Fatty acids, C18-22

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The available information comprises adequate, reliable studies (Klimisch score 2) and consistent studies from reference substances with similar structure and intrinsic properties. Read-across is justified based on a common functional group, common precursors/breakdown products and similarities in toxicological properties (refer to endpoint discussion for further details). The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006. In accordance with Column 1 of REACH Annex IX the 2-generation reproductive toxicity study (required in Section 8.7.3) does not need to be conducted as no adverse effects on reproductive organs were observed in 28-day or 90-day repeated dose toxicity studies.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Human health effects on toxicity to reproduction are predicted from adequate and reliable data for source substances by read-across to the target substance within the group applying the group concept in accordance with Annex XI, Item 1.5, of Regulation (EC) No 1907/2006.

Fatty acids are found in all living organism fulfilling three fundamental roles. Besides their function as part of molecules like phospholipids and glycolipids important for the cell-structure, they are often precursors of signalling molecules such as prostanoids. The third and best understood role of fatty acid is their role as nutritional energy source. Based on their physiological function within the body no toxicity to reproduction of fatty acids is expected as demonstrated by animal studies with C7 fatty acid (heptanoic acid), C22 fatty acid (docosanoic acid) and fatty acids, tall oil.

 

The toxicity to reproduction of docosanoic acid (CAS# 112-85-6) was evaluated in a combined repeated dose and reproductive/developmental toxicity screening test performed under GLP according to OECD Guideline 422 (Nagao et al., 2002).Groups of 13 male and 13 female Sprague-Dawley rats received daily doses of 100, 300 and 1000 mg/kg bw of docosanoic acid by gavage. While the males were treated for 42 days, the females received the test substance from 14 days prior to mating until day 3 of lactation. As result, neither mortality nor abnormalities in general condition were observed. Since no difference in number of corpora lutea, implantation rate, number of implantations and all other reproductive parameters was noted in all treated groups compared to the control group, there were no indications of any adverse effects on reproduction. Thus, the NOAEL toxicity to reproduction was found to be ≥ 1000 mg/kg bw/day.

 

A combined reproduction / developmental toxicity screening test performed equivalently to OECD 421 is available for heptanoic acid (CAS# 111-14-8) (Hoberman, 1990). 10 female Sprague-Dawley rats were orally treated with 200, 1000 or 2000 mg/kg bw/day of the test substance dissolved in corn oil. Control animals received the vehicle only. Females were treated 7 days prior to mating and afterwards during mating, gestation and lactation until day 4 post-parturition. Male rats were used only as breeders (10 animals/group), and were not given the test substance. They were not considered to be a part of the test system and therefore not evaluated. Examinations on all dams comprised cage side observations, determinations of body weights and food consumption, gross necropsy, histopathology of gross lesions as well as determinations of reproductive indices.

One dam of the 1000 mg/kg bw/day dose group died following an intubation accident. In the 2000 mg/kg bw/day dose group 3 animals died, which was considered to be a treatment-related. Clinical signs included rales in significant numbers of rats of all treated groups over the whole study period. Possibly, this effect is due to gastroesophagal reflux and subsequent aspiration of dosing solution. Furthermore, excess salivation, urine-stained abdominal fur, ungroomed coat, chromorrhinorrhoea and/or decreased motor activity as well as ataxia were observed in the mid and high-dose group animals.

On day 1 to 7 of the study, mean body weight change in animals of the 1000 mg/kg bw/day dose group was lower than in controls (+ 7.8 g vs. + 8.8 g). Concomitantly, relative feed consumption was about 6% lower than in controls. During gestation, maternal body weight changes averaged +140.5 g (vs. 132.8 g in controls) although relative feed consumption was about 6% lower than in controls. During lactation, maternal body weight changes averaged +12 g (vs. +0.1 g in controls) and relative feed consumption was about 13% increased, when compared to controls. In the 2000 mg/kg bw/day dose group reduced body weight gain, reduced mean absolute body weights (about 2%) and reduced relative feed consumption (-17% compared to controls) were observed during the whole premating period. During gestation, maternal body weight changes were significantly lower than in controls (+ 126 g vs. + 132.8 g). Absolute body weights were about 7% lower when compared to controls and concomitantly, relative feed consumption tended to be reduced when compared to controls (about 2%). During lactation, maternal body weight changes averaged -14.5 g (vs. +0.1 g in controls). Absolute body weights were about 11% lower when compared to controls and the relative feed consumption was about 5% decreased, when compared to controls.

No effects on fertility and gestation indices as well as average number of implantations were observed. At gross pathology, the only treatment-related effect observed was red mottled lungs in one of the high-dose females.

