Fatty acids, tall-oil, compds. with N-[3-(dimethylamino)propyl]tall-oil amides

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese MAFF guideline No. 8147 (2000, amended 2001)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: 2004/73/EEC (2004)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, tall-oil, compds. with N-[3-(dimethylamino)propyl]tall-oil amides

EC Number:

295-714-4

EC Name:

Fatty acids, tall-oil, compds. with N-[3-(dimethylamino)propyl]tall-oil amides

Cas Number:

92128-22-8

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

Reaction products of tall-oil fatty acids and (3-aminopropyl)dimethylamine

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands B.V. (Horst, Netherlands)
- Age at study initiation: at least 12 weeks
- Weight at study initiation: females: 203-268 g
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.2-21.4
- Humidity (%): 62-67
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

administration volume: 2 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before the start of treatment, the suitability of the formulation was confirmed by the analysis of concentration and stability of dosage forms prepared in the same way as it was done in the study. Analyses were carried out before the start of the study. The respective analytical data confirmed that the formulations in corn oil at 5 to 500 mg/ml are stable at room temperature for at least 17 days. For content checks the concentrations of samples of control and each test item dosage formulation prepared were determined twice during the study.

Details on mating procedure:

The animals were mated by placing one female overnight into a Type IIIh cage together with one male rat. If sperm was detected in the vaginal smear taken on the morning following mating, this day was regarded as day 0 of gestation.

Duration of treatment / exposure:

day 6 to day 20 of gestation

Frequency of treatment:

once daily

Duration of test:

21 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

DOSE SELECTION RATIONALE:
The dose levels were selected based on the results of a previous dose toleration study in rats with Zusatzmittel VP PU 1748 in which two rats received the limit dose of 1000 mg/kg orally once daily from day 6 to day 20 of gestation (Study number: T3074534). In this orientating study no indications for maternotoxicity or fetotoxicity occurred. In addition, an orientating systemic toxicity study was conducted in male and female rats in which the doses of 100, 300 and 1000 mg/kg Zusatzmittel VP PU 1748 were orally administered over approximately two weeks (Study number: T8079921). Some female animals at the high dose level of 1000 mg/kg showed histologically slight forestomach erosion and ulceration. Correspondingly, the NOAEL for female rats was 300 mg/kg Zusatzmittel VP PU 1748 in this orientating study.

Examinations

Maternal examinations:

- Clinical observations: appearance, behaviour, excretory products and mortality, from day 0-21 p.c. once daily
- Body weights: on day 0 p.c. and daily from day 6-21 p.c.
- Food consumption: from day 0-21 p.c.
- Gross pathological examination: at the time of cesarean section on day 21  p.c. 

Ovaries and uterine content:

The following parameters were determined and assessed at cesarean section on day 21 p.c.:
- Number of corpora lutea
- Number of implantations
- Uterine weights
- Individual weight and appearance of the placentas
- Number of early resorptions
- Number of late resorptions
- Number of dead fetuses
- Number of live fetuses

Fetal examinations:

- Number of live fetuses
- Sex of live fetuses
- Individual weights of live fetuses
- External abnormalities (malformations/variations)
- Visceral abnormalities (malformations/variations)
- Skeletal and cartilage abnormalities (malformations/variations)

Statistics:

- Dunnett test:
mean feed consumption, mean body weights, mean body weight gains, and mean corrected body weight gains, mean uterine weights, mean number of Corpora lutea, mean number of implantations, mean number of resorptions, mean number of postimplantation losses, total number of fetuses, mean number of live/dead fetuses, mean placental weights, mean litter weight, mean fetal weights, mean fetal weight of male/female fetuses.

- Fisher’s exact test with Bonferroni-Holm correction:
fertility and gestation rate, percentage of preimplantation losses per group, percentage of postimplantation losses or dead fetuses per group, percentage of total resorptions, early and late resorptions per group, percentage of sex of male and female fetuses per group, percentage of fetuses or litters with external, visceral, skeletal and cartilage abnormalities (malformations and variations) findings per group.

- Wilcoxon-Mann-Whitney test with Bonferroni-Holm correction:
percentage of preimplantation losses per female, percentag of postimplantation losses, resorptions or dead fetuses per female, percentage of fetuses or litters with external, visceral, skeletal and cartilage abnormalities (malformations and variations) per female.

Historical control data:

no data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects: yes

Details on maternal toxic effects:
One female of the mid dose group of 300 mg/kg died due to misapplication. Increased sialorrhea occurred in all study groups including the control group and with a dose-dependently increased incidence at the low dose and higher. Due to local irritating properties of the test substance a compound-relation is considered likely. A dose-dependent effect on body weight gain was induced at the mid dose of 300 mg/kg and higher. The body weight gain was slightly lower at the end of the treatment period at 300 mg/kg and distinctly lower already after start of treatment at the high dose of 1000 mg/kg. This impairment was related to body weight loss at 300 mg/kg (individual animals) and at 1000 mg/kg and markedly reduced feed intake at the high dose level during the whole treatment period. No substance related macroscopic findings were observed up to and including 1000 mg/kg.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Remarks:

general toxicity (systemic)

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

other: food intake

2

Dose descriptor:

NOAEL

Remarks:

general toxicity (local)

Effect level:

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

Based on:

test mat.

