Fatty acids, tall-oil, compds. with oleylamine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Remarks:

The experimental study was performed with a structural analogue; read-across from results of this study is justified (see below).

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read-across from structural analogue

Reason / purpose for cross-reference:

reference to same study

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

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: Ca. 70 days.
- Weight at treatment start: Males: minimum 341 g, maximum 390 g,
Females: minimum 212 g, maximum 277 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 polypropylene 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.

Route of administration:

oral: gavage

Vehicle:

propylene glycol

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 body weight.

- 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 propylene glycol as a vehicle was established visually and by chemical analysis during the 7-day range-finding study:
Endpoint study record "7.5.1 Repeated dose toxicity: oral - 7d_range-finding_gavage_HLS_GAH0029".
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 14 days, until proof of pregnancy was confirmed. 
- 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).
- Concentrations (verified at first, second and last treatment week) of the test material formulations were lower than expected from nominal concentrations (see Table 1).
- Mean concentrations of WS400151 in prepared formulations attained by chemical analysis were 70.5% to 87.2% of the corresponding nominal concentration. Evaluation of a number of aspects of the analytical and formulation procedures did not lead to identification of the cause for the lower concentrations achieved by chemical analysis. In order to take a conservative approach, it was concluded that the concentrations determined by chemical analysis reflected the concentrations of test substance formulation administered to the animals.

Duration of treatment / exposure:

- Treatment period, males & toxicity phase females: Daily, for five consecutive weeks, in males commencing 14 days prior to mating
- Treatment period, main phase females (i.e. reproductive subgroup): 44 to 47 days (from 14 days prior to mating 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 13 weeks

Dose / conc.:

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

Remarks:

dose levels derived from chemical determination of administered dose formulations

Dose / conc.:

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

Dose / conc.:

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

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 7-day preliminary oral toxicity study in young adult rats (females nulliparous and non-pregnant) in which a NOAEL could not be established at dose levels of 100, 300 or 1000 mg/kg/day.

Positive control:

Not included in the study.

Parental animals: Observations and examinations:

Clinical observations performed and frequency:
- Clinical signs : At least twice a day (before and after administration)
- Detailed physical examination
and arena observations: Before treatment start and at least once a treatment week.
- Functional Observation Battery: During treatment week 5 (before dosing) on all toxicity subgroup animals (5 parental males/group inclusively)*.
- Body weight, all males: Weekly throughout the study.
Body weight, Toxicity Females: Weekly throughout the study.
Body weight, Repro. Females: Weekly for pre-pairing period; on gestation days 0, 6, 13, 20; on lactation days 1 & 7.
- Food consumption, all males: Weekly for pre-pairing period and for the period after mating.
Food cons., Toxicity Females: 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_GAH0030"

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 & 7
(No. of male pups in litter/No. of offspring in litter) x 100
- Gestation index (No. of live litters born on day 0/No. of living pregnant females) 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 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 and toxicity subgr. females: Killed in Week 6, after completion of the Treatment Week 5 investigations.
(2 high dose females died from intubation error on treatment day 32)
- reproductive subgr. females & offspring: Killed on Day 7 post partum.
(1 high dose female died from intubation error on treatment day 14)
(1 low and 1 high dose females without viable litter were killed on Day 25 after evidence of mating).
Gross pathology:
- adult/parental animals: Full macroscopic examination with tissue collection.
In addition, recording of the number of uterine implantation sites in reproductive subgroup females.

Organs Weights:
- main phase and tox. subgr. adults: Adrenals, brain, epididymides, heart, kidneys, liver, lungs & bronchi, ovaries, pituitary, prostate, seminal vesicles
& coagulation gland, spleen, testes, thymus, thyroid with parathyroids, uterus with cervix & oviducts.