Based on the results described above, the NOAEL for fertility was determined to be ≥ 2000 mg/kg bw/day. Based on mortality, clinical signs and reduced body weights during lactation in the high-dose group, the NOAEL for maternal toxicity is 200 mg/kg bw/day for females. In contrast no maternal toxic effects were found in two studies similar to OECD 414 performed with heptanoic acid in concentrations up to and including 1000 mg/kg bw/day (Serota et al., 1982 and 1983).

In a two-generation reproduction toxicity study similar to OECD Guideline 416, Sprague-Dawley rats (30 females per group and 15 males per group) were administered 5% and 10% fatty acids, tall oil (CAS# 61790-12-3) in the diet (corresponding to 2500 and 5000 mg/kg bw/day) as cited in a secondary source (Pine Chemical Association, 2004). The parental animals were fed the test substance, which consists predominantly of C18 unsaturated and saturated fatty acids, starting at 20 days pre-mating. Treatment of the parental generation was through the weaning of the first generation (F1). After weaning, 20 F1 males and 20 F1 females per group were maintained on the parental diet and mated at 100 days of age. The delivered F2 pups were followed until weaning. Parameters evaluated included F1 reproductive parameters, F1 fertility, viability, lactation and gestation indices. Haematology, serum chemistry, urinalysis, and organ weights for F1 animals, gross pathology of F1 and F2 animals and microscopic pathology of various organs of the F2 pups were performed.

There were no treatment-related effects on reproductive performance, the number of liveborn or still-born F1 litters and pups, or weaning weight of the F1 pups. No treatment-related changes in fertility, viability, lactation, or gestation indices were noted. No changes in haematology, clinical chemistry and urinalysis parameters and organ weights were found. Gross and microscopic pathology revealed no treatment-related effects. Thus, fatty acids, tall oil had no effect on the reproductive or developmental capabilities of rats at doses up to and including 5000 mg/kg bw/day.

 

No adverse effects on reproductive organs were observed in 28-day or 90-day repeated dose toxicity studies performed with members of the fatty acids category.

In a subchronic study rats received oleic acid (CA# 112-80-1) at dose levels up to 12500 mg/kg bw/day (Calandra, 1969). Gross pathology and histopathology included the determination of gonads weight as well as examination of testes, seminal vesicles, ovaries and uterus. No changes in absolute or relative gonads weight were noted and no gross and histopathological findings were observed in any of the examined reproductive tissues and organs. Moreover, in subacute toxicity studies with fatty acids, C8-18 and C18-unsatd, distn. residues (CAS# 70321-72-1) no adverse effects on reproductive organs up to and including the limit dose of 1000 mg/kg bw/day was noted, which was demonstrated by a lack of changes in gonads weight and the absence of histopathological findings in epididymides, seminal vesicles, testes, ovaries and uterus.

Since no effects on fertility were noted in the reproduction / developmental toxicity screening studies with heptanoic acid and docosanoic acid and since no adverse effects on reproductive organs were observed in 28- and 90-day repeated dose toxicity studies, no 2-generation reproduction toxicity study is required in accordance with Regulation (EC) No 1907/2006 Annex IX, Column 1, Section 8.7.3.

Since all the members of the category show similar structural and toxicological properties the results of individual members are also valid for other fatty acids within the category. Moreover, a substance-specific adjustment of the NOAEL for the individual category members is not required. An overall NOAEL for fertility for all fatty acids within the category of 1000 mg/kg bw/day is chosen as “worst case” among the available studies.

Short description of key information:
Key studies on reproductive toxicity by the oral route are available for the following category members:

Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (OECD 422), rat: NOAEL oral (fertility) ≥ 1000 mg/kg bw/day; CAS# 112-85-6, C22 (Nagao et al., 2002)

No hazard for reproductive toxicity was identified for fatty acids.

No data are available for reproductive toxicity after dermal exposure and inhalation, respectively.

Justification for selection of Effect on fertility via oral route:
Hazard assessment is conducted by means of read-across based on a category approach. The selected study is the most adequate and reliable study based on overall assessment of duration and dose-descriptor level (refer to the endpoint discussion for further details).

Effects on developmental toxicity

Description of key information

Key studies on developmental toxicity/teratogenicity by the oral route are available for the following category members:
Prenatal Developmental Toxicity Study (OECD 414), rat: NOAEL oral (develop. toxicity/teratogenicity) ≥ 1000 mg/kg bw/day; CAS# 111-14-8, C7 (Serota et al., 1983)
Prenatal Developmental Toxicity Study (OECD 414), rat: NOAEL oral (develop. toxicity/teratogenicity) ≥ 1500 mg/kg bw/day; CAS# 112-05-0, C9 (Serota et al., 1983)
Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (OECD 422), rat: NOAEL oral (developmental toxicity/teratogenicity) ≥ 1000 mg/kg bw/day; CAS# 112-85-6, C22 (Nagao et al., 2002)
No hazard for developmental toxicity/teratogenicity was identified for fatty acids. 
No data are available for developmental toxicity/teratogenicity after dermal exposure and inhalation, respectively.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

