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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

For the endpoint toxicity to reproduction (fertility) no one- or multigeneration reproductive toxicity studies are available. Data waiver is claimed.

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Effects on fertility - data waiver

The so far available information on reproductive parameters for the substance is a subacute rat toxicity study (OECD TG 407; Eiben, 2010), that gives no indication of adverse effects on organs of the male and female reproductive system after oral doses of up to 800 mg/kg. In addition, systemic absorption of the substance is assumed to be low after oral intake due to the physico-chemical characteristics of the test substance (limited water solubility of 10.3 mg/L at 20 °C, log Pow of 4.3 - 5.6 at 25 °C and a molecular mass of ca. 309 g/mol). Therefore, based on the available data, there is no reason to expect a specific reproductive toxicity of the test substance.

As confirmed by recent literature (Mangelsdorf et al., 2003; Ulbrich & Palmer, 1995; Janer et al., 2007; Dent, 2007; Sanbuissho et al., 2009) in rodents histopathological examinations in repeated dose toxicity studies of reproductive tissues are of high value and high sensitivity for evaluation of reproductive toxicity in males and females. Histopathological changes on the reproductive organs in repeated dose toxicity studies are indicative of effects on fertility. With this respect repeated dose toxicity studies should be considered sensitive and sufficient information to evaluate toxicity on fertility if histological examination of the reproductive organs is covered.

Taken together, although studies on fertility, respectively one- or multi-generation studies were not available for Fatty acids, tall-oil, compds. with N-[3-(dimethylamino)propyl] tall-oil amides, further testing is considered to be of low priority. In accordance to REACH Annex XI, 1.2., there is sufficient weight of evidence to conclude that Fatty acids, tall-oil, compds. with N-[3-(dimethylamino)propyl] tall-oil amides are not reproductive toxicants, and further testing on vertebrate animals for that endpoint shall be omitted.

Effects on developmental toxicity

Description of key information

The substance has a NOAEL for maternal toxicity (systemic effects) and developmental toxicity of 100 mg/kg bw/day after subacute oral administration to rats (Müller, 2010) or rabbits (van den Oetelaar, 2019). 

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: 2004/73/EEC (2004)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

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

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

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

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.

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

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.

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

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)

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

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.

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)

Abnormalities:

not specified

Developmental effects observed:

not specified

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)

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.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June to July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Chatillon sur Chalaronne, France
- Age at study initiation: 17-19 weeks
- Weight at study initiation (females): 3013-4254 g
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 2 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-20
- Humidity (%): 53-97
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

administration volume: 1 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

All concentrations analysed in the formulations of Groups 2 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). The mean accuracy of the formulation of Group 3 was slightly below the target concentration (i.e. 87% of target). Since the accuracy is only slightly below the criterion range of 90-110% and results of Groups 2 and 4 were acceptable, the lower accuracy of Group 3 was considered incidental and not an indication of an overall problem with the preparation of formulations. The results were therefore accepted.

In the Group 1 formulations, no test item was detected.

The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Details on mating procedure:

Untreated females were mated at the Supplier and arrived on Day 1-4 post-coitum (Day 0 post-coitum is defined as the day of successful mating).

Duration of treatment / exposure:

Day 6 to 28 post-coitum inclusive

Frequency of treatment:

once daily

Duration of test:

28 days

Dose / conc.:

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

Remarks:

Group 1

Dose / conc.:

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

Remarks:

Group 2

Dose / conc.:

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

Remarks:

Group 3

Dose / conc.:

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

Remarks:

Group 4

No. of animals per sex per dose:

