Fatty acids, C14-18 and C16-18-unsatd., maleated, reaction products with oleylamine

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented and reported study fully adequate for assessment. The study was conducted according to an internationally accepted technical guideline and in compliance with GLP in a recognized contract research organization.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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)

Version / remarks:

of 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

liquid: viscous

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Sprague Dawley rats, strain: Crl:CD(SD) with appropriate range of bodyweight at study start.
- Source: Charles River (UK) Ltd.
- Age at treatment start: 72 days.
- Weight at treatment start: Males: minimum 337 g, maximum 417 g,
Females: minimum 227 g, maximum 293 g.
- Housing Inside a barriered rodent facility:
all animals pre-pairing + toxicity subgroups: In groups up to 5 by sex in solid floor polycarbonate cages.
during pairing (1 male+1 female/cage): In RB3 modified polycarbonate cages with stainless steel grid-floor over absorbent paper-lined trays.
males after pairing: In groups up to 5 in solid floor polycarbonate cages.
females during gestation and lactation: Females housed individually (+litter) in solid floor polycarbonate cages.
- Bedding material (in solid floor cages): Wood based bedding, sterilised by autoclaving before use.
- Cage enrichment: Aspen chew block + plastic shelter (except during pairing or post Gestation Day 20).
- Diet (ad libitum): Standard rodent diet (SDS VRF1 Certified) without antibiotic, chemotherapeutic or prophylactic agent.
- Fasting (diet withheld): Main phase males and Toxicity phase females overnight before blood sampling for clinical pathology.
- Water (ad libitum): Potable drinking water from the public supply.
- Acclimation period: 7 days before treatment start, under laboratory conditions.

Routine analysis of the batch of diet used and water, chew blocks and bedding material did not provide evidence of contamination that might have prejudiced the study.

IN-LIFE DATES:
- Duration of test, males & toxicity phase females: Five weeks
Duration of test, main phase females (i.e. reproductive subgroup): From 14 days prior to pairing to day 7 of lactation.
Duration of test, offspring: From birth to day 7 of lactation.

ENVIRONMENTAL CONDITIONS

Air conditioned room kept at positve pressure without re-circulation of the filtered fresh air supplied to the room.
Controlled environment, environmental conditions were set at:
- Temperature (°C): 21 ± 2°C
- Relative Humidity (%): 40 to 70%
- Photoperiod (artificial lighting): 12 hrs day / 12 hrs night
- Rate of air exchange: At least 15 changes/h
Deviations from the target ranges for temperature and relative humidity were not evident.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Treatment of parental animals by oral gavage administration. Test substance was not directly administered to F1 animals.

- Concentration in vehicle: The concentration of the test material in vehicle varied between dose groups thus allowing constant dosage volume in terms of mL/kg bw/day.
- Amount (dose volume by gavage): 5 mL/kg bw/day.
Actual dose volumes were calculated at about weekly or shorter intervals accounting for the latest bodyweight.

- For concentrations of test material in vehicle at different dose levels, see Table 1 in "Any other information on materials and methods incl. tables"

- Justification for choice of vehicle:
The suitability of corn oil as a vehicle was established during the 14-day range-finding study. In addition, in the present main study, concentrations of dose formulations were chemically analysed.

Details on mating procedure:

- Male/female ratio per cage: 1/1
- Length of cohabitation: At the most 8 days (1 high dose female), until proof of successful mating was confirmed.
(1 control female had an acyclic oestrous and failed to mate)  
- Proof of successful mating: Formation of at least one copulation plug and a sperm positive vaginal smear.
The day this was found was referred to as day 0 of gestation.
(During cohabitation, females were checked every morning for pregnancy).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Chemical analysis of test material formulations by high performance liquid chromatography coupled with a mass spectrometer (HPLC-MS/MS).
- Mean concentrations of the test material formulations (verified for first and 7th treatment week) were confirmed at each dose level.
Chemical analysis confirmed that the mean concentrations of WS400107 in prepared formulations were within 93% to 100% of the corresponding
nominal concentration, thus confirming acceptable accuracy of formulation for dosing of the animals.
- Homogeneity and stability of test material formulations at 2 and 200 mg/L and at storage and handling conditions similar to those adopted for dosing of the animals were confirmed.