11 Mai - 24 Jun 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

test substance was administered solely in the period of GD 6-15

GLP compliance:

not specified

Limit test:

yes

Species:

rat

Strain:

other: Crl:COBS, CD® (SD) BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, New York, USA
- Age at study initiation: approx. 14 weeks (mating age)
- Housing: individually in elevated wire-mesh cages
- Diet: Purina Rodent Laboratory Chow ®, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 9 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approx. 24
- Humidity (%): 57 ± 4.8
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 11 Mai 1982 To: 24 Jun 1982

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared weekly by mixing appropriate amounts of the test item with corn oil on a magnetic stirrer (g/v) and transferred to amber bottles. The prepared dilutions were well shaken before use and animals were dosed while solutions were mixed on a magnetic stirrer.

Dosing volumes were based on individual body weights from the most weighing interval. Day 15 dosing was based on the body weight of Day 12.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1/2
- Length of cohabitation: up to 3 weeks
- After 10 days, females were rotated
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

Day 6 - 15 of gestation

Frequency of treatment:

daily, 7 days/week

Duration of test:

20 days (GD 0-20)

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

22 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The applied dose showed no maternal toxicity in a previous dose-range finding study (Serota et al., 1982).

Maternal examinations:

CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
- Parameters checked: e.g. alopecia, bloody crust, wheezing, labored respiration, urine stains, rough haircoat, stains on fur, salivation, corneal defect, wasting feed, water spillage, hunched, dyspnea, red discharge from vagina, soft feces

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 6, 9, 12, 15 and 20 of gestation

FOOD CONSUMPTION : Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food: Yes
- Time schedule for examinations: Days 6 - 8, 9 - 11, 12 - 14, 15 - 17 and 18 - 20 of gestation

WATER CONSUMPTION: Yes
- Time schedule for examinations: Days 6 - 8, 9 - 11, 12 - 14, 15 - 17 and 18 - 20 of gestation

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus, oavary, cecum, lung, kidney, stomach, liver, pancreas, adrenals, brain, pituitary, thymus, spleen, eyes, lymph nodes (mesenteric, cervical, mandibular)

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 resorptions: Yes (early and late resorptions)
- Other: Nongravid uterine weights (with ovaries attached): Yes

Fetal examinations:

- External examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [two-third per litter]
- Other: Visceral examinations: Yes [one-third per litter]; body weight and crown-rump distance [all per litter]

Statistics:

Mean values and standard deviations were calculated from the examined parameters.
Survival was analysed by the National Cancer Institute Package (Tomas, Breslow and Gart, 1977). The mean maternal body weight changes (days 0 - 6, 6 - 15, 15 - 20 and 0 - 20), mean maternal food and water consumptions), percent males per litter, mean fetal body weights and lengths, fetal viability, percent resorptions, implantation efficiency, gravid and non-gravid uterine weights and the incidence of visceral and skeletal anomalies and variants were analysed by Box´s test for homogeneity of variances. This test was followed by one-way classification analysis of variance (ANOVA) if the variances proved to be homogeneous. If the variances proved to be heterogeneous, a rank transformation of data was performed, which was followed by Box´s test and ANOVA. If ANOVA of untransformed or transformed data was significant, a Dunnett´s T-Test was used for control vs treatment group mean comnparisons. Pregnancy rates were analysed by a test of multiple proproportions using one degree of freedom Chi-square test with Yate´s continuity correction (Fleiss 1981). All pairwise comparisons were evaluated at the 0.5% probability (one-tailed) level.

Indices:

Mean incidence of resorptions (%): group mean of [(resorptions per litter/implantations per litter) x 100]
Mean incidence of fetal mortality (%): group mean of [(dead fetuses per litter/implantations per litter) x 100]
Mean incidence of fetal viability (%): group mean of [(live fetuses per litter/implantations per litter) x 100]
Mean incidence of visceral anomalies(%): group mean of [(number fo fetuses with anomalies per litter/number of fetuses examined viscerally per litter) x 100]
Mean incidence of visceral variants (%): group mean of [(number fo fetuses with variants per litter/number of fetuses examined viscerally per litter) x 100]
Mean incidence of skeletal anomalies (%): group mean of [(number fo fetuses with anomalies per litter/number of fetuses examined skeletally per litter) x 100]
Mean incidence of skeletal variants (%): group mean of [(number fo fetuses with variants per litter/number of fetuses examined skeletally per litter) x 100]

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
, MORTALITY AND CLINICAL OBSERVATIONS
One animal in the treatment group was found dead on day 8 of gestation. However, the survival rate of treated animals (95.5%) were comparable to control (100%).
No increased incidence of clinical observations was noted. Wheezing was observed in three treated rats during GD 6-15 and in two treated rats after treatment (GD 16-20). One treated rat had a rough haircout. In the control group, alopecia (neck), corneal defect, wasting feed and water spillage was each observed in one animal.


