Fatty acids, C18-unsatd., phosphates

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 23, 2017 - November 19, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

9-(dimethoxyphosphoryl)octadecanoic acid 10-(dimethoxyphosphoryl)octadecanoic acid

Molecular formula:

C20H41O5P

IUPAC Name:

9-(dimethoxyphosphoryl)octadecanoic acid 10-(dimethoxyphosphoryl)octadecanoic acid

Constituent 2

Reference substance name:

9,12-bis(dimethoxyphosphoryl)octadecanoic acid 9,13-bis(dimethoxyphosphoryl)octadecanoic acid 10,12-bis(dimethoxyphosphoryl)octadecanoic acid 10,13-bis(dimethoxyphosphoryl)octadecanoic acid

Molecular formula:

C22H46O8P2

IUPAC Name:

9,12-bis(dimethoxyphosphoryl)octadecanoic acid 9,13-bis(dimethoxyphosphoryl)octadecanoic acid 10,12-bis(dimethoxyphosphoryl)octadecanoic acid 10,13-bis(dimethoxyphosphoryl)octadecanoic acid

Constituent 3

Reference substance name:

Oleic acid

EC Number:

204-007-1

EC Name:

Oleic acid

Cas Number:

112-80-1

Molecular formula:

C18H34O2

IUPAC Name:

octadec-9-enoic acid

Constituent 4

Reference substance name:

Linoleic acid

EC Number:

200-470-9

EC Name:

Linoleic acid

Cas Number:

60-33-3

Molecular formula:

C18H32O2

IUPAC Name:

octadeca-9,12-dienoic acid

Constituent 5

Reference substance name:

Dimethyl (2-oxopropyl)phosphonate

EC Number:

224-110-5

EC Name:

Dimethyl (2-oxopropyl)phosphonate

Cas Number:

4202-14-6

Molecular formula:

C5H11O4P

IUPAC Name:

dimethyl (2-oxopropyl)phosphonate

Constituent 6

Reference substance name:

Dimethyl propylphosphonate

EC Number:

242-555-3

EC Name:

Dimethyl propylphosphonate

Cas Number:

18755-43-6

Molecular formula:

C5H13O3P

IUPAC Name:

dimethyl propylphosphonate

Constituent 7

Reference substance name:

Dimethyl methylphosphonate

EC Number:

212-052-3

EC Name:

Dimethyl methylphosphonate

Cas Number:

756-79-6

Molecular formula:

C3H9O3P

IUPAC Name:

dimethyl methylphosphonate

Test material form:

liquid

Specific details on test material used for the study:

Identification: Fatty acids, C18-unsatd., phosphates.
Appearance: Yellow liquid
Batch: 0101891886
Purity/Composition: 100% UVCB
Test item storage: At room temperature
Stable under storage conditions until: 02 November 2017 (retest date)
Test Facility test item number: 208073/A
Chemical name (IUPAC, synonym or trade name: Phosphorylated fatty acid
CAS number: 68604-99-9
pH (1% in water, indicative range): 2.28 – 2.21 (determined by Charles RiverDen Bosch)
Specific gravity / density: 1.04

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: (P) males were 10-12 weeks old and females were 12-14 weeks old
- Weight at study initiation: (P) Males: 241 and 298 g; Females:180 and 222 g
- Fasting period before study: F0-animals were fasted overnight with a maximum of approximately 24 hours before blood sampling, but water was available.
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not be left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study
- Water (e.g. ad libitum): ad libitum, Municipal tap water was freely available to each animal via water bottles.
- Acclimation period: a minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3°C
- Humidity (%): 55 ± 15%.
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 23-May-2017 (1st dosing) To: 12-Jul-2017 (last lactation day 4 necropsy)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

Test item dosing formulations for the dose range finder and main study were prepared and kept at room temperature under normal laboratory light conditions until dosing. Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily and dosed within 5 hours after adding the vehicle to the test item. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.

Dosing formulations were prepared at the test item concentrations indicated in the following table:
Group No. Test Item Id. Dose Level
(mg/kg/day) Dose Volume (mL/kg) Dose Concentration (mg/mL) Number of Animals Animal Numbers
Males Females Males Females
1 - 0 (Vehicle) 5 0 10 10 01-10 41-50
2 Fatty acids, C18-unsatd., phosphates. 100 5 20 10 10 11-20 51-60
3 Fatty acids, C18-unsatd., phosphates. 300 5 60 10 10 21-30 61-70
4 Fatty acids, C18-unsatd., phosphates. 1000 5 200 10 10 31-40 71-80


PREPARATION OF DOSING SOLUTIONS: The dosing formulations were prepared daily and dosed within 5 hours after adding the vehicle to the test item.


