Fatty acids, tall-oil, reaction products with 2-[(2-aminoethyl)amino]ethanol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-11-15 to 2013-04-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD guideline 421 in a GLP certified testing facility.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

other: liquid

Details on test material:

Batch no.: 0001232140
Receipt no.: 51684
Storage conditions: at room temperature, container kept tightly closed, in a dry and well-ventilated place
Stability (retest date): at least until October 31, 2013
Purity: 100%
Density: 0.9414 g/cm³
Colour: brownish
Consistency: liquid

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test species / Strain / Stock: Rat / CD® / Crl:CD(SD)
Breeder: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
Body weight (at 1st administration): males: 264.9 - 294.6 g; females: 194.3 - 223.2 g
Age (at 1st administration): males/females: approx. 7 to 8 weeks
Adaptation period: 5 days
Number and sex of animals: 96 animals (48 males and 48 females); 12 animals per sex and group

Feeding:
diet: ssniff® R-Z V1324, ssniff Spezialdiäten GmbH, Soest, Germany - ad libitum
water: tap water - ad libitum

Housing:
cages: MAKROLON cages (type III plus)
bedding material: granulated textured wood (Granulat A2, J. Brandenburg, Goldenstedt, Germany)
temperature range: 22°C ± 3°C
humidity: 55% ± 15%
photoperiod: 12 h light (about 150 lux at approx. 1.5 metres room height)/12 h dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Vehicle / Control: Corn oil (Caesar & Loretz GmbH, 40721 Hilden, Germany), storage at room temperature
The test item formulations were freshly prepared every day.
Frequency of administration: once daily
Administration volume: 2 mL/kg b.w./day
Batch no.: 12141505

Details on mating procedure:

- M/F ratio per cage: 1/1, randomly paired for mating
Mating during the dark period
The female was placed with the same male until pregnancy occurred or two weeks had elapsed.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear
If findings were negative, mating was repeated.
The day of conception (day 0 of gestation) was considered to be the day on which sperm was found. This procedure was repeated until at least 8 pregnant dams were available for each group.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for the analysis of the test item concentration and stability in the test item-vehicle mixtures were taken and stored at 20°C or colder until shipment.

Analysis of stability and concentration:
Immediately after preparation of the solution as well as after 8 and 24 hours of storage of the test item preparations at room temperature (3 samples per dose level group, number of samples: 9).
Analysis of concentration:
During the last administration of the test item to the group, always before administration to the last animal/dose level group (1 sample per dose level group, (number of samples: 3)

The analysis was in the responsibility of the Sponsor.

Duration of treatment / exposure:

males: once daily for up to 38 days (beginning 2 weeks prior to mating lasting up to the day before sacrifice until a minimum dosing period of 28 days was completed)
females: once daily, beginning 2 weeks prior to mating and continuing up to, and including, day 3 post-partum or the day before sacrifice

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

The test item was administered orally at a constant volume (2 mL/kg b.w./day) once daily.
The amount of test item actually administered was adjusted to each animal's current body weight daily.
The control animals received the vehicle at the same administration volume daily in the same way.

Examinations

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily (frequency was increased when signs of toxicity were observed)
Behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality, were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of dosing, weekly thereafter and at termination. During gestation, females were weighed on days 0, 7, 14 and 20, and within 24 hours of parturition (day 0 or 1 post-partum) and day 4 post-partum. Body weights were recorded individually for each adult animal.
The pups were weighed within 24 hours of parturition (day 0 or 1 post-partum) and on day 4 post-partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal was recorded weekly during the pre-mating period, daily during gestation and on day 4 post-partum.
- Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group upon completion of a treatment week (pre-mating and gestation) or treatment period (lactation). The relative food consumption (in g/kg b.w./day) was determined


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily

REPRODUCTIVE PARAMETERS:
- Number of pregnant females
- Pre-coital time
- Gestation length
- Corpora lutea: number per dam, distribution in the uterine horns, absolute number per group, mean per group
- Implantation sites: number per dam, distribution in the uterine horns, absolute number per group, mean per group

Litter observations:

- Number of pups absolute: at birth (alive and dead), after 4 days of life
- Number of pups per dam (male/female/total): at birth, after 4 days of life
- Number of stillbirths: absolute, per dam
- Number of pups with malformations: absolute per dam

- Live pups were counted and sexed within 24 hours of parturition (day 0 or 1 post-partum) and on day 4 post partum. Any abnormal behaviour of the offspring was recorded.