Duration of treatment / exposure:

- Treatment period, males & toxicity phase females: Daily, for five consecutive weeks, in males commencing 14 days prior to pairing
- Treatment period, main phase females (i.e. reproductive subgroup): 44 to 51 days (from 14 days prior to pairing to Day 6 of lactation)
- Offspring were not dosed

Frequency of treatment:

Daily, 7 days/week (during parturition, dosing omitted as appropriate)

Details on study schedule:

- Age at mating of the mated animals in the study: 12 to 14 weeks

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (vehicle control), 150, 400, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Toxicity phase animals: */ 5 females
Main phase animals (i.e. reproductive subgroups): 10 males / 10 females
*Explanatory note by the notifier:
Examinations assigned to the toxicity phase females to meet the requirements of a 28-day repeat dose oral toxicity study were also assigned to 5 (for some examinations to 10) main phase males per dose group. Therefore, these 5 main phase males per dose group are called also "toxicity subgroup" in the present robust study summary for clarification. After pairing with main phase females, all males were killed at the same time (Week 6).

Control animals:

yes, concurrent vehicle

Details on study design:

This study was conducted to examine both repeated dose toxicity and  reproductive/developmental toxicity as an OECD screening combined study
(OECD 422 test guideline).  Therefore, animals initially entering the study were divided into toxicity subgroup animals (toxicity phase) and reproductive subgroup animals (main phase), whereby 5 of the 10 F0 males (used for pairing) per dose group formed the toxicity male subgroups.

Dose selection was based on the results of a 14-day preliminary oral (gavage) toxicity study in the rat in which dose levels of 100, 300 or 1000 mg/kg/day did not have any severe toxic effects on young adult animals (females nulliparous and non-pregnant) preventing the choice of up to 1000 mg/kg/day in the present OECD 422 toxicity study.

Positive control:

Not included in the study.

Examinations

Parental animals: Observations and examinations:

Clinical observations performed and frequency:
- Clinical signs : At least twice a day
- Detailed physical examination
and arena observations: Before treatment start and at least once a treatment week (except possibly 1 female taking 5 to 8 days for pairing).
- Functional Observation Battery: During treatment week 5 (before dosing) on all toxicity subgroup animals (5 parental males/group inclusively)*.
- Body weight, all males: About weekly throughout the study.
Body weight, Toxicity Females: About weekly throughout the study.
Body weight, Repro. Females: Weekly for pre-pairing period; on gestation days 0, 6, 13, 20; on lactation days 1, 4 & 7.
- Food consumption, all males: Weekly for pre-pairing period and for the period after mating.
Food cons., Toxicity Females: About weekly throughout the study.
Food cons., Repro. Females: Weekly for pre-pairing period, during gestation for days 0-6, 6-13, 13-20, during lactation for days 1-4 & 4-7.
- Hematology: During treatment week 5 after functional observation battery*
- Blood (plasma) chemistry: During treatment week 5 after functional observation battery*

* Examinations confined to toxicity subgroup animals are marked above with an asterisk*
and are detailed in the separate endpoint study record "7.5.1 Repeated dose toxicity: oral - Repeat dose tox combined_gavage_rat_HLS_GAH0152"

Explanatory note
This study was conducted to examine both repeated dose toxicity and  reproductive/developmental toxicity as an OECD screening combined study
(OECD 422 test guideline).  Therefore, animals initially entering the study were divided into toxicity subgroup animals (toxicity phase) and reproductive subgroup animals (main phase), whereby 5 of the 10 F0 males (used for pairing) per dose group formed the toxicity male subgroups.

Oestrous cyclicity (parental animals):

Frequency of vaginal oestrus was determined by examination of vaginal smears taken daily from all main phase (i.e. reproductive subgroup) females from the beginning of the treatment period to the day of confirmed copulation.
- Regular: All observed cycles of 4 or 5 days
- Irregular: At least one cycle of 2, 3 or 6 to 10 days
- Acyclic: At least 10 days without oestrus

Sperm parameters (parental animals):

Parameters examined in male parental animals:
- testis weight,
- epididymis weight
- detailed qualitative histopathology examination of the testes taking into account the tubular stages of the spermatogenic cycle. This was to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.

Litter observations:

STANDARDISATION OF LITTERS: Not performed. The study ended on Lactation Day 7.

LITTER PARAMETERS EXAMINED
- From Day 20 post copulation 3 times a day checks for evidence of parturition, any difficulties and numbers of live and dead offspring.
- Total litter size on day 1 of age and mortality/live litter size on each day until 7 days after littering.
- Sex ratio expressed as percentage males and calculated for total offspring on Day 1 and for live offspring on Days 1, 4 & 7
(No. of male pups in litter/No. of offspring in litter) x 100
- Clinical signs, recorded daily
- Body weight of live pups (on days 1, 4 and 7 after littering) and weight change from Days 1-4, 4-7 and 1-7.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes, see below
WEIGHING OF ORGANS: Yes, see below
HISTOPATHOLOGY: Yes, see below

Terminal sacrifice
- all males (F0) and toxicity subgr. females: Killed in Week 6, after completion of the Treatment Week 5 investigations.
- reproductive subgr. females & offspring: Killed on Day 7 post partum.
(1 high dose female was killed after mating in poor clinical condition. It was confirmed to be non-pregnant)
(1 control female failed to mate and therefore was killed in Week 8 following 7 weeks of treatment)

Gross pathology:
- adult/parental animals: Full macroscopic examination.