BODY WEIGHT CHANGE, FOOD AND WATER CONSUMPTION
No effects on body weights, food and water consumption were noted. The mean body weight change (Day 0-20 of gestation) was 104.2 ± 12.5 g for the treated females compared to 106.9 ± 14.7 g for the control group. The treated rats had a total food consumption intake (Day 9 -20 of gestation) of 243.0 ± 31.3 g (controls: 249.9 ± 43.7).

GROSS PATHOLOGY
The effects noted in gross pathology examinations are present in control and test animals in low frequencies and are therefore not considered as adverse. In detail, control and test animals revealed anomalies of the kidneys (pelvises dilated, firm nodules on surface, depressed area in cortex) and red focal areas in the lung. Further, one test animal each showed yellow areas on the liver surface, reddened/enlarged cervical lymph nodes and a thin and pale nonglandular mucosa and smooth glandular mucosa. One control animal had clear raised areas on the surface of eyes.

OTHER
The pregnancy rate was 100% in the 1000 mg/kg bw/day dose group and in control animals.
No alterations in the number of corpora lutea, implantations and mean implantation efficiencies (number of implantations per number of corpura lutea) and resorptions were observed .
Gravid and nongravid uterine weights were comparable between the control and dose group (table 1).

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No alterations in the number of live and dead fetuses were observed. Fetal viability, size and sex remained unchanged.
Anomalies determined in gross pathology, visceral and skeleton examinations were present in both, control and test animals and are therefore not considered as treatment-related effects (for details, see table 2).

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1: Ovarian, Uterine and Litter Data

Parameter	Control	1000 mg/kg bw/day
Mean uterine weights Gravid	69.48 ± 13.953	67.63 ± 11.91
Mean uterine weights Nongravid	6.52 ± 1.424^a	7.04 ± 1.982^a
Corpura lutea (mean number)	15.9	14.7
Implantations (number)	13.5	12.8
Mean implantation efficiency (indeces)	85.7^a	88^a
Resorptions (number)	1.2	0.6
Mean incidence of resorptions (%)	9.2^a	4.3^a
Dead fetuses (number)	0.0	0.0
Mean incidence of fetal mortality (%)	0.0^a	0.0^a
Live fetuses (number)	12.4 ± 2.56	12.2 ±1.91
Mean incidence of fetal viability (%)	90.8^a	95.7^a

Incidences were calculated on a per litter basis.

Data are shown as means.

a: Data analysed following rank transformation

 

Table 2: Examination of the fetuses

Parameter	Control	1000 mg/kg bw/day
Live fetuses
Males
Mean body weight (g)	3.58 ± 0.201^b	3.46 ± 0.264^b
Mean crown-rump distance (cm)	3.8 ± 0.15^b	3.8 ± 0.19^b
Females
Mean body weight (g)	3.39 ± 0.186^b	3.32 ± 0.248^b
Mean crown-rump distance (cm)	3.7 ± 0.14^b	3.7 ± 0.18^b
Males per litter (Mean %)	54.0 ± 18.31	47.4 ± 13.62
Gross pathology findings:
Number of fetuses examined	191	175
Number of fetuses appearing normal	169	166
Skull	0	0
Palate	0	0
Cleft palate	0	0
Heart	0	0
Kidney	0	0
Ureter	0	0
Dilated	16	7
Hydroureter	0	1
Undulated	2	1
Umbilical hernia	1	0
Situs inversus	1	0
Hindleg	0	0
Small	1	0
Exencephaly	0	0
Tail missing	0	0
Mean incidence values for visceral findings
Number of litters	22	22
Number with anomalies	0^a	0.05 ± 0.213^a
Incidence of anomalies (%)^c	0^a	1 ± 5.3^a
Number with variants	0.1 ± 0.29^a	0.1 ± 0.29^a
Incidence of variants (%)^d	3 ± 9.7^a	3 ± 11.7^a
Mean incidence values for skeletal findings
Number of litters	22	22
Number with anomalies	0^a	0^a
Incidence of anomalies (%)^e	0^a	0^a
Number with variants	3 ± 2.3^a	3 ± 2.2^a
Incidence of variants (%)^f	39 ± 25.2^a	31 ± 24.6^a
Visceral Findings
Number of fetuses examined/number of litters	81/22	80/22
Number of fetuses with anomalies/number of litters with anomalies	0/0	1/1
Number of anomalies	0	1
Hydroureter	0	1
Number of fetuses with variants/number of litters with anomalies	2/2	2/2
Number of variants	2	3
Dilated renal pelves	2	1
Dilated ureters	0	2
Fetus small	0	0
Small tongue	0	0
Skeletal Findings
Number of fetuses examined/number of litters	191/22	175/22
Number of fetuses with anomalies/number oflitters with anomalies	0/0	0/0
Number of anomalies	0	0
Number of fetuses with variants/number of litters with variants	73/19	57/18
Number of variants	110	89
Skull:
Intraparietal-incomplete ossification/nonossified	21	20
Supraoccipital-incomplete ossification/nonossified	14	11
Hyoid	25	22
Supraoccipital - nonfused	0	2
Supraoccipital - lopsided	0	0
Supraoccipital - small	0	0
Small	0	0
Nasal bones appear short	0	0
Rib cage:
Parletals – irregular edges	2	4
Ribs – angulated	2	1
Ribs – 13^thpair small	1	0
Ribs – first pair small	0	0
Ribs – forked	0	0
Vertebrae:
Centra – incomplete ossification / nonossified	32	19
Centra – nonfused	10	5
Centra small	0	0
Centra – missing	0	0
Sternabrae – bipartite	1	0
Hemivertebrae	0	0
Vertebrae – less than 3 ossified	0	0
No of vertebrae below 3^rdsacral	0	0
Sacral vertebrae – nonossified	0	0
Halaligned centra ans arches / caudals	0	0
Caudals – less than 3 ossified	0	1
Caudals – incomplete ossification	0	0
Arches – nonossified	0	0
Pelvis:
Pelvic girdle – incomplete ossification	1	2
Pelvic girdle – small	0	0
Pubis – incomplete ossification/nonossified	1	1
Pubis – small	0	0
Ischium – incomplete ossification / nonossified	0	0