Postmortem examinations (parental animals):

- macroscopically examination for any abnormalities or pathological changes at the time of sacrifice
females:
- numbers of implantation sites and corpora lutea
- apparently non-pregnant uteri (placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI)
males:
- testes and epididymides were weighed
males/females:
- ovaries (2), testes (2), epididymides (2), accessory sex organs (coagulating gland, preputial gland, prostate, seminal vesicle, uterus (incl. cervix and oviducts), vagina) and all organs showing macroscopic lesions were preserved (testes and epididymides were preserved in Bouin's fixative, the remaining tissues were preserved in 7% buffered formalin)

Detailed histopathological examination was performed on the ovaries, testes and epididymides (with special emphasis on the qualitative stages of spermatogenesis and histopathology of intestitial testicular structure) of the adult animals of the control and the high dose group following haematoxylin-eosin and PAS staining (testes and epididymides).

Postmortem examinations (offspring):

All pups sacrificed at day 4 post-partum and all prematurely deceased pups (pups found dead in the cage) were carefully examined externally for gross abnormalities.

Statistics:

For all numerical values homogeneity of variances was tested by using the BARTLETT chi-square test. If the variances were homogeneous, the DUNNETT test (p ≤ 0.01) was used to compare the experimental groups with the control group. In case of heterogeneity of variances, the STUDENT's t-test was carried out, limit of significance was p ≤ 0.01.
For the comparison of classification measurements the following statistical methods were employed (the limits of significance were p ≤ 0.05 and p ≤ 0.01):
FISHER's exact test, n < 100 or chi2-test with Yates' correction for continuity, n ≥ 100

Reproductive indices:

- Fertility index female
- Gestation Index

Offspring viability indices:

- Birth Index
- Live Birth Index
- Viability Index
- Pre-implantation loss
- Post-implantation loss

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

salivation and breathing sounds at 60 and 200 mg 400112/kg for male and females; for one male rat also at 20 mg 400112/kg

Mortality:

mortality observed, treatment-related

Description (incidence):

Two males treated with 200 mg 400112/kg bw/day died prematurely. The deceased animals revealed pilo-erection, a decreased body temperature, reduced motility, a thickened abdomen, a pale appearance, an anus smeared with faeces, reduced water consumption
and/or pultaceous faeces on several days before death. Necropsy revealed gastric and/or intestinal lesions.

Three of 12 female rats treated with 60 mg 400112/kg b.w./day and 5 of 12 female rats treated with 200 mg 400112/kg b.w./day died prematurely. Pilo-erection, breathing sounds, a haemorrhagic nose and snout, slightly reduced motility, a thickened abdomen, pultaceous faeces and/or a pale appearance were observed for all animals or individual animals on one to several days before death. Necropsy revealed gastric and/or intestinal lesions in all prematurely deceased female animals.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight of male animals treated with 60 or 200 mg WS400112/kg b.w./day was below the body weight of the control group from approximately test day 15 onwards (by up to 12%, partly statistically significant at p ≤ 0.01 or p ≤ 0.05). The body weight at autopsy was reduced accordingly.
The body weight of female animals treated with 60 or 200 mg WS400112/kg b.w./day was reduced by up to 9% during gestation (p ≤ 0.01). During the lactation period, only the high-dose group was affected by a decrease of up to 9% (p ≤ 0.01). The body weight at autopsy was reduced accordingly.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

relative food intake: males treated with 60 or 200 mg 400112/kg bw/day - reduction by up to 12% compared to the control. Females treated with 200 mg 400112/kg bw/day: reduction by up to 17% compared to the control.

Food efficiency:

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:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

histomorphological examination of the reproductive organs testes, epididymides and ovaries did not reveal any morphological lesions considered to be related to the test item for the control and high dose group

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

no test item-related influence was noted on the pre-coital time, the female fertility index and the gestation length of the animals treated with 20, 60 or 200 mg 400112/kg bw/day compared to the control (see Tables 5, 6, and 7 in "overall remarsk, attached background material").

Details on results (P0)

Body weight: males treated with 60 or 200 mg 400112/kg bw/day - reduction by up to 12% compared to the control; females treated with 200 mg 400112/kg bw/day: reduction by up to 9% compared to the control in the pre-mating and mating period and also in the lactation period; females treated with 60 or 200 mg 400112/kg bw/day: reduction by up to 9% compared to the control in the gestation period.

Mortality:
Two males treated with 200 mg 400112/kg bw/day died prematurely. Three of 12 female rats treated with 60 mg 400112/kg b.w./day and 5 of 12 female rats treated with 200 mg 400112/kg b.w./day died prematurely.

Macroscopic changes:
Macroscopic changes were noted in form of an dilated stomach filled with yellowish liquid and a dilated and empty intestine for one surviving male animal treated with 200 mg 400112/kg b.w./day. Further, a haemorrhagic nose, a thickened abdomen and an anus soiled with faeces were noted for this animal. These findings were probably caused by the corrosive properties of the test item.

