Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to internationally accepted test guidelines and is considered relevant, adequate and reliable.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 422 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine

EC Number:

270-279-3

EC Name:

Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine

Cas Number:

68424-19-1

Molecular formula:

Not applicable as the substance is a UVCB

IUPAC Name:

Fatty acids, C16-18 (even numbered) and C18-unsatd., compds. with triethanolamine.

Test material form:

semi-solid (amorphous): gel

Remarks:

paste

Details on test material:

- Name of test material (as cited in study report): Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine.
- Physical state: Viscous, translucent yellowish waxy paste
- Analytical purity: >95%
- Impurities (identity and concentrations):See confidential details
- Composition of test material, percentage of components: See confidential details
- Purity test date: 01/2013
- Lot/batch No.: DDR02/91
- Expiration date of the lot/batch: October 2014
- Storage condition of test material: At +10° to +25°C
- Other: Manufacturer/Supplier: OLEON NV, Assenedestraat 2, 9940 Ertvelde, Belgium

Test animals

Species:

rat

Strain:

other: CD/Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at first administration: 63 days
- Weight at first administration: Males: 319.6 – 369.4 g; Females: 212.7 – 246.3 g
- Housing: With exception of the mating period, the animals were kept singly in MAKROLON cages (type III plus)with a basal surface of approximately 39 cm x 23 cm and a height of approximately 18 cm. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages. The cages were changed and cleaned once a week.
- Diet (e.g. ad libitum): ssniff® R-Z V1324, (ssniff Spezialdiäten GmbH, 59494 Soest, Germany) served as food. This food was offered ad libitum, with exception of the night before the day of blood withdrawal for laboratory examination. Food residue was removed and weighed.
- Water (e.g. ad libitum): Tap water was offered daily ad libitum.
- Acclimation period: 5 adaptation days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C (maximum range)
- Humidity (%): 55% ± 15% (maximum range)
- Air changes (per hr): Not provided
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: February 27, 2013 To: Males: April 4, 2013; Females: April 26, 2013

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in the vehicle propylene glycol to concentrations of 50, 150 and 500 mg test item/mL. The test item formulations were freshly prepared and adjusted to the animal's current body weight on each administration day.
The test item was administered orally at a constant dose volume of 2 mL/kg bw/day per animal. The animals of the control group received the vehicle (propylene glycol) at a constant volume of 2 mL/kg bw in the same way.
In addition, the stability, homogeneity and concentration of the test item mixture was monitored

VEHICLE
- Justification for use and choice of vehicle (if other than water): propylene glycol (best solvent for test item)
- Concentration in vehicle: 50 - 150 - 500 mg/mL
- Amount of vehicle (if gavage): 2 mL/kg bw/day
- Lot/batch no. (if required): Batch no.: MKBK1806V, Sigma-Aldrich, USA, storage at room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the analysis of the test item-vehicle mixtures, samples of approx. 5 mL were taken at the following times and stored at -20°C or colder until analysis at LPT.

At study initiation:
Analysis of stability and concentration
Immediately after preparation of the solution as well as after 8 and 24 hours storage of the test item preparations at room temperature (3 samples/dose level group).
Number of samples: 3 x 3 = 9
Homogeneity
At the start of administration, during (middle) administration and before administration to the last animal of the dose level group (3 samples/dose level group).
Number of samples:3 x 3 = 9
At termination of the administration period at a time point when the majority of animals is dosed:
Analysis of concentration
During treatment with the test item always before administration to the last animal of the dose level group (1 sample/dose level group).
Number of samples: 1 x 3 = 3
Sum of all samples: 21

The samples were labelled with the study number, test item, test species, type of sample, aliquot number, concentration, test day, sampling time and date.
The validation of the analytical method is described in LPT Study 29660.

