the laboratory background control range.

Terminal studies

Unselected F1 offspring at 8 weeks of age, terminal necropsy of F1 offspring not selected to form the F1 parental generation revealed a small number of abnormalities in all groups, but these were of types and incidences which have previously been found to occur spontaneously in this strain of rat in these laboratories.

F1 males, terminal necropsy of Fi males after Day 20 of gestation revealed no macroscopic changes that could be related to treatment of the F0 females.

Conclusions:
This rat prenatal developmental toxicity study was conducted by oral gavage at dose levels of 50, 100 or 200 mg/kg/day of Surfactant A. Surfactant A is a 30% aqueous solution of the source substance linear C12 amine oxide. Each group consisted of 32 pregnant Sprague-Dawley (CD) female rats. Dosing was performed daily on gestation days 7 to 17. Twenty-one rats per group were sacrificed on gestation day 20 and their fetuses were examined for morphological development. The remaining 11 females per group were allowed to give birth and their offspring were evaluated for viability, growth, and the attainment of a number of developmental milestones as well as neurobehavioral effects and fertility. Treatment of the F1 dams was continued through weaning, but the F1 animals were not subsequently dosed.

Maternal toxicity was evaluated by regular measurement of body weight, food consumption and water consumption, and cageside observations over the course of the study. Prenatal developmental observations included fetal weight, number of resorptions and fetal deaths, sex ration, and morphological observations, including soft tissue evaluation and skeletal evaluations. Postnatal observations included gestation length, viability, and growth, as well as the time of attainment of a number of developmental milestones: eye opening, pinna detachment, fur growth and tooth eruption. The investigators also evaluated offspring for auditory startle reflex, visual reflexes, motor activity, muscle strength and coordination using a variety of techniques. A subset of F1 animals, 22 males and 22 females from each dose group, were mated upon reaching sexual maturity (ten weeks of age) to assess fertility. These animals were sacrificed on gestation day 20 and the offspring evaluated for viability, weight, and external morphology.

Maternal toxicity in the form of decreased body weight and food consumption was observed in the 200 mg/kg/day group. This group also had an increase in water consumption. There were no effects observed at the two lower dose levels. Developmental effects, consisting of decreased fetal weight and delayed ossification of some skeletal elements, was observed in the 200 mg/kg/day group. These are common observations in the presence of maternal toxicity and do not represent a reproductive hazard. There were no effects on growth, viability, behavior, or fertility in the F1 litters in any dose group. In summary, the 200 mg/kg/day group produced some maternal toxicity and associated developmental effects; 100 mg/kg/day was a clear no observed adverse effect level. Because the test material contained 30% active, the developmental NOAEL of 100 mg/kg/day was equivalent to 30 mg/kg/day of amine oxide.

In conclusion, oral administration of Surfactant A to pregnant rats, from Day 7 to Day 17 of gestation, at nominal dose levels of 50, 100 and 200 mg/kg/day, was associated, at the highest dose level, with a slight reduction of maternal bodyweight gain and food intake and with elevated water consumption. In females killed on Day 20 of gestation, mean foetal weight was depressed at the highest dose level, and this was associated with a slight retardation of foetal ossification. In females allowed to litter, parturition, and survival, growth and development of F1 offspring, were unaffected by previous treatment with Surfactant A. The subsequent growth, mating performance and fertility of F1 animals was similar in all groups, but in F1 females derived from F0 females that received 200 mg/kg/day, foetal and placental weights were slightly elevated compared with the concurrent controls.
Executive summary:

The influence of Surfactant A upon the organogenesis phase of pregnancy was assessed in rats of the Charles River CD strain. For this purpose, Surfactant A was administered by oral gavage to groups of female rats at nominal dose levels of 50, 100 and 200 mg/kg from Day 7 to 17 of gestation. A fourth group, serving as controls, received the vehicle, distilled water, on the same days of gestation and at the same volume-dosage. On Day 20, approximately two-thirds of the females from each group were killed for examination of their uterine contents; the remainder were allowed to give birth naturally and to rear their young to weaning. Randomly selected offspring of the F1 generation were then reared, untreated, to maturity at approximately ten weeks of age, when they were mated within groups. The F1 females were killed and examined on Day 20 of gestation, at which point the study was terminated. The offspring from these F1 females were evaluated for viability, weight, and external morphology.