22 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

DOSE SELECTION RATIONALE:
A dose range finder (Test Facility Study No. 20168377) was conducted to select dose levels for the Prenatal Developmental Toxicity Study (Test Facility Study No. 20168378). In this study dose levels of 0, 30, 100 and 300 mg/kg/day were tested in 6 pregnant rabbits per dose from Day 6 to 28 by oral gavage.
Maternal findings:
No maternal toxicity was observed up to 100 mg/kg/day. At 300 mg/kg/day, Female Nos. 21 and 23 were sacrificed for animal welfare reasons. These females were sacrificed between Days 15-16 post coitum, based on limited or absent food consumption from start treatment onwards and body weight loss (up to 12%) on Day 15 post coitum. These females showed reduced faeces production (up to severe) during the treatment period, combined with piloerection on the day of necropsy. For Female No. 23, a lean appearance was noted on the day of necropsy as well. At necropsy, no abnormalities were noted. One control female (No. 5) was sacrificed for animal welfare reasons on Day 19 post coitum. This female presented with severe clinical signs: piloerection, hunched posture and lean appearance for 2-5 consecutive days and up to severe reduction in faeces production for more than one week. She was also transiently noted with (slight) labored respiration and rales. In addition, she presented with persistent body weight loss from Day 12 post-coitum onwards (up to 10% on Day 18 post-coitum vs start of dosing) and very low/absent food consumption from Day 9 post-coitum onwards. Reduced faeces production was observed during the treatment period in most animals across the dose groups, in absence of a clear dose-related response. Other clinical signs were observed in animals that were sacrificed in extremis or were considered unrelated to treatment (i.e. scabs, broken upper incisors, red staining vagina, swelling, scars and wounds) as these occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At 300 mg/kg/day, females showed on average body weight loss from start treatment up to Day 15 post coitum. No statistical significance was achieved. Mean body weight and body weight gain remained lower during the complete treatment period. At this dose level, mean food consumption was reduced when compared with concurrent control up to Day 18 post coitum. No statistical significance was achieved. The two animals of which food consumption was most severely reduced were sacrificed between Days 15-16 post coitum. Food consumption of the females that remained in the study recovered to levels that were on average similar to concurrent control. The weight gain corrected for gravid uterus weight was considered unaffected by treatment up to 300 mg/kg/day. Macroscopic observations at necropsy did not reveal any alterations that were considered to be toxicologically relevant. One female in each of the 30, 100 and 300 mg/kg/day groups was non-gravid. All remaining females were pregnant. At 300 mg/kg/day, only 3 litters were available for evaluation due to one non-gravid female and two preterm sacrifices. A higher mean pre-implantation loss (% per litter) was noted for females at 300 mg/kg/day when compared with control females (23.4% vs 3.3%). This higher incidence was caused by Female no. 19, which had 8 corpora lutea but only 3 implantation sites (resulting in 62.5% pre-implantation loss). No toxicologically relevant changes were noted in the numbers of corpora lutea and implantation sites, post implantation loss and early and late resorptions.
Fetal Findings:
Litter sizes were within normal limits for all groups. The fetal weights (combined) were comparable to the controls for the 30, 100 and 300 mg/kg/day dose group and remained within the historical control range of the Test Facility. External examination of the fetuses did not show any abnormalities.
Conclusion:
Based on the results of the dose range finder, selected dose levels for the main study were 30, 100 and 300 mg/kg/day.

Maternal examinations:

- Clinical observations: at least once daily, beginning on Day 6 p.c. and lasting up to the day prior to necropsy
- Body weights: on Days 6, 9, 12, 15, 18, 21, 24, 27 and 29 p.c.
- Food consumption: on Days 6-9, 9-12, 12-15, 15-18, 18-21,21-24, 24-27 and 27-29 p.c.
- Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
- Post-mortem examinations: Sacrifice on gestation day 29

Ovaries and uterine content:

Each ovary and uterine horn of all animals was dissected and examined as quickly as possible to determine:
- The number of corpora lutea
- The weight of the (gravid) uterus (not for animals sacrificed before planned necropsy)
- The number of implantation sites
- The number and distribution of live and dead fetuses
- The number and distribution of embryo-fetal deaths

Fetal examinations:

Litters of females surviving to scheduled necropsy were subjected to detailed external, visceral and skeletal examinations, as described in the following sections. External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

External Examinations:
Each viable fetus was examined in detail to detect macroscopic visible abnormalities and their weight was determined (not for fetuses of animals sacrificed before planned necropsy). Nonviable fetuses (the degree of autolysis was minimal or absent) were examined and weighed. For recognizable fetuses of females euthanized in extremis, a gross external examination was performed.

Visceral Examinations:
All fetuses were internally sexed and examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar. The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination of all groups using the Wilson sectioning technique. After examination, the tissues without variation or malformations were discarded. Tissues with variations or malformations were stored in 10% formalin. The heads from the remaining one-half of the fetuses in each litter of all groups were examined by a mid-coronal slice. All carcasses, including the carcasses without heads, were eviscerated, skinned, labeled and fixed in 96% aqueous ethanol for subsequent examination of skeletons.