Basis for effect level:

clinical signs

Remarks on result:

other: increased sialorrhea (local irritating effect)

3

Dose descriptor:

NOAEL

Remarks:

maternal developmental toxicity

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: no treatment-related effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: yes

Details on embryotoxic / teratogenic effects:
The gestation rate, number of corpora lutea, postimplantation loss, litter size, fetal sex distribution, placental weights and placental appearance were not affected by treatment up to and including 1000 mg/kg. The spontaneous occurring slightly higher mean number of fetuses at the mid dose level of 300 mg/kg was consequently accompanied by marginally lower fetal body weight. Incidence of structural findings classified as malformations observed in this study at levels up to and including 1000 mg/kg was low and dose-relation was not evident. The types of fetal malformations are considered a representative sample of spontaneous malformations in the rat strain used. Thus treatment-relation for malformations was not evident up to an including 1000 mg/kg. Furthermore, no substance related fetal structural findings classified as external, visceral or cartilage variations were noted up to and including 1000 mg/kg. At the mid dose of 300 mg/kg and higher skeletal findings indicating a retarded ossification occurred. These changes were seen at the 300 mg/kg level together with incidentally higher litter size which may have contributed to the marginally retarded ossification. A further sign of a retarded fetal development was a reduced fetal weight at the high dose level of 1000 mg/kg. The more progressed ossification in sternebrae as well as the mandibular and the zygomatic bone at1000 mg/kg is considered toxicologically irrelevant as it is highly unlikely that more progressed and retarded ossification occurs simultaneously. In addition, the fused cartilage (processus spinosus) of the 10th and 11th thoracal vertebral arches noted without dose-dependence at the low dose of 100 mg/kg and higher is highly likely a spontaneous effect.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Remarks:

fetal developmental toxicity

Effect level:

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

Based on:

test mat.

Sex:

not specified

Basis for effect level:

fetal/pup body weight changes

other: skeletal findings (retarded ossification)

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Table‎ 1: Mean Body Weight Gain (females during the treatment and gestation period)
Dose [mg/kg bw/day]	0	100	300	1000
mean body weight gain [g]
days 6 - 20 p.c.	98.1	100.5	91.0	61.2**
days 0 - 21 p.c.	134.1	141.5	130.8	91.7**
days 15 – 18 p.c.	35.9	34.2	27.0*	25.2**
days 18 – 21 p.c.	35.1	35.6	30.3	23.1*
corrected body weight gain [g]
days 6 - 21 p.c.	20.3	23.0	16.0	-14.1**
Statistically significant difference to control * p < =.05 and ** p < 0.01

Table‎ 2: Mean Feed Intake
Dose [mg/kg bw/day]	0	100	300	1000
Mean feed consumption [g/female/day]
days  0 -  3 p.c.	20.7	20.8	21.8	21.1
days  3 -  6 p.c.	22.4	23.2	23.9*	22.7
days  6 -  9 p.c.	20.7	22.1	21.7	16.0**
days  9 - 12 p.c.	22.8	22.7	22.3	18.0**
days 12 - 15 p.c.	22.5	23.7	23.4	17.3**
days 15 - 18 p.c.	25.7	25.8	24.7	18.9**
days 18 - 20 p.c.	24.5	25.3	21.5	18.4**
days  6 - 21 p.c.	23.2	23.9	22.9	17.7**
days  0 - 21 p.c.	22.7	23.4	22.9	18.9**
Statistically significant difference to control * = p < 0.05 and ** = p < 0.01

Table‎ 3: General Reproduction Data
Dose [mg/kg bw/day]	0	100	300	1000
Number inseminated females (total):	25	25	25	25
- Number died during gestation	0	0	1^a	0
- Number delivered prematurely	0	0	1^b	0
Number surviving females with defined day 0 p.c.:	25	25	23	25
- Number not pregnant	0	1^c	1^c	0
- Number surviving females pregnant	25	24	22	25
- Number surviving females with viable fetuses	25	24	22	25
- Number surviving females with total resorptions	0	0	0	0
Values per female with viable fetuses:	25	24	22	25
- Corpora lutea (mean)	16.2	16.17	17.05^d	16.04
- Preimplantation loss (% per group)	10.37	9.79	10.89^d	10.22
- Implantations (mean)	14.52	14.58	15.13^d	14.40
^a one female (no. 67) died ^b one female (no. 75) delivered prematurely on day 21 p.c. ^cdams not pregnant: nos. 49 (100 mg/kg) and 62 (300 mg/kg) ^d21 dams were used for calculation (excluding dam 66)