Histopathology:
- toxicity subgroups: The following organs were microscopically observed for the control and 1000 mg/kg bw/day groups:
Brain, eyes, Harderian glands, optic nerves, pituitary gland, thyroid with parathyroids, heart, thymus, liver, spleen,
adrenals, kidneys, testes, epididymides, ovaries, lung, trachea, esophagus, 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, skin with mammary glands, sternum with marrow,
seminal vesicle & coagulation gland, prostate. In addition, the jejunum, mesenteric lymph nodes and duodenum
were also examined for the 7.1 and 21.9 mg/kg bw/day toxicity subgroups and any gross lesions for all adult animals.
- reproductive subgroups: Gross lesions.

Postmortem examinations (offspring):

Pup survivors were killed on Day 7 post partum.
Full macroscopic examination of decedent and surviving pups including assessment of the presence of milk in the stomach, where possible.
(Missing or grossly autolysed or cannibalised pups 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_GAH0030"

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 and 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. or 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)
- 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"

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

two deaths, intubation error

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

only at 75 mg/kg/day

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

only at 75 mg/kg/day

Food efficiency:

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

only at 75 mg/kg/day; counts of monocytes slightly high, of total white cells and neutrophils marginally high

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

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

at 21.9 and 75 mg/kg/day; in jejunum, mesenteric lymph node and/or duodenum

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS, NEUROBEHAVIOUR AND MORTALITY
Three high dose females (2 of the toxicity subgroup and 1 of the reproductive subgroup) were found dead following intubation errors. One low and one high dose females without viable litter were killed on Day 25 after evidence of mating. Mortality or clinical signs attributable to treatment with the test material or effects on sensory reactivity, grip strength or motor activity were not evident.

BODY WEIGHT AND WEIGHT GAIN
At 75 mg/kg/day, overall bodyweight gain was low in males during the 5-week treatment period and in females during the first two treatment weeks and during gestation.

FOOD CONSUMPTION
At 75 mg/kg/day, food consumption was slightly low in both sexes during the first two treatment weeks, and in dams it was lower than concurrent controls, overall during gestation and lactation. At lower dose levels (7.1 and 21.9 mg/kg/day) foodconsumption was unaffected by treatment with the test material.

REPRODUCTIVE ENDPOINTS
Oestrous cycles, pre-coital interval mating performance, fertility, gestation length, gestation index and litter size were not affected by treatment.

GROSS PATHOLOGY
After five treatment weeks at 75 mg/kg/day, the jejunum of all surviving animals (main phase males and toxicity phase females) was thickened, the mesenteric lymph nodes were enlarged in 2 males and 2 females and were pale in in 3 males, and the duodenum was thickened in 1 male and 2 females. At lower dose levels after 5 treatment weeks and at all dose levels in dams killed on Lactation Day 7, toxicologically relevant macroscopic lesions were not evident. Mean numbers of uterine implantations were considered unaffected by treatment with the test substance.

ORGAN WEIGHTS
Following lactation, adjusted mean kidney weight was low with relationship to treatment. This was considered to be fortuitous and toxicologically not significant, as there was no such change in toxicity phase animals after 5 treatment weeks.

HISTOPATHOLOGY
There were no histopathology findings indicative of adverse effects on reproductive organs. 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, in general, were not histopathologically examined.

After five treatment weeks, the presence of foamy macrophages in the mucosa of the jejunum in males at 21.9 mg/kg/day and both sexes at 75 mg/kg/day and in the duodenum in two females at 75 mg/kg/day, and foamy histiocytosis in the sinuses of the mesenteric lymph nodes in both sexes at 21.9 and 75 mg/kg/day were attributed to treatment with the test material and were considered potentially adverse, but were not considered to represent reproductive toxicity endpoints.

Further details of histopathology findings in adult animals (main phase males and toxicity subgroup females) of the present study are given in separate endpoint study record "7.5.1 Repeated dose toxicity: oral - Repeat dose tox combined_gavage_rat_HLS_GAH0030".