A dosage of 200 mg/kg bw/day to pregnant rats during organogenesis was associated with maternal toxicity manifest as impaired body weight performance, reduced food consumption and increased water intake. A slight retardation of foetal growth was also seen at the same dosage, but was without adverse effect upon pre- and post-natal survival and development, or upon the subsequent mating performance and fertility of the F1 offspring. There was a slight effect on the ossification of some skeletal elements secondary to the observed F0 maternal toxicity. However no adverse effects were seen on any on the post-natal development parameters of the F1 generation throughout the study. 

Dosages of 50 and 100 mg/kg/day (nominal) were tolerated without discernible influence upon the dam or upon the course and outcome of the pregnancy.

The substance was not teratogenic and the few slight effects seen on skeletal ossification were secondary to maternal toxicity at 200 mg/kg bw/day. The developmental NOAEL in this study was 100 mg/kg/day (nominal) or 30 mg/kg/day (actual, based on the 30% active test material).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
07-11-2007 to 28-05-2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD 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)
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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first day of treatment samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (4 h and 7 days). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 ºC) and delivered on dry ice to be stored at -20 ± 5 ºC until analysis.
The samples were analysed by HPLC coupled to an ELSD detector following an analytical procedure developed at RCC and quantified with the area under the peak. The test item was used as the analytical standard. Analysed samples were not discarded without written consent from the study director.
The analytical part of the study was conducted at RCC Ltd, Itingen, Switzerland under GLP-compliant conditions. The identity of Lauramine oxide was confirmed by its retention time, which was similar to that measured in the working standards. The test item content in all samples was found to be in the accepted range of ± 20 % of the nominal content. In addition, the homogenous distribution of Lauramine oxide in highly purified water was demonstrated. The results of the analytical phase confirmed the correct preparation and sotrage of appliation formulations during the conduct of this study.
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.
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
Duration of test:
See table
Dose / conc.:
0 other: mg AO/kg bw/day (actual dose received)
Dose / conc.:
40 other: mg AO/kg bw/day (actual dose received)
Dose / conc.:
100 other: mg AO/kg bw/day (actual dose received)
Dose / conc.:
250 other: mg AO/kg bw/day (actual dose received)
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous dose range finding toxicity study in Han Wistar rats, RCC study No. B51592 in which dose levels of 30, 60, 120, 500 and 1000 mg/kg/day corrected for purity were tested. Both the 1000 and 500 mg/kg/day resulted in lethality after a single or two doses, respectively. The overall NOEL was 120 mg/kg/day (corrected for purity).
- Rationale for animal assignment: Computer-generated random algorithm was used, with body weights taken into consideration in order to ensure similar mean body weights in all groups.
10 mL/kg bw was administered.
Maternal 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, reighting reflex and reaction to handling.
c) Open field observations: Level of ambulatory activity, including rearing (one minute evaluation) responsiveness to sharp noise, paw pinch, gait evaluation, quantity of urine and fecal pellets voided.
d) Categorical observations (could be made at any time during the FOB): Hair coat, behaviour, respiration, muscle movements, eyes, hearing ability (Preyer's reflex) urine or faeces, soiling, general abnormalities, posture.
e) Measurements/counts: Hind limb/fore limb grip strength, landing food splay, rectal temperature.
Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151. Activity of the animals (based on beam count) was recorded for 6 minute intervals over a period of 30 min. These data and the total activity over 30 min were reported.

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

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

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

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

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

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

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: See section above on clinical signs.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data
Fetal examinations:
- External examinations: Yes: All per litter
- Soft tissue examinations: No data
- Skeletal examinations: No data
- Head examinations: No data
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.
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.
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.
Historical control data:
No data
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
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 rate were not affected by the treatment with the test item. At 250 mg/kg/day gestation index was reduced since two dams did not deliver any pups and one dam delivered only one dead pup.
Implantation rate was unaffected. At 250 mg/kg/day, a statistically significant increase of post-implantation loss was observed. Although the higher postnatal loss was in the range of historical control data, this resulted in a reduced number of pups.

