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Toxicological information

Toxicity to reproduction

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Administrative data

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
Cross-reference
Reason / purpose:
read-across: supporting information

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1983
Report Date:
1983

Materials and methods

Test guidelineopen allclose all
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.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid - liquid: aqueous solution
Details on test material:
Alkyl chain length distribution: C8 - 0.1%; C10 - 0.1%; C12 - 96.4%; C14 - 2.9%; C16 - 0.1%; others - 0.4%
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.

Test animals

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.

Administration / exposure

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.
Doses / concentrationsopen allclose all
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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Details on results (P0)

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.

Effect levels (P0)

open allclose all
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

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

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.

Details on results (P1)

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.

Effect levels (P1)

open allclose all
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

Target system / organ toxicity (P1)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

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.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

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.

Effect levels (F1)

open allclose all
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

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

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.

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

open allclose all
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

Target system / organ toxicity (F2)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
750 ppm
Treatment related:
no

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.