VEHICLE

- Amount of vehicle (if gavage): 5ml/kg
- Lot/batch no. (if required): S7263585

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- If no evidence of copulation is obtained after 14 days, the animals will be separated without further opportunity for mating
- After successful mating each pregnant female was caged (how): housed individually in a plastic cage containing ground corncob bedding material (Bed O'Cobs®) and remained in these cages until euthanasia on lactation day 4.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed by using a validated analytical procedure (Analytical work instruction AWI 4224, entitled: Fatty acids, C18-unsatd., phosphates in formulations using LC-MS/MS, validated in ABL validation study no. ABL17062).

Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.

Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was  10%.

Duplicate sets of each sample (approximately 500 mg) were sent to the analytical laboratory. Stability results were considered acceptable if the sample analysis results were within or equal to ±10% of the concentration determined by the initial analysis of each formulation.

Duration of treatment / exposure:

Males: At least 14 days prior to mating and continuing throughout mating for a minimum of 28 days. Total of 29 doses.
Females: For 14 days prior to mating, throughout mating and continuing until one day prior to termination (lactation day 4 for females that deliver, post-mating day 25 or post-cohabitation day 25 for females that do not deliver). Total of 39-45 doses.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Remarks:

actual ingested

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day

Remarks:

actual ingested

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

actual ingested

Doses / concentrations 5

Remarks:

Doses / Concentrations:

Basis:
other: Corresponding to 0, 2.1, 4.2, 8.3 mg available Cl2/kg bw/day

No. of animals per sex per dose:

10 males and 10 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 10-day dose range finder with oral administration of Fatty acids, C18-unsatd., phosphates in rats, and in an attempt to produce graded responses to the test item.
Experimental design of DRF
Group No. Test Item Id. Dose Level
(mg/kg/day)a Dose Volume (mL/kg) Dose Concentration (mg/mL) Number of Females Animal Numbers
1 Fatty acids, C18-unsatd., phosphates 500 5 100 3 1-3
2 Fatty acids, C18-unsatd., phosphates 1000 5 200 3 4-6

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 10 days.
The dose levels were selected based on information provided by the Sponsor. No toxicity data was available for this substance but for a similar substance the acute oral LD50 was > 5000 mg/kg.

No signs of systemic toxicity were noted at any dose level.
Parameter 500 mg/kg 1000 mg/kg
Mortality No mortality. No mortality.
Clinical appearance Salivation at several occasions. Salivation at several occasions. Pale faeces observed by the end of treatment.
Body weight Normal. Normal.
Food consumption Normal. Normal.
Macroscopic examination No abnormalities noted. No treatment-related abnormalities noted.
Organ weights Liver and kidney weights considered to be normal. Liver and kidney weights considered to be normal.

Based on the results of the dose range finder, selected dose levels for the main study were 100, 300 and 1000 mg/kg.

Main study:
The objectives of this study were to determine the potential toxic effects of Fatty acids, C18-unsatd., phosphates when given orally by gavage for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development.
In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.

Group No. Test Item
Identification Dose Level
(mg/kg/day) Dose Volume (mL/kg) Dose Concentration (mg/mL) Number of Animals
Males Females
1 - 0 (Vehicle) 5 0 10 10
2 Fatty acids, C18-unsatd., phosphates 100 5 20 10 10
3 Fatty acids, C18-unsatd., phosphates 300 5 60 10 10
4 Fatty acids, C18-unsatd., phosphates 1000 5 200 10 10

Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability over 5 hours.
The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, functional observations (for 5 selected animals/sex/group),body weight and food consumption, estrous cycle determination, clinical pathology (for 5 selected animals/sex/group), measurement of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examinations.
In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 13-15 pups)).

Positive control:

Not needed

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

OTHER: Arena Observations, Water Consumption, Functional tests, Estrous cycle determination, general reproduction data were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day.

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.

Sperm parameters (parental animals):

Not investigated. Only testis weights were recorded.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, other: hematology parameters and clinical chemistry parameters were analysed]

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; No animals died during the course of the study.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE
Animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
- Male animals: All surviving animals; Following completion of the mating period (a minimum of 28 days of administration).

- Maternal animals: All surviving animals on PND 14-16.

GROSS NECROPSY
- Gross necropsy consisted of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera.

The organs identified in the table below were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated.

