Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1980
Report Date:
1980

Materials and methods

Test guidelineopen allclose all
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

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 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.

Administration / exposure

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.
Doses / concentrationsopen allclose all
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.

Examinations

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.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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.

Maternal developmental toxicity

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.

Effect levels (maternal animals)

open allclose all
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

Results (fetuses)

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.

Effect levels (fetuses)

open allclose all
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.

Overall developmental toxicity

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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).