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 receiveing 40 or 100 mg/kg/day since they showed the same severity grade as control males. An increase in severity grade of both findings was only recorded in males treated at 250 mg/kg/day.
Forestomach: Hyperkeratosis, parakeratosis, squamous cell hyperplasia and submucosal inflammation were recorded in all males and females at 100 and 250 mg/kg/day.
Submucosal oedema was recorded in males (3/5) and in females (3/5) at 250 mg/kg/day and in females at 100 mg/kg/day.
Erosions were recorded in males (3/5) and females (3/5) at 250 mg/kg/day.
Ulcerations were recorded in females at 100 (1/5) and 250 mg/kg/day (1/5).
Pustules were recorded in males at 250 mg/kg/day (2/5) and females at 100 (1/5) and 250 mg/kg/day.
Microscopic examination of the reproductive organs of males and females treated at 250 mg/kg/day failed to find any abnormality.

OTHER FINDINGS (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:
act. ingr.
Basis for effect level:
other: At doses of ≥100 mg AO/kg bw/day: reduced activity, body weight gain and food consumption noted; forestomach lesions observed. At 250 mg AO/kg bw/day: increased liver weights and microscopic changes in liver and kidney.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
VIABILITY (OFFSPRING)
Litter size, sex ratio and abnormalities at first litter check, postnatal loss Day 0 - 4 post pertum: 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.

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.
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: 250 mg AO/kg bw/day: reduction in number of pups; mean pup weight development reduced.
Abnormalities:
no effects observed
Developmental 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.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
23-03-2010 to 19-05-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD 422
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. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
At study initiation:
Analysis of stability and concentration: Immediately after preparation of the mixtures as well as 8 and 24 hours after storage of the test item preparations at room temperature (3 samples/dose level group). Total number of samples: 9.
Analysis of Homogeneity: At the start of administration, during (middle) administration and before administration to the last animal of each dose level group (3 samples/dose level group). Total number of samples: 9.

At study termination:
Analysis of concentration: During treatment with the test item always before administration to the last animal/dose level group (1 sample/dose level group). Total number of samples: 3.
Test item-vehicle mixtures (in total 21 vials), 4 further vials containing the test item or vehicle of test days 1 and 42 were dispatched on dry ice by courier for analysis.
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 pres-ence 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.
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
Duration of test:
Up to 56 days
Dose / conc.:
40 other: mg AO/kg bw/day (nominal)
Dose / conc.:
100 other: mg AO/kg bw/day (nominal)
Dose / conc.:
200 other: mg AO/kg bw/day (nominal)
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.
Maternal 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.
Ovaries and uterine content:
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.
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

Fetal examinations:
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.
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
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
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 sev-eral 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 rela-tionship 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, ges-tation 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:
40 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: Histopathological changes noted in the forestomach and the mesenteric lymph nodes and signs of systemic toxicity (salivation) observed at dose levels of 100 and/or 200 mg AO/kg bw/day
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
External examinations at dissection revealed no external abnormalities in any of the pups examined.
Dose descriptor:
NOEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no effects noted
Abnormalities:
not specified
Developmental 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. 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 NOAEL for reproductive toxicity was 100 mg AO/kg bw/day, p.o. via gavage.
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:
guideline 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
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.
Dose / conc.:
25 other: mg AO/kg bw/day (nominal)
Dose / conc.:
100 other: mg AO/kg bw/day (nominal)
Dose / conc.:
200 other: mg AO/kg bw/day (nominal)
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
Based on:
act. ingr.
Basis for effect level:
other: Adverse clinical observations, reduction in body weight gain, and reduced feed consumption were observed in the 100 and 200 mg AO/kg group in parent animals.
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.
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Reduced fetal body weights and associated delays in skeletal ossification were observed at 200 mg/kg dose level. Delays in skeletal ossifications were also observed in the 100 mg AO/kg/day dose group.
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:
no adverse effect observed
Dose descriptor:
NOAEL
25 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Reliability 1 for the rat and rabbit developmental toxicity studies.
Reliability 1 for the database..
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

In addition to the key study, there are several supporting studies to establish developmental toxicity potential.

In a combined repeat dose toxicity study with the reproduction/developmental toxicity screening test conducted with C12-14 AO 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 AO/kg bw/day [Ceccatelli et al (2008)]. 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. At doses of ≥100 mg AO/kg bw/day, reduced activity, body weight gain and food consumption were noted in males and/or females. Doses of 250 mg AO/kg bw/day also resulted in increased liver weights and microscopic changes in liver and kidney. Forestomach lesions were observed at both 100 and 250 mg AO/kg bw/day. The parental toxicity NOAEL was 40 mg AO/kg bw/day. Evaluation of reproductive and developmental parameters demonstrated that litter size and mean number of pups at first litter check were not affected by treatment. The sex ratio was also not affected by treatment. No abnormal pups were noted at any dose level. However, at 250 mg AO/kg bw/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. 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 100 mg AO/kg bw/day.