Organs Weighed at Necropsy for all selected animals:
Brain
Cervix
Epididymisa
Gland, adrenala
Gland, coagulationa, b
Gland, parathyroidc
Gland, prostate
Gland, seminal vesicle a
Gland, thyroid
Heart
Kidney a
Liver
Ovariesa
Spleen
Testesa
Thymus
Uterus
Epididymisa
Gland, coagulationa, b
Gland, parathyroidc
Gland, prostate
Gland, seminal vesiclea
Gland, thyroid
Testes a

a Paired organ weight.
b Weighed together with the seminal vesicles.
c Weighed together with the thyroid.


HISTOPATHOLOGY / ORGAN WEIGHTS
Animal identification
Artery, aorta
Body cavity, nasopharynx
Bone marrow
Bone, femur
Bone, sternum
Brain (seven levels)
Cervix
Epididymisa
Esophagus
Eyea
Gland, adrenal
Gland, coagulation
Gland, harderiana, b
Gland, lacrimal
Gland, mammary
Gland, parathyroidc
Gland, pituitary
Gland, prostate
Gland, salivary
Gland, seminal vesicle
Gland, thyroid
Gross lesions/masses
Gut-associated lymphoid tissue
Heart
Kidney
Large intestine, cecum
Large intestine, colon
Large intestine, rectum
Larynx
Liver
Lung
Lymph node (mandibular and mesenteric site)
Muscle, skeletal
Nerve, optica, b
Nerve, sciatic
Ovaries
Pancreas
Skin
Small intestine, duodenum
Small intestine, ileum
Small intestine, jejunum
Spinal cord
Spleen
Stomach
Testesa
Thymus
Tongue
Trachea
Urinary bladder
Uterus
Vagina

The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin:
Selected animals: Tissues identified in Text Table 10 (except animal identification, aorta, nasopharynx, esophagus, harderian gland, lacrimal gland, salivary gland, larynx, optic nerve, pancreas, skin and tongue).
Remaining animals: Gross lesions/masses.

Postmortem examinations (offspring):

SACRIFICE
Pups, younger than 7 days were euthanized by decapitation.
All remaining pups (PND PND13-15), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%).
The pups selected for blood collection on PND 13-15 were anesthetized using isoflurane followed by exsanguination.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 2 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

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

Non-Parametric
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
The motor activity data set was compared using an overall Kruskal-Wallis.

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 Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

Mating (%): Number of females mated x 100
Number of females paired

Precoital time: Number of days between initiation of cohabitation and confirmation of mating

Fertility index (%): Number of pregnant females x 100
Number of females mated

Gestation index (%): Number of females with living pups on Day 1 x 100
Number of pregnant females

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Post-implantation survival index (%): Total number of offspring born x 100
Total number of uterine implantation sites

Lactation index (%): Number of live offspring on Day 13 after littering x 100

Offspring viability indices:

Percentage live males at First Litter Check (%): Number of live male pups at First Litter Check x 100
Number of live pups at First Litter Check

Percentage live females at First Litter Check (%): Number of live female pups at First Litter Check x 100
Number of live pups at First Litter Check

Viability index (%): Number of live offspring on Day 4 before culling x 100
Number live offspring on Day 1 after littering

Lactation index (%): Number of live offspring on Day 13 after littering x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Salivation seen after dosing among animals of all dose groups, including controls, during the treatment period was considered to be a physiological response related to taste of the test item and/or vehicle rather than a sign of (systemic) toxicity.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The mean total protein levels in males and females at 1000 mg/kg were slightly lower, reaching a level of statistical significance in males, when compared to controls. Due to the minimal magnitude of the change (less than 5% lower than in controls), in the absence of correlating morphologic findings and because the mean values were within normal limits for rats of this strain and age , this finding was considered not to be toxicologically relevant.

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

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment with Fatty acids, C18-unsatd., phosphates were noted in the thyroid gland of the 1000 mg/kg/day group males.
Thyroid glands, an increased incidence and severity of follicular cell hypertrophy and colloid alteration was present in males treated at 1000 mg/kg/day up to slight degree. This was considered to be non-adverse at the incidences and severities recorded.
The findings in the male kidneys, in the form of hyaline droplet accumulation, was recorded in all dose groups including controls. In the absence of a dose response this finding was considered incidental and not related to treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

Parental Data

Mortality
No mortality occurred during the study period.