Data are shown as mean ± standard deviations.

^a: Incidences were calculated on a per litter basis.

^b: Data analysed following rank transformation

^c: Mean Incidence of Visceral Anomalies (%) – Group mean of ((number of fetuses with anomalies per litter/number of fetuses examined viscerally per litter)x100)

^d: Mean Incidence of Visceral Variance (%) – Group mean of ((number of fetuses with variants per litter/number of fetuses examined viscerally per litter)x100)

^e: Mean Incidence of Skeletal Anomalies (%) – Group mean of ((number of fetuses with anomalies per litter/number of fetuses examined skeletally per litter)x100)

^f: Mean Incidence of Skeletall Variance (%) – Group mean of ((number of fetuses with variants per litter/number of fetuses examined skeletally per litter)x100)

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The available information comprises adequate, reliable studies (Klimisch score 2) and consistent studies from reference substances with similar structure and intrinsic properties. Read-across is justified based on a common functional group, common precursors/breakdown products and similarities in toxicological properties (refer to endpoint discussion for further details). The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.

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

Human health effects on toxicity to reproduction are predicted from adequate and reliable data for source substances by read-across to the target substance within the group applying the group concept in accordance with Annex XI, Item 1.5, of Regulation (EC) No 1907/2006.

Fatty acids are found in all living organisms fulfilling three fundamental roles. Besides their function as part of molecules like phospholipids and glycolipids important for the cell-structure, they are often precursors of signalling molecules such as prostanoids. The third and best understood role of fatty acids is their role as nutritional energy source.

Based on this, no developmental toxicity/teratogenicity is expected for fatty acids which is demonstrated by studies with C7 fatty acid (heptanoic acid), C8 fatty acid (octanoic acid), C9 fatty acid (nonanoic acid and azelaic acid), C18:2 unsaturated fatty acid (linoleic acid and conjugated linoleic acid), C22 fatty acid (docosanoic acid) and fatty acids, tall oil, respectively.

In prenatal developmental toxicity studies similar to OECD Guideline 414, groups of 22 pregnant Crl:COBS, CD® (SD) BR rats were administered via gavage 1000 mg/kg bw/day of heptanoic acid (CAS# 111-14-8) or 1500 mg/kg bw/day of nonanoic acid (CAS# 112-05-0) from day 6 through 15 of gestation (Serota et al., 1983). A further group of 22 animals served as control group and received the vehicle corn oil.

Survival rate, body weight gain, food and water consumption were comparable between treated and control groups. Gross pathology findings were considered to be incidental in nature and showed no relationship to treatment. In addition, gravid uterus weights, the mean number of corpora lutea and implantations, mean implantation efficiency and mean incidence of resorptions revealed no evidence of maternal toxicity both in rats given heptanoic acid and nonanoic acid.

No alterations in the number of live and dead fetuses were observed. Foetal viability, size and sex remained unchanged in both groups receiving either heptanoic or nonanoic acid. In the heptanoic acid group, anomalies determined in gross pathology, visceral and skeleton examinations of pups were present in both control and treated animals and are therefore not considered as treatment-related effects. In the nonanoic group, a higher mean incidence of visceral anomalies was noted due to a cleft palate observed in two fetuses from the same litter. However, this increase was not statistically significant. Thus, heptanoic acid and nonanoic acid are not considered to induce maternal toxicity, developmental toxicity or teratogenicity in a concentration up to and including 1000 mg/kg bw/day and 1500 mg/kg bw/day, respectively.