In a rat two-generation reproductive toxicity study with a 30% aqueous solution of C12 AO conducted according to Japan Ministry of Health and Welfare guidelines (comparable to the OECD 416 study design) and fully quality assured by the GLP certified testing facility, the test substance was administered continuously in the diet over two generations at levels of up to 750 ppm [Tesh JM (1993); see Table 25]. The test substance had no adverse effect on the general condition of male and female rats but was associated with slight reductions in rate of bodyweight gain during maturation and gestation at levels of 375 ppm and above. Litter size at birth and viability of offspring up to weaning showed no consistent treatment-related effects. On Day 25 post-partum only, there was a dosage-related reduction in the rate of bodyweight gain of F1 and F2 offspring at 375 and 750 ppm. Thereafter there were no demonstrable effects on body weights. At terminal necropsy of parents and offspring of both generations some variations in organ weights were found, but these were largely related to the deficits in bodyweight and no macroscopic or microscopic abnormalities were detected that could be related to ingestion of the substance. The study report concludes no effects on development at up to the high dose NOAEL of 750 ppm in diet. Based on chemical intake data provided for the study, this translates to a dose range of ≥ 37 -128 mg AO/kg/day in males and 47 – 119 mg AO/kg bw/day in females.

In a prenatal developmental toxicity study with a 30% aqueous solution of C12 AO conducted in a GLP-certified laboratory according to Japan Ministry of Health and Welfare guidelines (similar to OECD TG 414), doses of 50, 100 or 200 mg/kg bw/day(equivalent to 15, 30 or 60 mg AO/kg bw/day)were administered by gavage to pregnant Sprague-Dawley rats on days 7 to 17 of gestation[Tesh JM (1980)]. Each group consisted of 32 pregnant animals: 21 per group were sacrificed on gestation day 20 and their fetuses were examined for morphological development. The remaining 11 females per group were allowed to give birth and their offspring were evaluated for viability, growth, and the attainment of a number of developmental milestones as well as neurobehavioral effects and fertility. Treatment of the F1 dams was continued through weaning, but the F1 animals were not subsequently dosed. Maternal toxicity in the form of decreased body weight and food water consumption was observed in the 200 mg/kg bw/day group. This group also had an increase in water consumption. There were no effects observed at the two lower dose levels. Developmental effects consisting of decreased fetal weight and delayed ossification of some skeletal elements were observed in the 200 mg/kg bw/day group. There were no effects on growth, viability, behavior, or fertility in the F1 litters in any dose group. In summary, the 200 mg/kg bw/day group produced maternal toxicity and associated developmental effects; 100 mg/kg bw/day was a clear no observed adverse effect level. Therefore, the LOAEL for maternal and developmental effects was 200 mg/kg bw/day (equivalent to 60 mg AO/kg bw/day) and the NOAEL was 100 mg/kg bw/day (equivalent to 30 mg AO/kg bw/day)

In a prenatal developmental toxicity study with a 30% aqueous solution of C12 AO conducted in a GLP-certified laboratory according to Japan Ministry of Health and Welfare guidelines (similar to OECD TG 414), doses of 40, 80, or 160 mg/kg bw/day (equivalent to 12, 24 or 48 mg AO/kg bw/day) were administered by gavage to pregnant New Zealand White rabbits on days 6 to 18 of gestation [Tesh JM (1981)]. Each group consisted of 14-15 pregnant animals. Caesarean sections were performed on gestation day 29. Fetal weights, number of resorptions, fetal deaths and fetal morphology (soft tissue and skeletal) were evaluated. There were three deaths in the 80 and 160 mg/kg bw/day groups but these deaths were not related to treatment. Maternal effects in the form of transient decreased body weight and food and water consumption were observed at some point during dosing in all three treatment groups, but only food consumption remained slightly reduced at the end of the study in all treated groups. One animal in the low dose group had a fully resorbed litter, but this was not dose-responsive or treatment-related. There were no other developmental effects observed in any dose group in the study. Therefore, the maternal toxicity and developmental toxicity NOAEL was ≥160 mg/kg bw/day (equivalent to ≥48 mg AO/kg bw/day).

