Reaction products of fatty acids, tall oil and fatty acids, C18 unsaturated, trimers and fatty acids, C18 unsaturated, dimers with (9Z)-octadec-9-en-1-amine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study, evaluated as reliable in the EU RAR report, but only secondary sources available

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

other: sesame oil

Details on exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): sesame oil (solubility reasons)

ADMINISTRATION VOLUME: 10mL/kg b.w.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: a maximum of 14 days

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

males: max. 28days (14 days prior to mating and until the end of the mating period)
females: 14 days prior to mating, through mating, gestation and 3 days of lactation.

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
12.5, 50, 150 mg/kg b.w.
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION: Yes

Sperm parameters (parental animals):

PAS-hematoxylin staining of testes sections

Litter observations:

number and sex of pups
stillbirths
live births
postnatal mortality
presence of gross anomalies
weight gain (day 0, day 4)

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals on the day after the end of treatment
- Maternal animals: All surviving animals on day 4 of lactation with their pubs

GROSS NECROPSY: yes

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination and weighed, respectively:
- ovaries
- uteri (including horns, cervix, and vagina), only uteri of non pregnant females were stained for implantation sites
- testes (with special emphasis on stages of spermatogenesis and histopathology of interstitial cell structure)
- epididymides
- accessory sex organs
- all gross lesions

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed on day 4 of lactation and necropsied

GROSS NECROPSY: yes

Reproductive indices:

- Pregnancy index: the percent ratio of females with live births to the pregnant females.
- Pre-coital interval: calculated on the dams which proved pregnant and was expressed for each group as the mean time lapse (in days) between the beginning of the mating period and the ascertainment that copulation had occurred.
- Pregnancy period: the duration of pregnancy was determined for all those dams that reached term of pregnancy as being the time that elapsed between the day vaginal smear proved positive and the day of parturition.
- The post-implantation losses were calculated for each litter m the following manner: Post- implantation losses equals (No. implantations) negative (No. live pups) divided by (No. implantations) x 100

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- 150 mg/kg: salivation, hunched posture, soft stools, piloerection; 6/10 male and 5/10 female animals died (5 males and 3 females during premating)
- 50 mg/kg: salivation; 1/10 male and 1/10 female animals died
- 12.5 mg/kg: no changes observed

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- 150 mg/kg: body weight loss of about 22g (m) and 17g (f)
- 50mg/kg: reduced body weight gain during premating together with lower food consumption in both sexes

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- mating and fertility indices, mean pre-coital time and parturition were unaffected up to 50 mg/kg/day (a slightly reduced fertility index at 50 mg/kg was not statistically significant and is within the normal variation for this rat strain. When looking at the percentages only, one also should keep in mind that 2 animals of this dose group died.)
- in the high dose group, only 3 out of the 7 mated females had positive vaginal smears, and only 1 female became pregnant, but didn't deliver. This finding is due to the excessive maternal toxicity of the test substance in this group.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- lower absolute weight of epididymides and higher value of testis weight relative to body weight at 150 mg/kg/day, corresponding to decreased body weight in this group
- no differences in ovary weights among the experimental groups

HISTOPATHOLOGY (PARENTAL ANIMALS)
- histology of testes, epididymides and ovaries did not show any compound-related changes in all dose groups
- no changes in testicular staging performed in the PAS-hematoxylin stained sections of control and high dose groups

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on death and reduced body weight in mid dose animals

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: mortality of more than 50% of parental animals was observed in the high dose

VIABILITY (OFFSPRING)
- no litters present at 150 mg/kg/day
- no effects on postnatal survival up to day 4 of lactation and no effects on sex ration at 50 mg/kg/day

CLINICAL SIGNS (OFFSPRING)
- no litters present at 150 mg/kg/day
- no significant effects up to 50 mg/kg/day

BODY WEIGHT (OFFSPRING)
- no litters present at 150 mg/kg/day
- slightly lower pup body weight seen in 50 mg/kg/day group
- no effects seen at 12.5 mg/kg/day

GROSS PATHOLOGY (OFFSPRING)
- no abnormalities observed in any pub either at birth or at autopsy on day 4 of lactation neither of the 50 or of the 12.5 mg/kg/day group

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: slightly lower pub weight on day 4 only was considered secondary to maternal toxicity, especially since no effects were observed in two developmental toxicity studies in rats and rabbits.

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Additional information

No data are available for the Reaction products of Fatty acids, tall-oil, compds. with oleylamine and Fatty acids, C18-unsatd., trimers, compds. with oleylamine. But since the substance is a salt, it is expected to dissociate into fatty acids C18 -unsatd., trimers, fatty acids, tall oil, and oleylamine. So the test substance was assessed based on it ions, following the approach outlined below.