Epididymides weights:
Absolute epididymides weights of the animals treated with 200 mg 400112/kg b.w./day appeared to be slightly reduced by 15% for the left and by 7% for the right epididymis. However, this slight change is not considered to be a specific effect on the reproduction organs but to be caused by the stress exerted by the general toxicity of 400112 and not by the test item per se.

Reproduction parameters of the dams:
No differences were noted on any of the reproduction parameters such as the number of corpora lutea, implantation sites, pups at birth (alive and dead), stillbirths and live-born pups of the female animals treated with 20, 60 or 200 mg 400112/kg b.w./day. No changes were noted for the birth index, the live birth index, and the pre- and post-implantation loss in the dosed groups.
No runts were observed in the litters of the F0 generation in any dose group. One runt noted in the control group is considered as coincidental. No abortion or any malformed foetuses were noted in any of the tested dose groups including the control.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

food consumption and compound intake

2

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Remarks:

the NOAEL of 200 mg/kg/d represented the highest dose tested at which toxic effects were observed in parental animals

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

colon

duodenum

ileum

jejunum

stomach

other: the effects on the gastrointestinal tract very likely are due to the surface active properties (corrosivity) of the test substance.

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

see Table 14 in "Overall remarks, attachments"

Gross pathological findings:

no effects observed

Details on results (F1)

Mortality: No differences were noted on the survival of the male and female pups of any dose group after treatment of the parental animals with 20, 60 or 200 mg 400112/kg b.w./day.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Remarks:

the dose of 200 mg/kg/d applied to parental animals was the highest dose tested.

Sex:

male/female

Basis for effect level:

other: overall effects (growth and development of the offspring )

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

The aim of the study was to obtain information on possible effects of 400112 on reproduction and/or development following oral administration to rats. Therefore an OECD 421 guideline study was performed.
No differences were noted on any of the reproduction parameters up to a dose level of 200 mg 400112/kg b.w./day.
No differences were noted up to a dose level of 200 mg 400112/kg b.w./day on the growth and development of the offspring until sacrifice on day 4 postpartum.

The following no-observed-adverse-effect Ievels were noted:
Effects on the parental animals
NOAEL (no-observed-adverse-effect Ievel): 20 mg 400112/kg b.w./day, p.o.
Effects on reproduction
NOAEL (no-observed-adverse-effect Ievel): > 200 mg 400112/kg b.w./day, p.o.
Effects on the F1 pups
NOAEL (no-observed-adverse-effect Ievel): > 200 mg 400112/kg b.w./day, p.o.

Executive summary:

The aim of the experiment was to obtain information on possible effects of the test item 400112 on reproduction and/or development. The test item was administered orally to rats at dose levels of 20, 60 and 200 mg/kg b.w./day during the pre-mating, mating and post-mating periods to parental males as well as during the pre-mating, mating, gestation and lactation periods until day 3 post-partum to parental female animals.

 

Effects on the parental generation

Three of 12 female rats treated with 60 mg 400112/kg b.w./day and 2 of 12 male and 5 of 12 female rats treated with 200 mg 400112/kg b.w./day died prematurely. The deaths are regarded as test item-related as necropsy revealed gastric and/or intestinal lesions in nearly all deceased animals. Several signs of systemic toxicity were noted from the intermediate dose of 60 mg 400112/kg b.w./day onwards among which the most frequent were salivation, pilo-erection and breathing sounds. Further, the body weight and the food consumption were slightly but partly statistically significantly reduced. The macroscopic inspection of the surviving male and female animals at the end of the treatment period did not reveal any test-tem related changes except for gastrointestinal lesions in one high-dose animal only. No specific effect of on the reproduction organs was noted for 400112. Slightly reduced absolute epididymides weights at 200 mg/kg b.w./day are considered to be caused by the stress exerted by the general toxicity and not by the test itemper se. The histomorphological examination of the ovaries, testes and epididymis of the high-dose animals did not reveal test-item related changes.

 

Effects on reproduction

No differences were noted on any of the reproduction parameters up to a dose level of 200 mg 400112/kg b.w./day.

 

Effects on the F1 pups

No differences were noted up to a dose level of 200 mg 400112/kg b.w./day on the growth and development of the offspring until sacrifice on day 4 postpartum.

 

The following no-observed-adverse-effect Ieveis were noted:

Effects on the parental animals

NOAEL (no-observed-adverse-effect level): 20 mg 400112/kg b.w./day, p.o.

Effects on reproduction

NOAEL (no-observed-adverse-effect level): >200 mg 400112/kg b.w./day, p.o.

Effects on the F1 pups

NOAEL (no-observed-adverse-effect level): >200 mg 400112/kg b.w./day, p.o.