The heptanoic acid and nonanoic acid doses applied in the prenatal developmental toxicity study by Serota et al. (1983) were determined in previous dose-range finding studies (Serota et al., 1982). In these so called maternal tolerance studies, pregnant rats (6 females per dose) were exposed to 100, 500 and 1000 mg heptanoic acid/kg bw/day and to 200, 1000 and 2000 mg nonanoic acid/kg bw/day from gestational day 6 – 15, respectively. Criteria evaluated were mortality, clinical signs, body weights, food and water consumption and after sacrifice on gestational day 16 gross pathology, and ovarian and uterine data.

No adverse effects in groups dosed with heptanoic acid were noted compared to control. An apparent treatment related effect for nonanoic acid was a non-increase of body weight on day 9 compared to day 6 in the 2000 mg/kg bw/day group. However, the mean body weight change from gestation day 6-15 for the high-dose group was comparable to the control animals. No alterations in the number of implantations or resorptions, nor in the number of live and dead foetuses were observed between the control and the groups treated with heptanoic acid and nonanoic acid, respectively.

 

Moreover a reproduction / developmental toxicity screening test performed equivalently to OECD 421 is available for heptanoic acid (CAS# 111-14-8) (Hoberman, 1990). 10 female Sprague-Dawley rats were orally treated with 200, 1000 or 2000 mg/kg bw/day of the test substance dissolved in corn oil. Control animals received the vehicle only. Females were treated 7 days prior to mating and afterwards during mating, gestation and lactation until day 4 post-parturition. Male rats were used only as breeders (10 animals/group), and were not given the test substance.

The offspring was examined for numbers, viability, body weights, sex ratios and external morphology of the pups. Delivered pups were additionally observed for viability, clinical observations and body weights during a four-day post-parturition period. Afterwards, they were sacrificed and subjected to post-mortem examination of malformations and gross lesions of the thoracic and abdominal viscera.

In the offspring, there were no adverse clinical signs and no effects on viability and body weights. Gross pathology revealed no malformations or gross lesions up to and including the highest dose tested. Based on these results the NOAEL for developmental toxicity was determined to be ≥ 2000 mg/kg bw/day.

The toxicity to reproduction of docosanoic acid (CAS# 112-85-6) was evaluated in a combined repeated dose and reproductive/developmental toxicity screening test performed under GLP according to OECD guideline 422 (Nagao et al., 2002). Groups of 13 male and 13 female Sprague-Dawley rats received daily doses of 100, 300 and 1000 mg/kg bw of docosanoic acid by gavage. While the males were treated for 42 days, the females received the test substance from 14 days prior to mating until day 3 of lactation. Since the viability, the body weights, and the results of pathological examination of the pups did not reveal any differences between the dose groups and the control groups, there was no indication for any adverse effects on development. Thus, the NOAEL for developmental toxicity and teratogenicity was ≥ 1000 mg/kg bw/day based on the absence of treatment-related effects.

In a two-generation reproduction toxicity study similar to OECD Guideline 416 Sprague-Dawley rats (30 females per group and 15 males per group) were administered 5% and 10% fatty acids, tall oil (CAS# 61790-12-3) in the diet (corresponding to 2500 and 5000 mg/kg bw/day) (Pine Chemical Association, 2004). The parental animals were fed the test substance starting at 20 days pre-mating until the weaning of the first generation (F1). After weaning, 20 F1 males and 20 F1 females per group were maintained on the parental diet and mated at 100 days of age. The delivered F2 pups were followed until weaning. Haematology, serum chemistry, urinalysis, and organ weights for F1 animals, gross pathology of F1 and F2 animals and microscopic pathology of various organs of the F2 pups were performed.

There were no treatment-related effects on the number of liveborn or still-born F1 litters and pups, or weaning weight of the F1 pups. In the pups no changes in haematology, clinical chemistry and urinalysis parameters and organ weights were found and gross and microscopic pathology revealed no treatment-related effects. Thus, the NOAEL for developmental toxicity of fatty acids, tall oil was ≥ 5000 mg/kg bw/day.

There are several publications available, which also point out, that fatty acids do not have properties of inducing developmental toxicity or teratogenicity. In general, the developmental studies reported in these publications were not performed according/similar to current guidelines.

The effect of octanoic acid (CAS# 124-07-2) on foetal development was analysed in a developmental screening study with a Chernoff/Kavlock assay (Narotsky et al., 1994). Groups of 16 Sprague-Dawley rats received a daily dosage of 1125 and 1500 mg/kg bw/day in corn oil on gestation days 6 - 15 via gavage. The dose levels were based on preliminary range-finding studies with nongravid female rats. Maternal body weights were determined and the animals were observed for clinical signs throughout the experimental period ending on postnatal day 6. Uterine implantation sites were recorded. Pups in each litter were examined externally, counted and weighed. 2 pups per litter were selected on postnatal day 6 for skeletal examinations.