Concentration and stability:
The measured concentrations of the test item in the test item vehicle mixtures were between 100.3% and 104.7% of the nominal concentration. These values indicated correctly prepared test item vehicle mixtures, which were stable at room temperature for at least 24 hours.
Further samples to control the concentration of the test item in the test item vehicle mixtures were taken on test day 30. The concentrations of the test item were between 101.9% and 105.8% of the nominal concentration, indicating correctly prepared test item vehicle mixtures.

Homogeneity
The measured concentrations of the test item in the test item vehicle mixtures were between 100.4% and 104.9%. These values indicated that there was no phase separation between the test item and the vehicle during the procedure of administration of the test item vehicle mixtures to the animals.

Details on mating procedure:

- M/F ratio per cage: Mating was monogamous: 1 male and 1 female animal were placed in one cage during the dark period.
- Length of cohabitation: The female was placed with the same male until pregnancy occurs or 2 weeks have elapsed.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After approx. 2 weeks of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: yes, the re-mating procedure is repeated until at least 8 pregnant dams are available for each group.
- After successful mating each pregnant female was caged (how): singly in MAKROLON cages (type III plus) with a basal surface of approximately 39 cm x 23 cm and a height of approximately 18 cm.

Duration of treatment / exposure:

Males: beginning 2 weeks prior to mating lasting up to the day before sacrifice until a minimum dosing period of 28 days was completed.
Females: 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

Duration of test:

5 adaptation days
54 test days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels have been selected in agreement with the Sponsor based on the results of a 14-day dose-range-finding study in rats dosed at 100, 300 and 1000 mg (active ingredient)/kg b. by oral gavage (LPT Study No. 29626).
- Rationale for animal assignment (if not random):
The animals were randomly allocated to the test groups.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the test period, each animal was observed for clinical signs at least once daily. Mortality was recorded twice daily. In addition, animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals were checked regularly from 7:00 a.m. to 11 :00 a.m. with a final check performed at approximately 3:30 p.m.
- Individual animals were observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness. Cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Additionally, once before the first exposure (to allow for within-subject comparisons) and once a week thereafter, detailed clinical observations were made in all animals; in test week 4 these observations were performed prior to any laboratory investigations. These observations were made outside the home cage in a standard arena and at the same time, each time. Signs observed included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes. Body weights were recorded individually for each adult animal.
- 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.

FOOD CONSUMPTION: Yes
Food consumption of 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). From these data the relative food consumption (in g/kg bw/day) was determined using the following formula:
Relative food consumption (g/kg bw/day) = (Total food given (g) - Total food left (g)) / Number of animal days# x Body weight (kg)

# The term 'animal days' counts one animal day for each animal alive for a whole
day; it is assumed that on the day of death an animal does not eat.

WATER CONSUMPTION:
- Time schedule for examinations: Water consumption was monitored daily by visual appraisal throughout the study.

HAEMATOLOGY: see Section 7.5.1

CLINICAL CHEMISTRY: See Section 7.5.1

NEUROLOGICAL OBSERVATIONS: see Section 7.5.1

REPRODUCTIVE PARAMETERS:
Number of pregnant females
Pre-coital time
Gestation length calculated from day 0 of pregnancy
Corpora lutea
lmplantation sites

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: Dams with offspring were sacrificed on day 4 post-partum, or shortly thereafter. Females showing no evidence of copulation were sacrificed 24 days after the last day of the mating period.
- Organs examined:
At the time of sacrifice or death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes . Apparently non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI.
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter. Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter, were carefully examined externally for gross abnormalities.
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: No
- Head examinations: No

Statistics:

Toxicology and Pathology data were captured, whenever possible, using the departmental computerized systems (Provantis® Integrated preclinical software, Instem LSS Ltd.). Raw data not fully compatible with the computerized systems were maintained on paper according to appropriate SOPs.
The test item-treated groups (2 - 4) were compared with the control group (1).
The following statistical methods were used:
-STUDENT's t-test: All numerical functional tests (p ≤ 0.05 or p ≤ 0.01)
-Multiple t-test based on DUNNETT, C. W., New tables for multiple Comparisons with a control, Biometrics, 482-491 (Sept 1964): Body weight / Food consumption / Haematology / Clinical chemistry / Absolute and relative organ weights(p ≤ 0.05 or p ≤ 0.01)
- Exact test of R. A. FISHER: Histopathology, if applicable (p ≤ 0.05)

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 is p ≤ 0.01.