Skeletal Examinations:
All eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson. Subsequently, the skeletal examination was done on all fetuses from Groups 1, 3 and 4. Since possible treatment related effects in the mid dose group were observed, skeletal examination was extended to all fetuses from the low dose group. All specimens were archived in glycerin with bronopol as preservative. A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and Study Director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.

Statistics:

Parametric:
Datasets with at least 3 groups (the designated control group and at least 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

Non-Parametric:
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution were compared using the Mann Whitney test. Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

Incidence:
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant. No statistics were applied for data on maternal survival, pregnancy status and group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Indices:

For each litter the following calculations were performed:

Pre-implantation loss (%) = (number of corpora lutea - number of implantation sites) divided by the number of corpora lutea x 100

Post-implantation loss (%) = (number of implantation sites - number of live fetuses) divided by the number of implantation sites x 100

The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:

Viable fetuses affected/litter (%) = number of viable fetuses affected/litter divided by the number of viable fetuses/litter x 100

Historical control data:

available

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg/day, a total of 11 females presented with piloerection during 2-6 consecutive days; for Female Nos. 69, 81 and 82 this clinical sign was only noted on the day of necropsy. Other clinical signs observed in females at 300 mg/kg/day were mainly noted in animals that were sacrificed in extremis or were considered unrelated to treatment (i.e. rales and scabs). Reduced faeces production was observed during the treatment period in most animals across the other dose groups, in absence of a clear dose-related response.

Any other clinical signs noted during the treatment period (i.e. alopecia, rales, scales, scabs, pale faeces, salivation, swelling and broken/bent teeth) occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

mortality observed, treatment-related

Description (incidence):

At 300 mg/kg/day, 10/22 females were sacrificed for animal welfare reasons before the day of scheduled necropsy (i.e. Nos. 69, 74, 75, 76, 79, 81, 82, 83, 86 and 88). These females were sacrificed between Days 15-18 post coitum, based on limited or absent food consumption from start treatment onwards and body weight loss (up to 12%). These females showed reduced faeces production (up to severe) during the treatment period, combined with a hunched posture, lean appearance and/or piloerection on the day of necropsy. For Female No. 79, a pale appearance was noted on the day of necropsy in addition to the previously mentioned clinical signs. At necropsy, no abnormalities were noted in any of the preterm decedents.

One control female (No. 8) was sacrificed for animal welfare reasons on Day 17 post coitum, based on the absence of food consumption from start treatment onwards. This female had lost 6% body weight vs Day 6 post coitum and showed reduced faeces production (up to severe) from Day 9 post coitum onwards. In addition, on Day 17 post coitum this female had a hunched posture and a lean appearance. No abnormalities were noted at necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg/day, an overall body weight loss was noted up to and including Day 15 post coitum. A total of 15 females lost weight during the first six days of treatment and for
remaining females body weight gain was absent or minimal during this period. Females with the most body weight loss on Day 15 post coitum (between 5-12%) were sacrificed between Days 15-18 post coitum, resulting in an on average similar body weight gain to concurrent controls from Day 18 post coitum onwards. Mean body weight of females at 300 mg/kg/day was lower than concurrent control throughout the treatment period, reaching statistical significance on Days 12-24 post coitum.

Body weights and body weight gain were considered to have been unaffected by treatment up to 100 mg/kg/day.

For females at 100 mg/kg/day, mean body weight was slightly lower than the concurrent control mean throughout the treatment period. As body weight gain of these females
remained similar to concurrent control and these females had a lower mean body weight on Day 6 (measured prior to start treatment) this change was considered unrelated to treatment with the test item.

Mean weight gain corrected for gravid uterus of treated animals was considered unaffected by treatment with the test item up to 300 mg/kg/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg/day, mean food consumption was reduced when compared with concurrent control up to Day 18 post coitum. Mean food consumption was statistically significantly
lower during Days 6-9 and 9-12 post coitum (absolute and relative) and Days 15-18 post coitum (absolute only). The animals of which food consumption was most severely reduced
were sacrificed between Days 15-18 post coitum. Food consumption of the females that remained in the study recovered to levels that were on average similar to concurrent control.