A maternal mortality rate of 31% and 44% was found in the low- and high-dose group. Most of the deaths occurred shortly after dosing and were attributed to the respiratory effect of the treatment. However, the high percentage of deaths could have been also due to tracheal intubation according to the authors. Maternal toxicity like rale and dyspnoea was noted in 95% of the test animals and reduced weight gain was observed in the animals of both dose groups taken into account gestation day 6 – 20. Decreased progeny viability on post-gestational day 6 occurred in dams with peripartum respiratory symptoms of the high-dose group, while all pups gained weight and no malformations including lumbar ribs were found. Based on these findings, the NOAEL for teratogenicity was determined to be ≥ 1500 mg/kg bw/day, whereas the NOAEL for developmental toxicity was considered to be 1125 mg/kg bw/day and the NOAEL for maternal toxicity was found to be below 1125 mg/kg bw/day.

Developmental toxicity of octanoic acid (CAS# 124-07-2) was also evaluated in a published study, where 12 Sprague-Dawley rats received a single dose of 18.75 mmol/kg bw by gavage on day 12 of gestation (Scott et al., 1994). As a result of the dosing, severe maternal toxicity was noted, but not further specified. The analysis of foetuses was performed on day 20 of gestation. In addition, the content of octanoic acid in foetuses and in maternal fluid was analysed 0.25, 0.5, 1, 2, 4, 8, and 24 hours after dosing. According to the authors the observed maternal toxicity was the reason for the slight reduction in the foetal body weight when compared to control values. However, no significant differences to control values were found regarding number of living foetuses, number of implantation sites, number of resorptions and number of malformations, respectively. Thus, the NOAEL for developmental toxicity was found to be 18.75 mmol/kg bw (total dose) corresponding to 2704 mg octanoic acid /kg bw/day.

In another study which is insufficient for assessment due to limited documentation, octanoic acid (CAS# 124-07-2) was injected subcutaneously once to mice at a dose of 600 mg/kg bw/day on day 8 of gestation (Nau and Loescher, 1986). According to the authors, no significant differences compared to control were found for octanoic acid. No further information was provided.

Developmental toxicity and teratogenicity of azelaic acid(CAS# 123-99-9) were investigated in rats and rabbits by Mingrone et al. (1983). One group of female rats and female rabbits was administered azelaic acid via feed (rat: 140 mg/kg bw/day; rabbit: 200 mg/kg bw/day) during pregnancy. Rats were sacrificed on gestation day 19 and rabbits on gestation day 25, respectively. No abnormalities were found in either the macro- or microscopic appearance of uteri, placentae and ovaries and no abortions or foetal malformations were detected in both species. The number and the weight of the foetuses were comparable to the control groups.

In a second group of female rabbits and female rats, the treatment with azelaic acid was extended for a period of 3 months, including the period of suckling before sacrifice. No significant differences were noted in the number or weight of animals born from mothers fed with azelaic diet. No still-born animals were found and new-born animals did not present any malformations. Thus a NOAEL for developmental toxicity and teratogenicity of ≥ 140 mg/kg bw/day for rats and ≥ 200 mg/kg bw/day for rabbits was determined, respectively.

Linoleic acid (CAS# 60-33-3) was evaluated in a Chernoff/Kavlock developmental toxicity screening test among 55 other test substances (Seidenberg and Becker, 1987). 26 - 30 timed-pregnant mice were administered by gavage linoleic acid on gestation days 8 – 12. The concentration was not specified in the publication, however, in general a single minimally toxic dose was used or if test substances did not produce toxicity in a preliminary range-finding study, the animals were exposed to a dose level of 10 g/kg bw/day. Since no statistically significant difference was seen in the parameters examined (average number of live neoantes per litter, average number of dead neonates per litter, average neonatal body weight) in comparison to the control group, linoleic acid was designated as nonteratogen/nonembryotoxin.

The effect of conjugated linoleic acid (CLA) on neonatal growth and development in Fischer rats was examined in two experiments by Chin et al. (1994).

In the first experiment rats were either fed a basal diet (control group) or basal diet supplemented with 0.5% CLA (corresponding to 250 mg/kg bw/day) throughout gestation (~ 20 dams per group). On day 20 of gestation, 10 rats of the treatment group and 9 rats of the control group were sacrificed and liver, mammary gland, skeletal muscle and abdominal adipose tissue were collected. Fetuses were removed, weighed and examined grossly for abnormalities. 11 further dams of the control group and 13 further dams in the CLA-fed group were allowed to deliver at term.