For the comparison of classification measurements (for example the fertility index) the FISHER's exact test, n < 100 or chi-square test with Yates' correction for continuity, n ≥ 100(p ≤ 0.05 and p ≤ 0.01) were employed.
These statistical procedures were used for all data. Significantly different data were indicated in the tables of the report.
The mean values and standard deviations were calculated to the highest possible degree of accuracy and then rounded to the reported number of decimal places. Hence, deviations to the last decimal place of up to ±1 may occur caused by rounding.

Indices:

Reproductive indices:
Gestation Index
Fertility Index
Pre-implantation loss [%]
Post-implantation loss [%]

Offspring viability indices:
Birth Index
Live Birth Index
Viability Index

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

sporadic and transient salivation and breathing sounds during the pre-mating period for (2 male and) 2 female animals of the intermediate and the high dose group, not considered to be adverse.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

In the intermediate dose group one stillbirth each was noted from dam no. 63 and from dam no. 72. This is regarded to be spontaneous.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No test item-related differences were noted between the gestation length of the females of the control group and those of the treatment groups (100, 300 and 1000 mg test item/kg bw/day).
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): No test item-related differences were noted between the gestation length of the females of the control group and those of the treatment groups (100, 300 and 1000 mg test item/kg bw/day).

Details on maternal toxic effects:

Maternal toxic effects:no.
Remark: basophilic tubules (4 of 5 rats) and interstitial nephritis (3 of 5 rats) in the kidney of the male rats of the high dose group

Details on maternal toxic effects:
One of 10 male animals with moderate salivation on test days 8 and 9 was noted in the intermediate dose group (300 mg test item/kg/bw/day). In the high dose male group (1000 mg test item/kg/bw/day) one of 10 animals with slight salivation was noted on test day 14.
Signs like moderate salivation and/or breathing sounds were noted for two of 10 female animals of the high dose group (1000 mg test item/kg/bw/day) during the pre-mating period on one day each, however they were not considered to be adverse.
During microscopic examination, test item-related changes like basophilic tubules and interstitial nephritis were noted in the kidney of the male animals of the high dose group (1000 mg test item/kg bw/day). For the female rats no statistically significant changes were noted in the kidney. Basophilic tubules were not observed in the female rats and only 2 of 5 female rats showed a marginal interstitial nephritis.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: highest dose tested

Results (fetuses)

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Parameters on the body weight of pups with statistically significant differences in comparison to the control group which were considered as to be not test itemrelated.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Parameters on the body weight of pups with statistically significant differences in comparison to the control group which were considered as to be not test itemrelated.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No statistically significant differences were noted in the mean number of live born pups per dam between the control group and treatment groups (100, 300 and 1000 mg test item/kg bw/day).

Changes in sex ratio:

not examined

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

No differences were noted on the mean litter weight of the pups per dam between the control group and any of the treatment groups (100, 300 and 1000 mg test item/kg bw/day).
No test item-related differences were noted on the total litter weight per dam between the control group and any of the treatment groups (100, 300 and 1000 mg test item/kg bw/day).
The statistically significant increase in total litter weight per dam by 15.3% on lactation day 0/1 noted in the high dose group was not an adverse effect. Therefore, it was considered as not test item related.

External malformations:

no effects observed

Description (incidence and severity):

The external examinations of the pups at sacrifice on day 4 of lactation revealed no external visible changes in the control group and in any of the treatment groups (100 and 300 mg 1000 mg test item/kg bw/day).