Clinical Observations
No test item-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.
Salivation seen after dosing among animals of all dose groups, including controls, during the treatment period was considered to be a physiological response related to taste of the test item and/or vehicle rather than a sign of (systemic) toxicity.
Rales were incidentally noted for individual animals treated at 100, 300 or 1000 mg/kg. At the incidence observed and in the absence of a dose-relationship, this was not considered to be a sign of toxicological relevance.
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats 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.
Note to clinical signs tables: For males, “Repro period” represents the mating phase. For females, “Repro period” represents the mating, post coitum and lactation phase.

Body Weights and Body Weight Gains
Body weights and body weight gain were considered to have been unaffected by treatment.
The statistically significantly higher body weight gain in females at 1000 mg/kg on Day 1 of the mating period was considered to have arisen by chance and to be unrelated to treatment since no trend was apparent regarding duration of treatment.

Food Consumption
Food consumption before or after correction for body weight were considered to have been unaffected by treatment.
In females at 1000 mg/kg, mean food consumption and relative food consumption was slightly low during the lactation period, when compared to controls. This was considered to have arisen by slightly high control values and was considered not to represent a change of toxicological significance.

Functional Tests
Functional observation parameters were not considered to be affected by treatment.
Average hind grip strength in males at 1000 mg/kg was slightly lower when compared to controls, but individual values remained within the normal range to be expected for rats of this age and strain and this minimal difference was therefore considered to be unrelated to treatment.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.
The average number of total movements was slightly higher for males at 1000 mg/kg (mainly caused by slightly increased activity at intervals 6 and 7), but slightly lower for females at 1000 mg/kg (mainly caused by a slightly lower activity at interval 10) when compared to controls. These minimal differences were not considered to be related to treatment, as all individual values remained within the control range and/or the normal range to be expected for rats of this age and strain.

Haematology
Haematological parameters of treated rats were considered not to have been affected by treatment.

Coagulation
Coagulation parameters of treated rats were considered not to have been affected by treatment.

Clinical Chemistry
The mean total protein levels in males and females at 1000 mg/kg were slightly lower, reaching a level of statistical significance in males, when compared to controls. Due to the minimal magnitude of the change (less than 5% lower than in controls), in the absence of correlating morphologic findings and because the mean values were within normal limits for rats of this strain and age , this finding was considered not to be toxicologically relevant.
The other clinical biochemistry parameters in treated animals were considered not to have been affected by treatment.
Any other statistically significant changes in clinical chemistry parameters were considered to be unrelated to administration of the test item due to the minimal magnitude of the change, variation in direction of change, absence of a dose response, and/or absence of biological relevance.
Thyroid hormone analyses:
Serum levels of T4 in F0-males were considered not to be affected by treatment.

Gross Pathology
There were no test item-related gross observations.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Organ Weights
Test item-related higher liver weights (relative to body weights) were noted in the 1000 mg/kg/day group males as shown in the table below.

Mean Percent Liver Weight Differences from Control Groups
Males
Dose level (mg/kg/day): 100 300 1000

LIVER
Absolute -2 2 8
Relative to body weight 1 5 11*
*: P<0.05,
This minimal increase in relative liver weight, in the absence of any test item-related finding in the liver, was considered non-adverse.
Slightly higher heart weights were observed in the 300 mg/kg/day group females, achieving statistical significance for the heart weight relative to body weight. In the absence of a dose response relationship no toxicological significance was attached to this finding.
There were no other test item-related organ weight changes.

Histopathology
Test item-related microscopic findings after treatment with Fatty acids, C18-unsatd., phosphates were noted in the thyroid gland of the 1000 mg/kg/day group males and are summarized in the table below.
Summary Test Item-Related Microscopic Findings – Scheduled Euthanasia Animals
Males
Dose level (mg/kg/day): 0 100 300 1000

THYROID GLANDS a 5 5 5 5
Follicular Cell Hypertrophy
Minimal 1 1 2 3
Slight - - - 1
Colloid alteration
Minimal 1 - 1 2
Slight - - - 1
a = Number of tissues examined from each group.
Thyroid glands, an increased incidence and severity of follicular cell hypertrophy and colloid alteration was present in males treated at 1000 mg/kg/day up to slight degree. This was considered to be non-adverse at the incidences and severities recorded.
The findings in the male kidneys, in the form of hyaline droplet accumulation, was recorded in all dose groups including controls. In the absence of a dose response this finding was considered incidental and not related to treatment.
In a single male the forestomach showed signs of mild irritation which was considered to be due to the gavage procedure and therefore was not considered to be test item-related.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Reproductive performance
There were no couples without offspring.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

Reproduction Data
Estrous Cycle
Length and regularity of the estrous cycle were not considered to have been affected by treatment.
Most females had regular cycles of 4 to 5 days. An irregular cycle was noted for control female no. 42 and female nos. 53 and 56 at 100 mg/kg (all with normal litters). Given their incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.