Fatty acids, C18-unsatd., trimers, CAS 68937-90-6, has already been registered during the first phase of REACH. Extensive data exist either for fatty acids, C18-unsatd., trimers, or for the dimeric form, which has been used as a read across substance in the dossier. After acute oral exposure, no clinical signs or mortality was observed up to 5000mg/kg b.w. The substance did not cause skin or eye irritation, skin sensitization, or gene mutation. After repeated exposure via diet for 13 weeks, some changes in histopathology were observed at a dose just below 4000mg/kg, which is considerably higher than the limit dose of 1000mg/kg used in current protocols. No effects were observed in low dose animals, that received app. 800mg/kg. No effects on reproductive organs, fertility or developmental toxicity were observed in an OECD 421 screening study.

The main component of fatty acids, tall-oil is oleic acid (CAS 112-80-1), which is a natural constituent of vegetable and animal fat, e.g., 70-75% of olive oil are oleic acid dimers and trimers. In it monomeric form, oleic acid causes skin and eye irritation, is almost not toxic after acute exposure (LD50 app. 4200mg/kg, LD0 app. 2850mg/kg), and did not cause mutations in salmonella cells (NTP).

Because of the obvious non-toxicity of fatty acids, tall-oil and fatty acids, C18-unsatd., trimers, oleylamine was identified as the hazard inducing component and used for hazard assessment as a worst case approach. This substance is part of an EU defined category for primary alkyl amines (EU risk assessment for primary alkyl amines, 2008; CLH report for coco alkyl amine, 2010). Read across was performed to several members of this category, depending on availability of reliable studies. According to the CLH Dossier, which was adopted by the RAC in 2011, primary alkyl amines are classified to cause damage to the eyes, skin corrosion or irritation and respiratory tract irritation. In addition, organ toxicity (liver, GI-tract, immune-system) was observed, presumably secondary to the local irritant properties of the alkyl amine. In addition, similar effect as described after repeated exposure to oleylamine and tallow alkyl amine were also observed in the acute oral toxicity study for the reaction mass of fatty acids, tall-oil, compds. with oleylamine and fatty acids, C18 -unsatd., trimers, compds. with oleylamine at 2000mg/kg, i.e.,swollen lymph nodes and peyer's plaques in region of the small intestine and red discoloration of the small intestine and corresponding severe weight loss.

Tallow Alkyl Amine (CAS 61790 -33 -8)

In a GLP-conform reproduction/developmental toxicity screening test according to OECD 421 (APAG, 2000, data based on EU RAR, 2008) groups of 10 Crl:CD (SD) BR rats per sex were treated with 12.5, 50, and 150 mg/kg bw/d by gavage using sesame oil as a vehicle. Males were treated daily from 14 days prior to mating until the end of the mating period for a maximum of 28 days. Females were treated daily for 14 days before the start of the mating period, throughout mating, during pregnancy, and until day 3 of lactation. The animals were mated one male with one female.

At the highest dose, 6 males and 5 females died during the premating or mating period. Besides salivation, hunched posture, and in some cases soft stool and piloerection, body weight was decreased by 22g in males and by 17g in females. At 50mg/kg, 1 male and 1 female died prematurely, and body weight gain was reduced compared to control animals. Both groups showed lower food consumption. Histopathology of testes and epididymides (carried out for high dose and control animals) did not reveal any substance related changes. In particular, no changes were seen in the testicular staging performed in PAS haematoxylin stained sections. A moderately increased frequency of athropic corpora lutea was seen in high dose females, but is considered secondary to decrease in body weight and growth.

Pre-coital interval, mating index, fertility index, parturition, number of pubs, number of stillborns, and pub sex ratio were unaffected at the low and mid dose. An adverse impact of test substance on fertility was only observed at the highest dose level of 150 mg/kg bw/d, which was lethal for more than half of the treated dams. Since no histopathological changes were observed in the reproductive organs, absence of litters in high dose animals was considered secondary to the severe toxicity at this dose level. In addition, in the 28 -day study with this test substance, histopathology on reproductive organs was also performed, and no adverse effect was detected up to 150mg/kg b.w.

Pub weight was slightly lower in the mid dose group. This dosage also resulted in two deaths and emaciation of the maternal animals, and does not indicate developmental toxicity. Furthermore, as also discussed below, no teratogenic or developmental effects were observed in two OECD 414 studies in rats and rabbits with the structurally related oleylamine.

Thus the NOAELs for developmental toxicity and fertility were set to 50mg/kg b.w., while the NOAEL for systemic effects was 12.5mg/kg b.w.