Key result

Dose descriptor:

NOEL

Effect level:

>= 75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: reproductive toxicity

Remarks:

highest dose tested

Key result

Dose descriptor:

NOAEL

Effect level:

>= 7.1 - < 21.9 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

duodenum

ileum

intestine

mesenteric lymph node

Treatment related:

yes

Dose response relationship:

yes

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

Histopathological findings:

not examined

No treatment-related abnormality was observed.

No abnormality in sex ratio.

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

>= 75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks:

F1 animals were not dosed. Exposure to the test substance may have occurred in utero and via lactation. Animals were killed on day 4 post partum.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

Key result

Reproductive effects observed:

no

Conclusions:

In the present study the no-effect-level (NOEL) for reproductive toxicity has been 75 mg/kg/day, i.e. the highest dose level tested. Dose levels substantially higher than this were not selected for the present study, because of general toxicity. Effects indicative of reproductive or developmental toxicity of WS400151 were not evident. Based on the read across approach applied also for the target substance WS400101 no reproductive and developmental toxicity is expected. Therefore, classification and labelling regarding reproductive/developmental toxicity according to EU classification rules [ REGULATION (EC) 1272/2008] was not inferred.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Justification for selection of Effect on fertility via oral route:
The substance WS400101 is the salt (physical mixture) of fatty acids, tall oil, and octadec-9-enylamine. This chemical composition is similar to the substance WS400151 (CAS 147900-93-4) which is the salt (physical mixture) of fatty acids, C18-unsaturated, trimers and octadec-9-enylamine. Based on the result of the oral repeat dose study including reproduction and developmental toxicity screening performed with WS400151 it is concluded that the toxic effects observed in this study were due to octadec-9-enylamine; fatty acids, C18-unsaturated, trimers do have low toxicological potential. Because WS400101 also is a fatty acids salt of octadec-9-enylamine it is expected that the same toxicological effects would be observed in an oral repeat dose study including reproduction and developmental toxicity screening due to octadec-9-enylamine; the fatty acids do have low toxicological potential.

The oral repeat dose study performed with WS400151 included reproduction and developmental toxicity screening. No effects on fertility or on development of the F1 generation were observed up to the highest doses of WS400151 exhibiting strong toxicity to parental animals. With regard to reproduction toxicity, in the harmonised classification and labelling report on octadec-9-enylamine it is concluded that all available studies on different long-chain alkylamines “do not call for specific classification / labelling for this endpoint. This result is supported by several repeat-dose studies in which no adverse effects on reproductive organs were seen after treatment with primary alkyl amines” (ECHA 2011, ECHA/RAC/CLH-O-0000002197-73-01/F and ECHA 2011, Annex 1; ECHA/RAC/CLH-O-0000002197-73-01/A1).

Effects on developmental toxicity

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Remarks:

The experimental study was performed with a structural analogue; read-across from results of this study is justified (see below).

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read-across from structural analogue

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) of 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

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: Ca. 70 days.
- Weight at treatment start: Males: minimum 341 g, maximum 390 g,
Females: minimum 212 g, maximum 277 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 polypropylene 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.

Route of administration:

oral: gavage

Vehicle:

propylene glycol

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 body weight.

- 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 propylene glycol as a vehicle was established visually and by chemical analysis during the 7-day range-finding study:
Endpoint study record "7.5.1 Repeated dose toxicity: oral - 7d_range-finding_gavage_HLS_GAH0029".
In addition, in the present main study, concentrations of dose formulations were chemically analysed.

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).
- Concentrations (verified at first, second and last treatment week) of the test material formulations were lower than expected from nominal concentrations (see Table 1).
- Mean concentrations of WS400151 in prepared formulations attained by chemical analysis were 70.5% to 87.2% of the corresponding nominal concentration. Evaluation of a number of aspects of the analytical and formulation procedures did not lead to identification of the cause for the lower concentrations achieved by chemical analysis. In order to take a conservative approach, it was concluded that the concentrations determined by chemical analysis reflected the concentrations of test substance formulation administered to the animals.