Organs Weights:
- all males (F0) +
toxicity subgroup females: Adrenals, brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles with coagulation gland,
spleen, testes, thymus, uterus with cervix & oviducts.
- dams: Ovaries

Histopathology:
- toxicity subgroups: The following organs were microscopically observed for the control and 1000 mg/kg bw/day toxicity subgroups:
Brain, eyes, pituitary gland, thyroid with parathyroids, heart, thymus, liver, spleen, adrenals, kidneys, testes,
epididymides, ovaries, lung, trachea, oesophagus, stomach, duodenum, jejunum, ileum, caecum, rectum, colon,
Peyer's patch, lymph node (axillary, mesenteric), urinary bladder, uterus (with cervix & oviducts), vagina,
spinal cord, sciatic nerve, skeletal muscle, sternum with marrow, seminal vesicle & coagulation gland, prostate.
In addition, the duodenum, jejunum, ileum, mesenteric lymph nodes, seminal vesicles and prostate
were also examined by light microscopy for the remaining F0 males of the control and 1000 mg/kg bw/day
groups and all adult males and toxicity subgroup females of the 150 and 400 mg/kg bw/day groups and any
gross lesions for all adult animals.

- reproductive subgroups All above organs/tissues from a premature death (1 high dose female). Gross lesions from adult dams comprised only focal hairloss not necessating any examination by light microscopy.

Postmortem examinations (offspring):

Pup survivors were killed on Day 7 post partum and had a careful external macroscopic examination for gross abnormalities.

Externally abnormal offspring and premature deaths had an internal macroscopic examination including assessment of the presence of milk in the stomach, where possible. (Missing or grossly autolysed or cannibalised offspring could not be examined).

Statistics:

As detailed in Endpoint study record "7.5.1 Repeated dose toxicity: oral - Repeat dose tox combined_gavage_rat_HLS_GAH0152"

Reproductive indices:

- Pre-coital interval (pairing days until detection of mating)
- No. of animals mating (evidence of successful copulation, i.e. at least one copulation plug or a sperm positive vaginal smear)
- No. of animals achieving pregnancy
- Percentage mating (No. of animals mating/No. of animals paired) x 100
- Fertility index (No. of animals achieving pregnancy/ No. of animals paired) x 100
- Conception rate (No. of animals achieving pregnancy/No. of animals mated) x 100
- Gestation length (time elapsing between detection of mating and commencement of parturition)
- Gestation index (No. of live litters born/No. of living animals having achieved pregnancy)
- No. of living pregnant females
- For further reproductive parameters, see also the above section "Litter observations" and section "Offspring viability indices" below.

Offspring viability indices:

- Post-implantation survival index  (Total no. of pups born/Total no. of uterine implantation sites) x 100
- Live birth index (No. of live pups on Day 1 after littering/Total no. of pups born) x 100
- Viability index (No. of live pups on Day 4 after littering /No. of live pups on Day 1 after littering) x 100
- Lactation index (No. of live pups on Day 7 after littering /No. of live pups on Day 1 after littering) x 100
- For further parameters indicative of the viability of the offspring, see also the above section "Litter observations"

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

one high dose female was killed for animal welfare reasons; it had very low eight gain and was not pregnant.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

body weight gain was affected in males and females

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

body weight gain was affected in males and females

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

but not representing toxicity or toxicological significance unclear

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
There were no premature deaths, clinical signs or adverse effects on neurobehaviour attributable to treatment with the test material. One high dose female of the reproductive subgroup was killed during the gestation phase, because of its poor clinical condition (thin built, hunched posture, piloerection, ungroomed fur and 13 g bodyweight loss over 14 days, confirmed to be not pregnant at necropsy). One control female of the reproductive subgroup failed to mate and therefore was killed after 7 weeks of treatment (Week 8).

BODYWEIGHT GAIN AND FOOD CONSUMPTION
A dose related adverse effect on bodyweight gain was evident in males over the 5 weeks of treatment attaining statistical significance at 400 and 1000 mg/kg bw/day. Nulliparous females were unaffected by treatment, but in dams treated with 1000 mg/kg bw/day bodyweight gain was statistically significantly lower than in concurrent controls during gestation Days 6 to 13 and overall during gestation Days 0 to 20. Food consumption was lower than in concurrent controls in males at 1000 mg/kg/day during the 5-week treatment period and in dams at 1000 mg/kg/day on lactation Days 4 to 7.