Effects on developmental toxicity

Description of key information

rat, oral: not teratogenic (CMA (1989), OECD 414, GLP)
rabbit, oral: not teratogenic (TSCAT (1989), OECD 414, GLP)

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Toxic Substances Control Act (TSCA) Guidelines (40 CFR Part 798.4700, September 1985, and revised edition May 1987)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Portage, Michigan
- Age at study initiation: females: 14 weeks, males used for pairing: 9-11 months
- Weight at study initiation: females: 229-316g
- Fasting period before study: no
- Housing: individually in stainless steel wire mesh cages
- Diet (e.g. ad libitum): Purina Certified Rodent Meal #5002 ad lib.
- Water (e.g. ad libitum): deionized tap water ad lib.
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 3°C
- Humidity (%):55% +/- 15%
- Air changes (per hr): 10-12
- Photoperiod (hrs dark / hrs light): 12h/12h

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dose solutions were prepared daily. Appropriate amounts of the test article for each dose group were weighed into volumetric flasks. Corn oil was added to achieve the final concentrations. The flasks were inverted several times to ensure adequate mixture. The dosing solutions were stored under a nitrogen blanket at room temperature.

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility
- Concentration in vehicle: 2-16mg/ml
- Dosage volume: 5ml/kg b.w.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first day of dosing and near the end of the dosing period, a subsample from each dosing solution, including the control, was taken and analyzed for verification of the test article concentration.The average recovery for all dosing solutions was within 10% of the nominal concentrations.

Details on mating procedure:

- Impregnation procedure: cohoused
- Male rats: resident Sprague Dawley Crl:COBS CD BR VAF/PLUS
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

10 days (from gestation days 6 to 15)

Frequency of treatment:

daily

Duration of test:

Animals were sacrificed on gestation day 20.

Remarks:

Doses / Concentrations:
10, 40, 80mg/kg b.w.
Basis:
actual ingested

No. of animals per sex per dose:

28

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on a range-finding study using 50, 100, 150, 250 mg/kg/day.
Mortality occurred in the 100, 150 and 250 mg/kg/day groups. Outward clinical signs of toxicity and body weight losses or reduced weight gain occurred at the 50, 100, 150 and 250 mg/kg/day levels. A dose level of 100 mg/kg/day was considered to be excessive for a high dose level of the definitive teratology study due to the induced mortality. Conversely, 50 mg/kg/day did not produce sufficient maternal toxicity to be considered suitable as a high dose level. Thus, 80 mg/kg/day was selected in anticipation of producing sufficient maternal toxicity. Graduated doses of 40 and 10 mg/kg/day were selected as the mid and low dose levels, respectively. A dose level of 40 mg/kg/day was expected to induce minimal maternal toxicity while the 10 mg/kg/day dose level was selected to determine a no effect level for maternal and developmental toxicity.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: check for clinical signs, physical or behavioral abnormalities, mortality

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days 0, 6, 9, 12, 16, 20

FOOD CONSUMPTION: Yes
- Measured on gestation days 0, 6, 9, 12, 16, and 20.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 (2 animals per group were sacrificed after end of treatment on gestation day 15 to determine severity of gastrointestinal irritation.)
- Organs examined: The thoraic, abdominal, and pelvic cavities were opened and examined. The uterus was removed, weighed, and opened. Uteri with no macroscopic evidence of implantations were stained with 10% aqueous ammonium sulfate solution.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data

Historical control data:

Cesarean section data, fetal malformation data, fetal variation data were provided.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Clinical signs:
Rats receiving 40 or 80mg/kg b.w. exhibited rales, salivation, soft stools, diarrhea, few and abnormal colored feces, fecal and /or urine stain, and unkempt appearance. At the highest dose, also emaciation, rough coat, and dark red material around the eyes, nose and or mouth was observed.

Body weight:
Maternal body weights were reduced in high dose animals from gestation day 9 until scheduled sacrifice. In the mid dose, body weights were also reduced, but body weight gain returned to normal or even exceeded control values after the end of treatment. Food consumption was reduced in both groups during the entire treatment period.

Necropsy:
No substance related findings.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOAEL

Effect level:

>= 80 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

80 mg/kg bw/day

Species:

rat

Additional information

No data are available for the Reaction products of Fatty acids, tall-oil, compds. with oleylamine and Fatty acids, C18-unsatd., trimers, compds. with oleylamine. As a worst case approach, read across was performed to the group of primary alkyl amines, since coco alkyl amine is considered the hazard inducing component of the salt. Further justification for the read across approach can be found in the endpoint summary for fertility and and more detailed in the attached document in IUCLID chapter 13.

