Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Tridecylamine-Isomerengemisch (CAS 86089-17-0)
- Test substance No.: 00/0693-1
- Purity 99.6%
- Batch No.: Tank 66 from November 16, 2000
- Physical state/appearance: Liquid/colorless-clear
- Stability: proven by reanalysis
- Storage conditions: Room temperature, under N2

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Age at study initiation: 70 - 84 days
- Weight at study initiation: 146.4 - 183.2 g
- Housing: single housing from day 0- 20 post coitum in type DK III stainless steel wire mesh cages (BECKER & CO., Castrop-Rauxel, FRG; height: 15 cm x 37.5 cm x 21 cm; floor area about 800 cm²).
- Diet: ground Kliba maintenance diet rat/mouse/hamster meal (PROVIMI KLIBA SA, Kaiseraugst, Switzerland) ad libitum
- Water: Tap water ad libitum
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12 (6.00 pm - 6.00 am dark; 6.00 am - 6.00 pm light)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% Carboxymethylcellulose in doubly distilled water with 0.05% Cremophor EL
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The aqueous test substance emulsions were prepared at the beginning of the administration period and thereafter at intervals which took into account the analytical results of the stability verification. For the preparation of the emulsions, an appropriate amount of the test substance was weighed depending on the dose group in calibrated beakers, topped up with 0 .5% Carboxymethylcellulose in doubly distilled water with 0.05% Cremophor EL and subsequently thoroughly mixed using a magnetic stirrer. This devise was also used to keep the emulsions homogeneous during treatment of the
animals.


ADMINISTRATION VOLUME:
10 ml/kg bw/d
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test substance emulsions were analyzed by GC. Samples of the test substance emulsions were sent to the analytical laboratory twice during the study period (at the beginning and towards the end) for verification of the concentrations. The samples which were taken for the first concentration control analyses at the beginning of the administration period were also used to verify the homogeneity for the samples of the low and the high concentrations (5 and 80 mg/kg body weight/day). 3 samples (one from the top, middle and bottom in each case) were taken for each of these concentrations from the beaker with a magnetic stirrer running.
Details on mating procedure:
The animals were mated by the breeder ("time-mated") and supplied on day 0 post coitum (p.c. = detection of vaginal plug / sperm).
Duration of treatment / exposure:
gestation days 6-19
Frequency of treatment:
daily
Duration of test:
3 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 5, 20, 80 mg/kg bw/d
Basis:

No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
The animals were examined at least daily for clinical symptoms. Mortality was checked twice a day or once on Saturday/Sunday. Food consumption and body weight on gestation days 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20 were recorded. Corrected body weight gain was calculated after terminal sacrifice as [bw on gestation day 20 - (uterus weight+bw gestation day 6)]. Dams were sacrificed on gestation day 20. Necropsy included gross pathology assessment. Uterus and ovaries were removed with subsequent examination.
Ovaries and uterine content:
Examination and recording of: unopened uterus weight; no. of corpora lutea; no. and classification of implantation sites as live fetuses or dead implantations (early or late resorptions, dead fetuses). Conception rate and pre- and postimplantation losses were calculated.
Fetal examinations:
Fetuses were weighed and sexed by anogenital distance observation (which was later confirmed by internal examination of those fetuses which were preserved in BOUIN's solution), and macroscopically examined (viability, condition of placenta, fetal membranes, umbilical cords, placental weight). Approx. 50% of all fetuses were subjected to soft tissue examinations after fixation in BOUIN's solution, the other 50% of fetuses was examined for skeletal changes.
Statistics:
Statistical evaluation of data included FISHER's Exact Test for conception rate, mortality of the dams, and number of litters with fetal findings; WILCOXON Test for proportions of fetuses with malformations and/or variations in each litter; and DUNNETT's Test for all other data including water consumption and body weight gain.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
Only pregnant dams or pregnant dams sacrificed at schedule were used for calculations. Animals which were totally or partially excluded were 2 non-pregnant animals in each of the groups except the high dose group.
Mortality: none in any of the groups.
Clinical data and signs:
Low dose, 5 mg/kg bw/d: no substance related effects seen.
Intermediate dose, 20 mg/kg bw/d: no substance related effects seen, except transient salivation in two dams during gestation days 8-19. Salivation started immediately after dosing and persisted for some minutes.
High dose, 80 mg/kg bw/d: Transient salivation was noted in 24/24 animals during gestation days 6-19 starting shortly after dosing and lasting few minutes. Additionally, urine smeared fur (4/24 animals) and piloerection (1/24) was noted. Salivation was regarded to be treatment-related due to bad taste or local affection of the upper digestive tract, but was not assessed as an adverse or toxic effect. Food intake was significantly reduced on most of the days. If calculated for the entire treatment period it was 8% less than in the concurrent control. Statistically significant effects on body weight parameters were noted: lowered mean body weight on gestation day 13 (-5%) and lowered corrected body weight gain (-21%) whereas body weight was comparable in all groups throughout the treatment period.
Terminal examination of dams: The mean gravid uterus weight was not influenced by the administration of the test substance. There were no substance-related findings at necropsy in any of the dams. Conception rates reached 92% in control, low, and intermediate dose groups; it was 100% in the high dose group. However, there were no substance-related and/or biologically relevant differences between the test groups in conception rate, mean number of corpora lutea and implantation sites or in the values calculated for the pre- and postimplantation losses, the number of resorptions and viable fetuses.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
Prenatal developmental toxicity (delayed ossification) only at maternally toxic doses. No signs of teratogenicity up to and including the highest dose tested.
Examination of fetuses: Sex distribution was unchanged in all treatment groups. In high dose animals, placental weight was statistically significantly increased (+12% vs. concurrent control), but well in the historical control range. Therefore this was considered to be spontaneous in nature and without toxicological relevance. Mean fetal body weight was unchanged.
The isolated and scattered occurrence of external malformations (anophthalmia, absent tail, cleft palate) in all animal groups including controls did not attain statistical significance in any of the test groups and did not suggest any relationship to treatment. No external variations were noted.
No soft tissue malformations nor unclassified soft tissue observations were seen. The noted soft tissue variations noted (uni- or bilateral dilations of the renal pelvis) were found in fetuses from all test groups without significant differences between the test groups.
Skeletal malformations were noted in single fetuses from all test groups in proportions of 4/111 fetuses (3.6%; control group), 2/108 fetuses (1.9%), 2/109 fetuses (1.8%), and 4/116 fetuses (3.4%) in the low, mid, and high dose group, respectively. No relation to dosing was seen, and no difference to historical control data was noted.
Statistically significantly increased incidences of some skeletal variations were seen in the high dose group fetuses as follows: general delays in skeletal ossification (9.2% fetuses/litter vs. 0% in controls); incomplete ossification of supraoccipital (28.6% fetuses/litter vs. 16.5% in controls); bipartite ossification of thoracic centrum (4.2% fetuses/litter vs. 0% in controls); incomplete ossification of sacral arch (57.4% fetuses/litter vs. 25.7% in controls). The overall incidence of skeletal variations was not different from historical control data.
Thus, there are slight indications of substance-induced effects on skeletal maturation without effects on fetus weight.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
>= 80 mg/kg bw/day
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Individual external malformations in fetuses:

Test group

(mg/kg bw/d)

Number and sex of fetuses

Malformation

Incidence (%) on the basis of

fetuses

litters

0 (Control)

1, f

Acaudate (+)

0.5

4.5

5

1, m

Anophthalmia

0.5

4.5

20

-

0

0

80

2, m

Cleft palate

0.9

8.3

f = female; m = male; (+) = fetus with associated skeletal malformations

.

Individual fetal skeletal malformations:

Test group

(mg/kg bw/d)

Number and sex of fetuses

Malformation

Incidence (%) on the basis of

fetuses

litters

0 (Control)

1, f

Misshapen lumbar vertebra, absent sacral vertebra, absent caudal vertebra (+)

3.6

18

3, m

Misshapen humerus

5

1, f

Misshapen humerus

1.9

9.1

1, m

Misshapen humerus

20

1, f

Misshapen humerus

1.8

9.1

1, m

Misshapen humerus

80

1, m

Cleft sternum (changed cartilage), absent tuberositas deltoidea

3.4

17

3, m

Misshapen humerus

f = female; m = male; (+) = fetus with associated external malformations

.

Occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter):

Test group (mg/kg bw/d)

HCD

Mean %

(range)

Finding

0

(Control)

5

20

80

General delays in skeletal ossification

0.0

0.0

0.0

9.2*

Not present

Incomplete ossification of supraoccipital; unchanged cartilage

16.5

24.5

14.0

28.6*

14.0

(5.4 – 21.2)

Bipartite ossification of thoracic centrum; dumbbell-shaped cartilage of centrum

0.0

0.9

0.0

4.2*

1.1

(0.0 – 3.2)

Fused sacral centrum and arch; unchanged cartilage

0.0

5.5**

3.9*

0.0

2.5

(0.0 – 5.9)

Incomplete ossification of sacral arch; cartilage present

25.7

37.4

18.6

57.4**

15.3

(0.0 – 46.1)

Unilateral ossification of sternebra; unchanged cartilage

0.0

3.1*

0.0

3.5*

1.5

(0.0 – 3.6)

Total fetal skeletal variations

98.1

96.5

89.5

95.6

94.1

(87.0 – 99.2)

* = p ≤ 0.05, ** = p ≤ 0.01; HCD = historical control data

Applicant's summary and conclusion

Conclusions:
Maternal toxicity was noted in dams at 80 mg/kg body weight
per day as substantiated by statistically significant
impaired body weight gain and reduced corrected terminal
body weight (-21% vs. control). Since transient salivation
was not regarded as an adverse effect but resulting from bad
taste no substance-related effect of toxicological relevance
was noted at the low and the intermediate dose level.
Gestational parameters were unchanged at all dose levels up
to and including 80 mg/kg body weight per day.
Fetal parameters were unchanged at all dose levels with the
exception of some skeletal variations in the high dose
fetuses. Transient delays in the skeletal maturation were
noted which affected the skull and the ventral column. The
overall incidence was not different from historical control
data. Therefore these skeletal variations were regarded as
marginal effects and not indicative for selective effects on
fetuses, probably due to maternal stress. No signs of
teratogenicity were noted.
The NOAEL for maternal toxicity was 20 mg/kg body weight per day in this study. The NOAEL for prenatal development was
equally 20 mg/kg body weight per day. No signs of
teratogenicity were noted up to and including the highest
dose tested, i.e. 80 mg/kg body weight per day.