ORGAN WEIGHTS
After 5 weeks of treatment at 1000 mg/kg/day, prostate and seminal vesicle weights were statistically significantly lower than in concurrent controls. The toxicological significance of this finding remained unclear.

GROSS PATHOLOGY AND HISTOPATHOLOGY (NON-NEOPLASTIC)
The effects on prostate and seminal vesicle weights were consistent with small prostates and small seminal vesicles macroscopically apparent and reduced colloid microscopically seen in these tissues in four of ten high dose males. The toxicological significance of this finding remained unclear. Testes, ovaries and epididymides were unaffected by treatment. Evaluation of the seminiferous testicular tubules regarding their stage in the spermatogenic cycle and regarding the integrity of the various cell types present within the different stages did not reveal any cell or stage abnormalities. Main phase females (reproductive subgroup) killed on Lactation Day 7 were not histopathologically examined.

Thickening of the jejunum, ileum and duodenum was macroscopically apparent in a number of animals from all test item treated groups and thickening of the colon in two of ten males of the high dose group (1000 mg/kg/day) after 5 weeks of treatment, and were attributed to treatment with the test material. Histopathology revealed foamy macrophages within the villi of the illeum and jejunum (at 150, 400 and 1000 mg/kg/day) and of the duodenum (at 400 and 1000 mg/kg/day). Macrophage aggregates in the mesenteric lymph nodes in all treated groups were considered to be secondary deposits of the foamy macrophages seen in the intestines. These pathology findings were considered not to represent toxicity, but to result from test material ingestion by macrophages.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

> 150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: A dose related adverse effect on bodyweight gain was evident in males over the 5 weeks of treatment attaining statistical significance at 400 and 1000 mg/kg bw/day.

2

Dose descriptor:

NOAEL

Effect level:

> 400 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Nulliparous females were unaffected by treatment, but in dams treated with 1000 mg/kg bw/day bodyweight gain was statistically significantly lower than in concurrent controls during gestation Days 6 to 13 and overall during gestation Days 0 to 20.

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):

on bodyweight or bodyweight gain to Day 7, the day of scheduled sacrifice of the pups.

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

disclosed by careful external macroscopic examination of all pups surviving to Day 7 and by necropsy of externally abnormal pups and decedents

Histopathological findings:

not examined

Details on results (F1)

There was no indication of any effect of parental treatment with WS400107 on survival, clinical condition, sex ratio, bodyweight, bodyweight gain or develpment of the offspring until Lactation Day 7. Adverse effects on number of implantations and litter size are addressed in "Details on results (parental animals)" in the present robust study summary.

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: NOEL = highest dose tested. Offspring development up to Day 7 of age.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

 

Table 2: No. of Implantations and Litter Size - Group Mean Values for Main Phase Females during Lactation
Group			1		2		3		4
Compound			Vehicle Control		WS400107		WS400107		WS400107
Dose (mg/kg/day)			0		150		400		1000
Group		Implantations		Total litter size		Live litter size
				Day		Day		Day		Day
				1≠		1		4		7
Statistical test:		Sh		Wi		Wi		Wi		Wi
1	Mean	16.8		16.1		16.0		15.3		15.2
	SD	1.4		1.7		1.7		1.6		1.6
	N	9		9		9		9		9
2	Mean	15.9		14.8		14.5		14.3		14.3
	SD	1.5		2.0		2.3		2.1		2.1
	N	10		10		10		10		10
3	Mean	16.2		15.2		15.1		14.9		14.8
	SD	1.7		1.7		1.5		1.4		1.4
	N	10		10		10		10		10
4	Mean	14.0*		12.8**		12.4**		12.1**		12.0**
	SD	4.2		3.7		3.5		3.4		3.3
	N	9		9		9		9		9
		Wi		Wi		Wi		Wi		Wi
4a	Mean	15.3		13.9*		13.5**		13.1*		13.0*
	SD	1.9		1.9		1.6		1.6		1.4
	N	8		8		8		8		8

 

Statistical test Wi = Treated groups compared to Control using Williams’ test,    * p<0.05,    ** p<0.01

Statistical test Sh = Treated groups compared to Control using Shirley’s test,     * p<0.05,    ** p<0.01

≠ Includes offspring that died prior to designated Day 1 of age

a Excludes Litter 4F, 41 which was atypically small

 

Applicant's summary and conclusion

Conclusions:

Reduced mean numbers of implantations and live litter sizes on Days 1-7 of age were observed at the highest dose level of 1000 mg/kg/day. However, this reduction of mean values could be traced back to a sub-population of females in the high dose group (4/10) which showed weight loss or retarded weight gain in the first two weeks of dosing. Thus, the observed effects are due to maternal toxicity, and not to a specific effect on reproductive parameters. Survival and growth of the offspring of all dose groups, their clinical condition and macropathology findings were comparable with control values.