OECD 414 in rats using oleylamine (CAS 112 -90 -3)

28 pregnant female Spraque Dawley rats were treated orally by gavage with 10, 40, or 80 mg/kg bw/d of the test substance in corn oil during gestation days 6 to 15. During the study,animals were examined daily. Any clinical signs of toxicity including physical or behavioural abnormalities were recorded. Individual body weight and food consumption were recorded on gestation days 0, 6, 9, 12, 16, and 20. Two animals in each group were selected to be sacrificed and necropsied after treatment on gestation day 15 to determine the appearance and severity of gastrointestinal tract irritation. On gestation day 20, caesarean section was performed on all

surviving animals. The number of viable foetuses, early and late resorptions, and corpora lutea was recorded. Foetuses were examined for external, visceral, and skeletal abnormalities.

All animals survived to scheduled sacrifice. Outward clinical signs of toxicity were observed at the 40 and 80 mg/kg bw/d dose levels. These observations most likely indicated a generalised irritant effect of the test substance as characterised by rales, salivation, unkempt appearance and changes in the amount, colour, and consistency of the faeces. However, no other signs of treatment-related

gastrointestinal irritation or other internal changes were observed at the gestation day 15 and 20 necropsies. Animals treated with 80mg/kg also exhibited emaciation, rough coat, and dark red material around the eyes, nose, and/or mouth. Dose-dependent body weight loss (during gestation days 6-9) or reduced weight gain (during gestation days 12-16), along with a corresponding reduction in food consumption occurred during the treatment period in the 40 and 80 mg/kg bw/d groups. Following cessation of treatment (days 16-20), increase in weight gain and food consumption were noted at both dose levels.

Caesarean section data obtained from the treated groups did not reveal any meaningful differences (concerning number of corpora lutea, implantation sites, viable foetuses, foetal sex and foetal weight) when compared with the controls. Foetal evaluations of type

and frequency of malformations and variations did not reveal any indications for a treatment related teratogenic effect.

The NOAEL for maternal toxicity was 10mg/kg b.w., and the NOAEL for teratogenicity was 80mg/kg b.w., the highest dose tested.

OECD 414 in rabbits using oleylamine (CAS 112 -90 -3)

22 inseminated female New Zealand White rabbits were treated orally by gavage with 3, 10, or 30 mg/kg bw/d of the test substance in corn oil during gestation days 6 to 18. Any clinical signs of toxicity including physical or behavioural abnormalities and food consumption were recorded daily. Two animals in each group were selected to be sacrificed and necropsied after treatment on gestation day 18 in order to determine the appearance and severity of gastrointestinal tract irritation. On gestation day 29 caesarean section was performed on all surviving animals. The number of viable foetuses, early and late resorptions, and corpora lutea was recorded. Foetuses were examined for external, visceral, and skeletal abnormalities.

As a result of the study, two females died in the 30 mg/kg bw/d group, one on gestation day 9 and the other on gestation day 25. In the 10 mg/kg bw/d dose group, rales and laboured breathing were noted. Additional findings at the 30 mg/kg bw/d level included few or no faeces and emaciation. Irritation of the snout area as observed in high dose females was characterised by swollen raised white areas, scab-like lesions and/or sloughing of the skin of the lips and the chin. No signs of treatment-related gastrointestinal irritation or internal changes were observed at gross necropsy at gestation days 18 and 29. Dose-dependent body weight loss or reduced weight gain, along with a corresponding reduction in food consumption occurred during treatment in the 10 and 30 mg/kg bw/d groups. Following cessation of treatment, weight gain increased in the 30 mg/kg bw/d group.

Caesarean section data obtained from treated groups did not reveal any meaningful differences (concerning number of corpora lutea, implantation sites, viable foetuses, implantation loss, foetal sex and foetal weight) when compared with the controls. Foetal evaluations of type and frequency of malformations and variations did not reveal any indications for a treatment-related teratogenic effect.

Thus the NOAEL for maternal toxicity was set at 3mg/kg b.w., and the NOAEL for teratogenicity was 30mg/kg b.w., the highest dose tested.

Justification for classification or non-classification

In the fertility/developmental toxicity screening test with the read across substance tallow alkyl amines an adverse impact on fertility was only seen at the highest dose level of 150 mg/kg bw/d, which was lethal for more than half of the treated dams. No adverse effects on spermatogenesis or reproductive organs were observed, so that lack of offspring is considered secondary to excessive toxicity. This result is supported by several repeat-dose studies, in which also no adverse effects on reproductive organs were seen after treatment with primary alkyl amines. The next lower dose also caused excessive maternal toxicity, i.e., two animals died, but fertility was unaffected in this group.

No adverse effects on rat or rabbit offspring up to and including dose levels evoking marked maternal toxicity were reported in the two developmental toxicity studies performed with oleylamine.

In conclusion, the available data on reproduction toxicity do not call for classification for this endpoint for the reaction mass of fatty acids, tall-oil, compds. with oleylamine and fatty acids, C18-unsatd., trimers, compds. with oleylamine according to 67/548/EEC or CLP/GHS_EU.