Table‎ 4: Gestation Rate
Dose	Females with viable fetuses		Females
[mg/kg bw/day]	N	in % of Females with Implantations	with Total Resorption
0	25	100.0	0
100	24	100.0	0
300	22^a	100.0	0
1000	25	100.0	0
^a female no. 67 (pregnant) died due to misapplication and one female (no. 75) with premature delivery was excluded from calculation

Table‎ 5: Mean Values for Parameters of Intrauterine Development
Dose [mg/kg bw/day]	0	100	300	1000
Number of females
with implantations	25	24	23^a	25
with viable fetuses	25	24	23^a	25
Values per female:
placental weight in g (mean)	0.67	0.68	0.68	0.67
number of live fetuses (mean)	13.48	13.21	14.22	13.64
postimplantation loss(mean)	1.04	1.38	0.91	0.76
% males	50.15	47.00	48.01	46.33
fetal weight in g (mean)	4.90	5.03	4.70	4.49**
^a including data on one female (no. 75) which delivered prematurely on day 21 p.c. Statistically significant difference to control ** = p < 0.01

Table 6: Fetal Malformations
Malformation (N)		Dose [mg/kg bw/day]
	0		100	300^a	1000
External malformations (Total):	0		0	0	0
Visceral malformations (Total):	1		1	1	2
- severely dilated renal pelvis	1		1	0	2
- absent eyes	0		0	1	0
Skeletal malformations (Total):
- sternoschisis	0		0	1	0
- supernumerary lumbar vertebra	1		0	0	0
- fused thoracal vertebral bodies	0		1	0	0
- absent ribs	1		0	1	0
Number of fetuses per group (Total)^b	338		317	329	342
Number of fetuses with malformations	3		2	3	2
Fetuses with malformations per group (%)	0.89		0.63	0.91	0.58
Number of litters per group	25		24	23	25
Number of litters with malformations	3		2	3	2
Litters with malformations per group (%)	12.0		8.3	13.0	8.0
^a including fetal data on one female (no. 75) which delivered prematurely on day 21 p.c. ^b including dead fetuses (1, 0, 2, 1 in control, low, mid and high dose, respectively)

Applicant's summary and conclusion

Executive summary:

In a prenatal developmental toxicity study according to OECD TG 414 25 inseminated female Wistar rats each were treated orally by gavage with the test substance using corn oil as the vehicle. Females were treated from day 6 to day 20 of gestation with dosages of 0, 100, 300 and 1000 mg/kg bw/day, respectively. The fetuses were delivered by cesarean section on day 21 of gestation. Examinations were performed on general tolerance of the test compound by the females and possible effects on intrauterine development. Increased sialorrhea occurred in all study groups including the control group and with a dose-dependently increased incidence at the low dose and higher. Due to local irritating properties of the test substance a compound-relation is considered likely. A dose-dependent effect on body weight gain was induced at the mid dose of 300 mg/kg and higher. The body weight gain was slightly lower at the end of the treatment period at 300 mg/kg and distinctly lower already after start of treatment at the high dose of 1000 mg/kg. This impairment was related to body weight loss at 300 mg/kg (individual animals) and at 1000 mg/kg and markedly reduced feed intake at the high dose level during the whole treatment period. No substance related macroscopic findings were observed up to and including 1000 mg/kg. The gestation rate, number of corpora lutea, postimplantation loss, litter size, fetal sex distribution, placental weights and placental appearance were not affected by treatment up to and including 1000 mg/kg. The spontaneous occurring slightly higher mean number of fetuses at the mid dose level of 300 mg/kg was consequently accompanied by marginally lower fetal body weight. Incidence of structural findings classified as malformations observed in this study at levels up to and including 1000 mg/kg was low and dose-relation was not evident. The types of fetal malformations are considered a representative sample of spontaneous malformations in the rat strain used. Thus treatment-relation for malformations was not evident up to an including 1000 mg/kg. Furthermore, no substance related fetal structural findings classified as external, visceral or cartilage variations were noted up to and including 1000 mg/kg. At the mid dose of 300 mg/kg and higher skeletal findings indicating a retarded ossification occurred. These changes were seen at the 300 mg/kg level together with incidentally higher litter size which may have contributed to the marginally retarded ossification. A further sign of a retarded fetal development was a reduced fetal weight at the high dose level of 1000 mg/kg. The more progressed ossification in sternebrae as well as the mandibular and the zygomatic bone at 1000 mg/kg is considered toxicologically irrelevant as it is highly unlikely that more progressed and retarded ossification occurs simultaneously. In addition, the fused cartilage (processus spinosus) of the10th and 11th thoracal vertebral arches noted without dose-dependence at the low dose of 100 mg/kg and higher is highly likely a spontaneous effect. Summarizing and evaluating all data investigated the no-observed-adverse-effect level (NOAEL) for maternal toxicity (systemic effects) and developmental toxicity is 100 mg/kg bw/day. Regarding the maternal toxicity for local irritating effect (sialorrhea) the NOAEL is < 100 mg/kg bw/day. Thus, developmental toxicity occurs only in the presence of clear maternal toxicity.