Food consumption before or after correction for body weight was considered unaffected by treatment with the test item up to 100 mg/kg/day.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Mean liver weight and liver:body weight ratios of treated animals were considered to be similar to those of control animals.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations in the dose groups up to 300 mg/kg/day that were considered to have arisen as a result of treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The numbers of pre- and post-implantation loss in the 30 and 100 mg/kg/days groups were in the range of normal biological variation and therefore considered unaffected by treatment with the test item.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

The numbers of early and late resorptions in the 30 and 100 mg/kg/days groups were in the range of normal biological variation and therefore considered unaffected by treatment with the test item.

The statistically significantly lower mean percentage of total resorptions in females at 100 mg/kg/day was attributed to the relatively high concurrent control mean and was considered unrelated to treatment with the test item as an opposite effect would be expected in case of toxicity.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Two females were non-gravid in the control, 30 and 100 mg/kg/day groups each. Except for one preterm decedent at 300 mg/kg/day, all remaining females were pregnant. The number of pregnant females was therefore considered unaffected by treatment with the test item. Due to the mortality observed, 19, 20, 20 and 12 litters with viable fetuses were available for evaluation of maternal pregnancy data and in the control, 30, 100 and 300 mg/kg/day groups, respectively.

The lower number of females and litters available at 300 mg/kg/day was considered insufficient for a thorough evaluation of maternal pregnancy data. In addition, as severe maternal toxicity was noted at this dose level, the fetal data was considered not representative for evaluation of developmental effects. The data of females and litters at 300 mg/kg was reported in the tables, but not evaluated nor taken into account for determination of the maternal and developmental No Observed Adverse Effect Levels (NOAELs).

Other effects:

no effects observed

Description (incidence and severity):

The numbers of corpora lutea and implantation sites in the 30 and 100 mg/kg/days groups were in the range of normal biological variation and therefore considered unaffected by treatment with the test item.

Details on maternal toxic effects:

The combination of clinical signs (hunched posture, lean appearance and/or piloerection), reduced food consumption, mean body weight loss, reduced faeces production and mortality (sacrificed animals for animal welfare reasons) at 300 mg/kg/day was considered treatment-related and adverse.

Due to the mortality observed at 300 mg/kg/day, only 12 litters with viable fetuses were available at this dose level. The lower number of litters was considered insufficient for a thorough evaluation of maternal pregnancy data and therefore not evaluated nor taken into account for the determination of the maternal and developmental No Observed Adverse Effect Levels (NOAELs).

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: the combination of clinical signs (hunched posture, lean appearance and/or piloerection), reduced food consumption, mean body weight loss, reduced faeces production and mortality (sacrificed animals for animal welfare reasons) at 300 mg/kg/day

Dose descriptor:

NOAEL

Remarks:

maternal developmental toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: the combination of clinical signs (hunched posture, lean appearance and/or piloerection), reduced food consumption, mean body weight loss, reduced faeces production and mortality (sacrificed animals for animal welfare reasons) at 300 mg/kg/day

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The fetal weights (male, female and combined) were comparable to the controls for the 30 and 100 mg/kg/day dose group and remained within normal limits.

Mean combined (male and female) fetal body weights were 36.8, 37.1 and 36.0 gram for the control, 30 and 100 mg/kg/day groups, respectively.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment up to 100 mg/kg/day.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no test item-related effects on litter size up to 100 mg/kg/day.

Mean litter sizes were 9.3, 9.1 and 9.6 fetuses/litter for the control, 30 and 100 mg/kg/day groups, respectively.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on external morphology following treatment up to 100 mg/kg/day.

In total six externally malformed fetuses were observed in this study. At 100 mg/kg/day, one fetus had gastroschisis and tarsal flexure and the dead fetus in the litter of one dam had a carpal flexure. At 30 mg/kg/day, flexure of both tarsals were observed for one fetus. The three remaining malformations affected control fetuses. One control fetus had distended abdomen that appeared to have a cardiac cause (large heart) that caused ascites. A second control fetus had omphalocele and the third one was grossly malformed (including related gross visceral and skeletal malformations).

Due to the single occurrence and/or occurrence in control fetuses, these malformations were considered to be chance findings. Moreover, the malformation of carpal and/or tarsal flexure that occurred among affected fetuses at 30 and 100 mg/kg/day is the most common finding in historical control fetuses.