Details on mating procedure:

- Male/female ratio per cage: 1/1
- Length of cohabitation: At the most 14 days, until proof of pregnancy was confirmed. 
- 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).

Duration of treatment / exposure:

- Treatment period, males & toxicity phase females: Daily, for five consecutive weeks, in males commencing 14 days prior to mating
- Treatment period, main phase females (i.e. reproductive subgroup): 44 to 47 days (from 14 days prior to mating to day 6 of lactation)
- Offspring were not dosed

Frequency of treatment:

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

Duration of test:

- Duration of test, all F0 males & toxicity subgroup females: Five weeks
Duration of test, reproductive subgroup females (F0): From 14 days prior to mating to day 7 of lactation.
Duration of test, offspring: From birth to day 7 of lactation.

Dose / conc.:

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

Remarks:

dose levels derived from chemical determination of administered formulations

Dose / conc.:

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

Dose / conc.:

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

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 7-day preliminary oral toxicity study in young adult rats (females nulliparous and non-pregnant) in which a NOAEL could not be established at dose levels of 100, 300 or 1000 mg/kg/day.

Maternal examinations:

Clinical observations performed and frequency:
- Clinical signs : At least twice a day (before and after administration)
- Detailed physical examination
and arena observations: Before treatment start and at least once a treatment week.
- Body weight: Weekly for pre-mating and mating period; on gestation days 0, 6, 13, 20; on lactation days 1 & 7.
- Body weight: Weekly for pre-pairing period; on gestation days 0, 6, 13, 20; on lactation days 1 & 7.
- Food consumption: Weekly for pre-pairing period, during gestation for days 0-6, 6-13, 13-20, during lactation for days 1-4 & 4-7.
- Frequency of vaginal estrus: Daily by examination of vaginal smears taken from the beginning of the treatment period to the day of confirmed copulation.
(During the pairing period, females were checked every morning for pregnancy).

Additional parameters examined:
- No. of animals mating (evidence of successful copulation, i.e. sperm positive vaginal smear and at least one copulation plug)
- Pre-coital interval (pairing days until detection of mating)
- No. of animals achieving pregnancy
- No. of living pregnant females
- From Day 20 post copulation 3 times a day checks for evidence of parturition, any difficulties and numbers of live and dead offspring.
- Gross pathology
- Organ weights
adrenals, brain, heart, kidneys, liver, lungs & bronchi, ovaries, pituitary, spleen, thymus, thyroid with parathyroids, uterus with cervix & oviducts.
- Histopathology (gross lesions).

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. Examinations confined to toxicity subgroup animals and/or to parental males (F0) are detailed in the separate endpoint study records,
"7.5.1 Repeated dose toxicity: oral - Repeat dose tox combined_gavage_rat_HLS_GAH0030" and
"7.8.1 Toxicity to reproduction - Tox to reproduction screening combined_gavage_rat_HLS_GAH0030"

Ovaries and uterine content:

The ovaries and uterine content were macroscopically examined after termination on day 7 of lactation, i.e. day 7 post partum.
Numbers of uterine implantation sites were recorded and post implantation survival determined.
In addition, gestation length (time elapsing between detection of mating and commencement of parturition) was recorded.

Fetal examinations:

Litters were examined post partum as follows (day of birth = day 0):
- Number:  Daily until day 7 post partum.
- Sex: In total litter: 1st day; in live litter 1st and 7th day
- Live births/mortality: Daily until day 7 post partum.
- Clinical signs: Daily until day 7 post partum
- Body weight of live pups: 1st, 4th, 7th day and weight change from days 1-4 and 1-7.
- Necropsy:  7th day (Full macroscopic examination of all pups including assessment of the presence of milk in the stomach, where possible.
(Missing or grossly autolysed or cannibalised pups 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_GAH0030"