Mating index
Mating index was not considered to be affected by treatment. All females showed evidence of mating.

Precoital time
Precoital time was not considered to be affected by treatment. All females showed evidence of mating within 5 days.

Number of implantation sites
Number of implantation sites was not considered to be affected by treatment.

Fertility index
Fertility index was not considered to be affected by treatment. All mated females were pregnant.

Developmental Data
Gestation index and duration
Gestation index and duration of gestation were not considered to be affected by treatment.

Parturition/maternal care
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Post-implantation survival index
The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment.
For female no.60 (at 100 mg/kg) and female nos.61 and 68 (at 300 mg/kg), the number of pups were higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during lactation. No toxicological relevance was attached to this finding in the current study.

Litter size
Litter size was not considered affected by treatment.

Live birth index
The number of live offspring on Day 1 after littering compared to the total number of offspring born was not considered to be affected by treatment.
In several litters a dead pup was observed at first litter check, i.e. one pup in litter 52 (at 100 mg/kg), in litter 69 (at 300 mg/kg) and in litters 75 and 80 (1000 mg/kg). Since the mortality incidence remained within the range considered normal for litters from rats of this strain and age, no toxicological relevance was attributed to these dead pups at birth.

Viability index
The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was not considered affected by treatment.
In several litters one or more pups went missing or died between birth and PND 4, i.e. in litters 53, 55 and 60 (at 100 mg/kg) a total of five and in litters 72 and 78 (at 1000 mg/kg) a total of eight pups. Pups missing were most likely cannibalised by the mother. This is a normal phenomenon in litters of rats of this strain and age and, in absence of a dose-related trend, considered not affected by treatment in this study.

Lactation index
The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not considered to be affected by treatment.
In one litter (no.75, at 1000 mg/kg), one pup was missing on PND 5. This missing pup was also most likely cannibalised by the mother (see also 9.4.6. Viability index above). Based on the single incidence, no toxicological relevance was attributed to this finding.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

(Systemic)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at the hifhes dose level tested

2

Key result

Dose descriptor:

NOAEL

Remarks:

(Reproduction)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at the highest dose level tested

3

Key result

Dose descriptor:

NOAEL

Remarks:

Development

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at the highest level tested

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Clinical signs
The nature and incidence of the clinical signs observed remained within the range considered normal for pups of this age, and were therefore considered not to affected by treatment and therefore of no toxicological relevance.
Note: Only days on which clinical signs were present between first and last litter check are presented in the table.

Body weights
Body weights of pups were not considered to be affected by treatment.

Sex ratio
Sex ratio was not considered to be affected by treatment.

Anogenital distance
Anogenital distance (absolute and normalized for body weight) in male and female pups was not considered to be affected by treatment.

Areola/nipple retention
Treatment up to 1000 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Clinical biochemistry (T4 levels)
Serum T4 levels in male and female PND 13-15 pups were not considered to be affected by treatment.

Macroscopy
No macroscopic findings were noted among pups that were considered to be related to treatment.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

(Development)

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at the highest dose tested

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following no-observed-adverse-effect level (NOAEL) of Fatty acids, C18-unsatd., phosphates were established:
Parental NOAEL: at least 1000 mg/kg
Reproduction NOAEL: at least 1000 mg/kg
Developmental NOAEL: at least 1000 mg/kg

Executive summary:

Wistar Han rats were treated with Fatty acids, C18-unsatd., phosphates by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg. The rats of the control group received the vehicle, Polyethylene glycol 400, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, duringpost-coitum, and at least 13-15 days of lactation (for 50-56 days).

Test formulations preparedwere considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels. Test formulations preparedwere consideredstable, for at least 5 hours at room temperature.

Parental results

No mortality occurred among the F0-males and females and no treatment related clinical signs were observed.

At 1000 mg/kg, treatment-related effects were limited to a minimal increase in relative liver weight (without microscopic correlate) and slightly increasedincidence and severity offollicular cell hypertrophy and colloid alteration in the thyroid glands of males. Both findings were considered to be non-adverse.

No treatment-related changes were noted in any of the remaining parameters investigated in this study (i.e.functional observations, body weight, food consumption, clinical laboratory investigations and macroscopic examination).

Reproductive results

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg).

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Developmental results

No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg).

No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

The study was considered reliable without restriction since the study was conducted according to the current guideline (OECD422) and in compliance with GLP.