External variations were not observed in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on skeletal morphology following treatment up to 100 mg/kg/day.

Only two skeletal malformations were observed. A rib anomaly was observed in one fetus at 30 mg/kg/day and a vertebral anomaly occurred in one control fetus. Due to the single occurrence and/or occurrence in a control fetus, these were considered chance findings.

Skeletal variations occurred at an incidence of 66.2%, 75.5% and 83.5% at control, 30 and 100 mg/kg/day, respectively.

The incidence of unossified sternebra nos. 5 and/or 6 was statistically significantly increased at 100 mg/kg/day. Mean litter incidences of this finding were 14.0%, 26.0% and 31.1% per
litter in control, 30 and 100 mg/kg/day groups, respectively. However, because the value at 100 mg/kg/day was lower than the historical control maximum range (38.9% per litter) and
the control incidence was lower than the historical mean value (21.6% per litter), the higher incidence of unossified sternebra nos. 5 and/or 6 was considered a chance finding and not to
be treatment-related.

Statistical significance was also reached for a higher incidence of malaligned sternebrae at 30 mg/kg/day. Mean incidences were 2.1%, 6.9% and 3.9% per litter in control, 30 and 100 mg/kg/day groups, respectively, but because there was no dose relationship and all incidences were within the historical control range (2.9% - 14.5% per litter), this was considered not to be treatment-related.

The litter incidence for unossified tarsals was 1.1%, 0.5% and 2.2% per litter in control, 30 and 100 mg/kg/day groups, respectively. The incidence at 100 mg/kg/day was higher than the historical control maximum range (2.1% per litter), which was caused by 5 fetuses with an unossified talus from 4 litters. The weight of these fetuses ranged from 19.6 – 26.3 grams which was lower than the mean litter weight of their dam. In addition, these mean litter weights (ranging from 26.0 to 30.5 grams) were lower than the group mean fetal body weight of 36.0 grams. Consequently, it was considered that this sign of delayed ossification in these 5 fetuses was a fetal weight effect and not a direct toxicological effect. Moreover, as it was only seen in 5 out of 192 fetuses, it was considered not toxicologically relevant.

All other variations noted, were not considered treatment-related as they occurred infrequently and/or at frequencies that were within the range of available historical control data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on visceral morphology following treatment up to 100 mg/kg/day.

One fetus at 100 mg/kg/day had an enlarged right atrium and an atrial septum defect. Both these malformations were noted before in historical control fetuses. Due to the occurrence among historical control fetuses or occurrence in a control fetus, the observed visceral malformations were considered spontaneous in origin.

All visceral variations noted were considered unrelated to treatment as they occurred in the absence of a dose-related trend, infrequently and/or at frequencies that were within the range of available historical control data

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

The numbers of fetuses (litters) available for fetal morphological examination were 177 (19), 182 (20), 192 (20) and 95 (12) in the control, 30, 100 and 300 mg/kg/day groups, respectively. External and visceral examinations were done for all fetuses, soft tissue cephalic examination was done for approximately half of the fetuses of the same litter for all groups, and skeletal examination was initially done for all fetuses of control group and dose group 300 mg/kg/day. However, due to high mortality in dose group 300 mg/kg/day, an insufficient number of litters was available for a thorough evaluation and it was decided to perform skeletal examination for all dose groups.

The lower number of litters and fetuses available at 300 mg/kg/day was considered insufficient for a thorough evaluation of fetal data. In addition, as severe maternal toxicity was noted at this dose level, the fetal data was considered not representative for evaluation of developmental effects. The data of litters and fetuses at 300 mg/kg was reported in the tables, but not evaluated nor taken into account for determination of the developmental No Observed Adverse Effect Level (NOAEL).

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:

other: mortality in maternal animals at 300 mg/kg/day

Remarks on result:

other: the lower number of litters and fetuses available at 300 mg/kg/day was considered insufficient for a thorough evaluation of fetal data

Abnormalities:

no effects observed

Developmental effects observed:

no

For further information on the results presented please see " Appendix 1 - Figures and Summary Tables" attached as background material in section "Overall remarks, attachments".