In a combined repeat dose toxicity study with the reproduction/developmental toxicity screening test conducted with C12-18 AO under GLP according to OECD TG 422 the substance was administered via gavage to 10 rats/sex/group at 0, 40, 100 or 200 mg AO/kg bw/day [Hansen B (2010)]. Males were dosed for two weeks prior to mating, during mating and approximately two weeks after mating. Females were dosed for two weeks prior to mating and continuing up to and including day 3 post-partum or the day prior to sacrifice. A satellite group of non-mated animals was treated in the same manner as the main study animals and kept without treatment at least 14 days after the first scheduled sacrifice of main study females. No test item related mortality or influence on body weight was noted in any group. No signs of systemic toxicity were noted for the male and female animals treated with 40 mg AO/kg bw/day. Effects noted were as follows: At ≥100 mg AO/kg bw/day, Increased salivation; dose dependent increase of macrophages with vacuolisation in the mesenteric lymph nodes. This effect was still noted at the end of the recovery period. At 200 mg/kg bw/day, periodic reduction in food intake (males & females); periodic reduction in food intake; laboured breathing (one male) piloerection (females); changes in haematological parameters; effects in the forestomach - squamous cell hyperplasia with submucosal inflammatory reaction and hyperkeratosis/parakeratosis in the stratum corneum (males and females). These lesions were reversible within the 16-day recovery period. The NOEL for parental toxicity from this study was 40 mg AO/kg bw/day.

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 increase in pre-implantation loss compared to controls. 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. 

Toxicity to reproduction: other studies

Additional information

No other studies concerning toxicity to reproduction are currently available.

It should be noted that in repeated dose studies with the registered substance (C12 -14 AO), treatment-related ocular changes were noted.  In the two-generation reproductive toxicity study with C12 AO, there were no treatment-related observations of ocular problems noted in any of the three generations (no effects noted clinically or in histopathology). One observation of a cataract was noted in a low-dose adult animal in the F1 generation by histopathology.  This single instance was not dose-related and therefore not treatment-related.  Given the high spontaneous rate of cataracts in albino rats (Durand et al. 2001), it is not surprising to see a single observation of a cataract in a study in which a large number of animals were evaluated.  It is also worth noting that the observation of a cataract provides evidence of the laboratory’s ability to detect cataract by histopathology.

1) Durand, G et al. Spontaneous polar anterior subcapsular lenticular opacity in Sprague-Dawley rats.  Comp Med. 2001 Apr;51(2):176-179.

Justification for classification or non-classification

In a two-generation reproduction study in the rat with the category member C12 AO administered in the diet, P0 parental toxicity was evident (moderately impaired body weight performance) at 750 ppm (70 mg/kg/ bw/day). There were no adverse effects of treatment on oestrous cyclicity, mating performance, fertility, gestation, parturition or development of the offspring through two successive generations. In addition, in a combined repeat dose and reproductive and developmental toxicity screening study in the rat with the registered substance (C12 -14 AO) administered by gavage, parental toxicity was evident at doses 100 mg/kg/day. No adverse effects of treatment were noted on mating performance, fertility, gestation or parturition. Postnatal effects related to pup mortality and mean body weight at the highest dose of 250 mg/kg/day were associated with frank parental toxicity and were within historical control range, thus not indicative of direct reproductive toxicity. Based on these two reproductive toxicity studies, no classification for reproductive toxicity is warranted.

In a prenatal developmental toxicity study in rats and in a combined repeat dose and reproduction/developmental toxicity screening study in rats with C12 -14 AO, maternal toxicity was observed at doses ≥ 100 mg/kg/day. Developmental effects secondary to maternal toxicity, including delayed ossification and reduced pup weights, were also observed at these doses. In another prenatal developmental toxicity study with the category member C12 amine oxide, maternal toxicity and similar secondary developmental effects were observed at the high dose of 60 mg/kg/day active. However, in a prenatal developmental toxicity study in rabbits with C12 AO at doses up to 48 mg/kg/day, no maternal or developmental effects were observed. And in a two-generation reproductive toxicity study in rats at doses of up to 37 -128 mg/kg/day, no treatment-related effects on litter size or viability of offspring were noted in the presence of slight body weight reductions in the parental animals at this dose. Based on the results in the prenatal developmental toxicity studies on C12 AO and C12 -14 AO, no classification for developmental toxicity is warranted.