Indices:

- Percentage mating (No. of animals mating/No. of animals paired) x 100
- Conception rate (No. of animals achieving pregnancy/No. of animals mated) x 100
- Fertility index (No. of animals achieving pregnancy/ No. or animals paired) x 100
- Gestation index (No. of live litters born on day 0/No. of living pregnant females) x 100
- 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
- Sex ratio expressed as percentage males and calculated for total offspring on Day 1 and for live offspring on Days 1 & 7
(No. of male pups in litter/No. of offspring in litter) 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

Historical control data:

Not included in the study report.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

two deaths, intubation error

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

reduced body weight gain, only at 75 mg/kg/day

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

reduced food consumption, only at 75 mg/kg/day

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

only at 75 mg/kg/day; counts of monocytes slightly high, of total white cells and neutrophils marginally high

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:

effects observed, treatment-related

Description (incidence and severity):

only at 75 mg/kg/day: jejunum and duodenum thickened, mesenteric lymph nodes enlarged

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

at 21.9 and 75 mg/kg/day; in jejunum, mesenteric lymph node and/or duodenum

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

not examined

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: Based mainly on histopathology in nulliparous and non-pregnant females after 5 weeks of treatment

Details on maternal toxic effects:
CLINICAL SIGNS, NEUROBEHAVIOUR AND MORTALITY
Three high dose females (2 of the toxicity subgroup and 1 of the reproductive subgroup) were found dead following intubation errors. One low and one high dose females without viable litter were killed on Day 25 after evidence of mating. Mortality or clinical signs attributable to treatment with the test material or effects on sensory reactivity, grip strength or motor activity were not evident.

BODY WEIGHT AND WEIGHT GAIN
At 75 mg/kg/day, overall bodyweight gain was low in females during the first two treatment weeks and during gestation.

FOOD CONSUMPTION
At 75 mg/kg/day, food consumption was slightly low during the first two treatment weeks and lower than concurrent controls overall during gestation and lactation. At lower dose levels (7.1 and 21.9 mg/kg/day) foodconsumption was unaffected by treatment with the test material.

REPRODUCTIVE ENDPOINTS
Oestrous cycles, pre-coital interval mating performance, fertility, gestation length, gestation index and litter size were not affected by treatment.

GROSS PATHOLOGY
After five treatment weeks at 75 mg/kg/day, the jejunum of all surviving nulliparous non-pregnant toxicity phase females was thickened, the mesenteric lymph nodes were enlarged in 2 of these females, and the duodenum was thickened 2 females. At lower dose levels after 5 treatment weeks and at all dose levels in dams killed on Lactation Day 7, toxicologically relevant macroscopic lesions were not evident. In addition, mean numbers of uterine implantations were considered unaffected by treatment with the test substance.

ORGAN WEIGHTS
Following lactation, adjusted mean kidney weight was low with relationship to treatment. This was considered to be fortuitous and toxicologically not significant, as there was no such change in toxicity phase animals after 5 treatment weeks.

HISTOPATHOLOGY
In general, tissues from maternal animals were not histopathologically examined. In nulliparous, non-pregnant females histopathology findings indicative of adverse effects on reproductive organs were not evident. After five treatment weeks, the presence of foamy macrophages in the mucosa of the jejunum and duodenum at 75 mg/kg/day, and foamy histiocytosis in the sinuses of the mesenteric lymph nodes at 21.9 and 75 mg/kg/day were attributed to treatment with the test material and were considered potentially adverse, but were not considered to represent reproductive toxicity endpoints.

Further details of histopathology findings in adult animals (main phase males and nulliparous, non-pregnant toxicity subgroup females) of the present study are given in separate endpoint study record "7.5.1 Repeated dose toxicity: oral - Repeat dose tox combined_gavage_rat_HLS_GAH0030".