Executive summary:

In a prenatal developmental toxicity study according to OECD TG 414 22 time-mated female New Zealand White rabbits each were treated orally by gavage with the test substance using arachis oil as the vehicle. Females were treated from Day 6 to 28 post-coitum with dosages of 0, 30, 100 and 300 mg/kg bw/day, respectively. Examinations were performed on general tolerance of the test compound by the females and possible effects on intrauterine development. Formulation analyses confirmed that formulations of test item in arachis oil were prepared accurately and homogenously. At 300 mg/kg/day, food consumption was reduced or absent for most females during Days 6-18 post coitum. A concurrent mean body weight loss was observed in these females up to Day 15 post coitum. Reduced faeces production (up to severe) was noted and a total of 11 females presented with piloerection on 2-6 consecutive days. The females for which these effects were most severe were sacrificed for animal welfare reasons between Days 15-18 post-coitum. On the day of necropsy, these females showed a hunched posture, lean appearance and/or piloerection. The combination of these effects at 300 mg/kg/day was therefore considered to be adverse. Due to the mortality observed at 300 mg/kg/day, only 12 litters with viable fetuses were available at this dose level. The lower number of litters was considered insufficient for a thorough evaluation of maternal pregnancy data. In addition, as severe maternal toxicity was noted at this dose level, the fetal data was considered not representative for evaluation of developmental effects. The data of females and litters at 300 mg/kg/day were reported in the tables, but not evaluated nor taken into account for determination of the maternal and developmental no-observed-adverse-effect levels (NOAELs). No maternal toxicity was observed in the 30 and 100 mg/kg/day groups. No developmental toxicity was observed in the 30 and 100 mg/kg/day groups. In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental no-observed-adverse-effect level (NOAEL) for Fatty acids, tall-oil, compds. with N-[3-(dimethylamino)propyl]tall-oil amides was established as being 100 mg/kg/day due to severe maternal toxicity at 300 mg/kg/day. Moreover, at 300 mg/kg/day insufficient litters were available for a thorough evaluation of developmental toxicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study is GLP compliant and is of high quality (Klimisch score=1)

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In a prenatal developmental toxicity study according to OECD TG 414 (Müller, 2010) 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 body weight 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

In a further prenatal developmental toxicity study according to OECD TG 414 (van den Oetelaar, 2019) 22 time-mated female New Zealand White rabbits each were treated orally by gavage with the test substance using arachis oil as the vehicle. Females were treated from Day 6 to 28 post-coitum with dosages of 0, 30, 100 and 300 mg/kg bw/day, respectively. Examinations were performed on general tolerance of the test compound by the females and possible effects on intrauterine development. Formulation analyses confirmed that formulations of test item in arachis oil were prepared accurately and homogenously. At 300 mg/kg/day, food consumption was reduced or absent for most females during Days 6-18 post coitum. A concurrent mean body weight loss was observed in these females up to Day 15 post coitum. Reduced faeces production (up to severe) was noted and a total of 11 females presented with piloerection on 2-6 consecutive days. The females for which these effects were most severe were sacrificed for animal welfare reasons between Days 15-18 post-coitum. On the day of necropsy, these females showed a hunched posture, lean appearance and/or piloerection. The combination of these effects at 300 mg/kg/day was therefore considered to be adverse. Due to the mortality observed at 300 mg/kg/day, only 12 litters with viable fetuses were available at this dose level. The lower number of litters was considered insufficient for a thorough evaluation of maternal pregnancy data. In addition, as severe maternal toxicity was noted at this dose level, the fetal data was considered not representative for evaluation of developmental effects. The data of females and litters at 300 mg/kg/day was reported in the tables, but not evaluated nor taken into account for determination of the maternal and developmental no-observed-adverse-effect levels (NOAELs). No maternal toxicity was observed in the 30 and 100 mg/kg/day groups. No developmental toxicity was observed in the 30 and 100 mg/kg/day groups. In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental no-observed-adverse-effect level (NOAEL) for Fatty acids, tall-oil, compds. with N-[3-(dimethylamino)propyl]tall-oil amides was established as being 100 mg/kg/day due to severe maternal toxicity at 300 mg/kg/day. Moreover, at 300 mg/kg/day insufficient litters were available for a thorough evaluation of developmental toxicity.

Justification for classification or non-classification

No classification required for toxicity to reproduction according to Regulation (EC) No. 1272/2008 (CLP).