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: no effects

Details on embryotoxic / teratogenic effects:
There were no effects on the number of live fetuses, weight of the pups, macroscopic observations and offspring viability indices.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Table 1. Fertility and Reproductive parameters Parental generation

Parameter	Group 1 Control	Group 2 60 mg/kg	Group 3 120 mg/kg	Group 4 300 mg/kg
No. females evaluated	12	12	12	12
Mean precoital time (days)	2.8	3.8	3.7	2.3
Number of pregnant dams	12	12	11	12
Fertility index (%)	100	100	92	100
Gestation length (days)	22.3	22.0	22.4	22.6
No. dams with live pups	12	11	11	12
Gestation Index (%)	100	92	100	100
No.Corpora lutea (total)	179	187	178	184
No.Corpora lutea (mean)	14.9	15.6	16.2	15.3
No. Implantation sites (total)	170	178	170	179
No. Implantation sites (mean)	14.2	14.8	15.5	14.9
Number of pups at birth (total)	162	166	156	175
Number of pups at birth (mean)	13.5	13.8	14.2	14.6
Birth Index (mean %)	95.2	86.2	92.0	97.8
Birth Index (total %)	95.3	93.3	91.8	97.8
Number of stillbirths	0	0	2	0
No. of dams with stillborn pups	0	0	2	0
Number of live born pups (total)	162	166	154	175
Number of live born pups (mean)	13.5	13.8	14.0	14.6
Live birth index (mean %)	100.0	100.0	98.7	100.0
Live birth index (total %)^#1	100.0	100.0	98.7	100.0
Pre-implantation loss (mean %)	5.6	8.0	3.9	2.5
Pre-implantation loss (total %)^#2	5.0	4.8	4.5	2.7
Post-implantation loss (mean %)	4.8	13.8	9.2	2.2
Post-implantation loss (total %)^#3	4.7	6.7	9.4	2.2

^#1: based on the total number of live born pups and the total number of pups at birth (alive and dead)

^#2: based on the total number of corpora lutea and the total number of implantation sites

^#3: based on the total number of implantation sites and the total number of live born pups

Applicant's summary and conclusion

Conclusions:

No effects on the development of the F1offspring (pups)
NOAEL (no-observed-adverse-effect level): above 1000 mg/kg bw/day, p.o.

Executive summary:

The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development according to OECD guideline 422. The test item was administered orally to rats at dose levels of 100, 300 or 1000 mg test item/kg bw/day. The application started two weeks before mating on test day one and ended on the day or one day before sacrifice. Day of sacrifice was on test days 31/32 for the male rats and on lactation day 4 or shortly thereafter for the female rats.

Systemic toxicity:

No premature deaths were noted in any treatment group.

Sporadic signs of behavioural toxicity in term of salivation and breathing sounds were noted during the pre-mating period for 2 male and 2 female animals of the intermediate and the high dose group (300 or 1000 mg test item/kg bw/day). These changes were not considered to be adverse. The macroscopic examination revealed no test item related changes, During microscopic examination, test item-related changes like basophilic tubules and interstitial nephritis were noted in the kidney of the male animals of the high dose group (1000 mg test item/kg bw/day).

NOAEL (no-observed-adverse-effect level) for repeated dose toxicity was 300 mg/kg bw/day in males and 1000 mg/kg bw:day in females.

Effects on reproductive toxicity:

No test item-related influence was noted on the reproduction parameters in any treatment group (100, 300 and 1000 mg/kg bw/day). Microscopic examination revealed no changes in the reproductive organs from the male and female rats of the high dose group (1000 mg/kg bw/day).

NOAEL (no-observed-adverse-effect level): >1000 mg/kg bw/day, p.o.

Effects on prenatal/postnatal development:

No test item related influence was noted on the survival rate and the mean and total body weights of the pups.

External examination of the pups revealed no visible changes related to the test item. There were no effects on offspring viability indices.

NOAEL (no-observed-adverse-effect level): >1000 mg/kg bw/day, p.o