Key result

Dose descriptor:

NOAEL

Effect level:

> 7.1 - <= 21.9 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

histopathology: non-neoplastic

Fetal body weight changes:

no effects observed

Description (incidence and severity):

please note: fetuses were not examined in this reproduction and developmental toxicity screening study. Pups were examined on day 7 of lactation. No effects were observed on pup number, sex ratio, pup body weight, litter size and weight, postnatal survival, external malformations.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

no effects observed

Key result

Dose descriptor:

NOEL

Effect level:

> 75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks:

highest dose tested

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

In the present study, the no-adverse-effect-level (NOAEL) for maternal toxicity was 7.1 mg/kg/day. This NOAEL was derived mainly from histopathology findings in nulliparous, nonpregnant females after 5 weeks of treatment, because, in general, histopathology was not conducted on dams. The no-effect-level (NOEL) for developmental toxicity has been 75 mg/kg/day, i.e. the highest dose level tested. Dose levels substantially higher than this were not selected for the present study, because of general toxicity. Effects indicative of reproductive or developmental toxicity of WS400151 were not evident.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Justification for selection of Effect on developmental toxicity: via oral route:
The substance WS400101 is the salt (physical mixture) of fatty acids, tall oil, and octadec-9-enylamine. This chemical composition is similar to the substance WS400151 (CAS 147900-93-4) which is the salt (physical mixture) of fatty acids, C18-unsaturated, trimers and octadec-9-enylamine. Based on the result of the oral repeat dose study including reproduction and developmental toxicity screening performed with WS400151 it is concluded that the toxic effects observed in this study were due to octadec-9-enylamine; fatty acids, C18-unsaturated, trimers do have low toxicological potential. Because WS400101 also is a fatty acids salt of octadec-9-enylamine it is expected that the same toxicological effects would be observed in an oral repeat dose study including reproduction and developmental toxicity screening due to octadec-9-enylamine; the fatty acids do have low toxicological potential.

The oral repeat dose study performed with WS400151 included reproduction and developmental toxicity screening. No effects on fertility or on development of the F1 generation were observed up to the highest doses of WS400151 exhibiting strong toxicity to the dams. With regard to developmental toxicity, in the harmonised classification and labelling report two teratogenicity studies performed with octadecenylamine in rats and rabbits are summarised. No indications of an embryotoxic, foetotoxic or teratogenic effect were observed at dose levels causing maternal toxicity. It is concluded that all available studies on different long-chain alkylamines “do not call for specific classification / labelling for this endpoint.” (ECHA 2011, ECHA/RAC/CLH-O-0000002197-73-01/F and ECHA 2011, Annex 1; ECHA/RAC/CLH-O-0000002197-73-01/A1).

Justification for classification or non-classification

In the reproduction and developmental toxicity screening study (OECD 422) performed with WS400151 - the substance used for read-across - the no effect level (NOEL) for reproductive toxicity was derived at the dose of 75 mg/kg/day, i.e. the highest dose level that could be applied to the animals based on general toxic effects. The no-adverse-effect-level (NOAEL) for adult animals and specifically maternal toxicity was 7.1 mg/kg/day. This NOAEL was derived mainly from histopathology findings in nulliparous, nonpregnant females after 5 weeks of treatment, because, in general, histopathology was not conducted on dams. The no-effect-level (NOEL) for developmental toxicity has been 75 mg/kg/day, i.e. the highest dose level tested. Dose levels substantially higher than this were not selected for the present study, because of general toxicity. Effects indicative of reproductive or developmental toxicity of WS400151 were not evident. Additionally, for the constituent, i.e. octade-9 -enylamine, exhibiting the systemic toxic effects after repeated oral dosing, it is concluded in the report on harmonised classification and labelling that all available studies on different long-chain alkylamines “do not call for specific classification / labelling for this endpoint."

Therefore, classification and labelling regarding reproductive/developmental toxicity of WS400101 according to EU classification rules [REGULATION (EC) 1272/2008] was not inferred.