The CLA consumption during gestation had no effects on maternal food intake, body weight and mammary gland or liver weight. There were no significant differences in litter sizes, foetal body weights, foetal liver and brain weights between the control and CLA treated group on day 20 of gestation. Additionally, no gross visible foetal abnormality was observed. In tissues of maternal and foetal liver, maternal muscle and mammary gland, the amount of CLA was increased significantly over control levels.

Milk collection on day 10 of lactation revealed, that CLA was incorporated into milk fat of dams fed the CLA-supplemented diet. Pups receiving CLA during gestation and lactation showed significant higher mean pup weights on day 10 of lactation than pups from control dams (P < 0.036).

In the second experiment (Chin et al., 1994) Fischer rats received either a basal diet (control group) or basal diet supplemented with 0.25% or 0.5% CLA (corresponding to 125 and 250 mg/kg bw/day) throughout gestation and lactation. A further group received basal diet during gestation followed by 0.5% CLA-supplemented basal diet during lactation. Pups were weaned on day 22 of lactation and fed for 8 (males) and 10 (females) weeks, respectively, the same diet their mothers had received. The concentration of CLA in maternal milk increased in proportion to the dietary CLA level. Litter size, pup development and survival rate were not affected in any of the treatment groups. However, pup body weights were significantly increased on day 10 of lactation in the 0.5% CLA-supplemented diet group and in pups fed 0.25 and 0.5% CLA for 8 to 10 weeks of age compared to control group. Although food intake was not affected by CLA treatment for the time after weaning to 8 and 10 weeks of age, the feed efficiency was significantly increased in male pups fed 0.5% CLA and in female pups fed 0.25 and 0.5% CLA.

Based on the result of these two experiments, the NOAEL for maternal and developmental toxicity was ≥ 0.5% CLA (corresponding to ≥ 250 mg/kg bw/day).

Moreover, the effect of conjugated linoleic acid (CLA; composition: 42.6% cis-9, trans-11 CLA, 45.6% trans-10, cis-12-CLA and 8.7% other CLA isomers) on body weight gain and body composition during pre- and postnatal period was investigated in rats by Poulos et al. (2001). Sprague-Dawley rats were fed either a basal diet or the basal diet supplemented with 0.5% CLA (corresponding to 250 mg/kg bw/day) from gestation day 7 until lactation day 21. Litters were normalized to 6 pups (3 per sex) within 24 hours of birth. At weaning (day 21), one male and female pup of each litter were sacrificed and inguinal fat pads removed. The remaining progeny were assigned to control or CLA supplemented diet (each 1 male and female pup per litter) and fed until 11 weeks of age.

Maternal body weights during gestation and lactation, feed intake and liver weight of dams were not affected by CLA treatment. There were no differences in the litter size between CLA and control group. At weaning (day 21), female pups exposed to CLA were significantly heavier than female pups of dams fed control diet. Male pup weight was not influenced by CLA treatment at weaning, although males exposed to CLA throughout gestation, lactation and post-weaning were the heaviest, fastest growing and most feed efficient of all treatment groups. Dam’s diet did not alter average daily body weight gain or feed intake in either male or female pups. CLA had no effect on liver weight in either male or female pups. The main long-term effect of the dam’s diet was the significantly heavier soleus and gastrocnemius muscles (P < 0.01) and increased tail lengths (control: 18.01 cm; CLA: 18.65 cm), which was an indication of skeletal growth, of male pups whose dams were fed CLA.

The NOAEL for developmental toxicity was considered to be ≥ 250 mg/kg bw/day based on the results of the study.

Since all the members of the category show similar structural and toxicological properties the results of individual members are also valid for other fatty acids within the category. Moreover, a substance-specific adjustment of the NOAEL for the individual members of the category is not required. An overall NOAEL for all fatty acids within the category of 1000 mg/kg bw/day is chosen as “worst case” among the available key studies. In addition, heptanoic acid and docosanoic acid, which elicit a NOAEL for developmental toxicity and teratogenicity of ≥ 1000 mg/kg bw/day are representing category members with a low and the highest molecular weight.

In conclusion, the available data do not provide any evidence of developmental toxicity/teratogenicity of fatty acids.

References:

Chin, S.F. at al (1994). Conjugated Linoleic Acid Is a Growth Factor for Rats as Shown by Enhanced Weight

Gain and Improved Feed Efficiency. J. Nutr. 124 (12): 2344-2349.

Narotsky, M.G. et al. (1994). Developmental Toxicity and Structure-Activity Relationships of Aliphatic Acids, Including Dose-Response Assessment of Valproic Acid in Mice and Rats. Fundamental and Applied Toxicology 22(2):251-265.

Nau, H. and Löscher, W. (1986). Pharmacologic evaluation of various metabolites and analogues of valproic acid: teratogenic potencies in mice. Fundam Appl Toxicol, May, 6(4): 669-676.

Mingrone, G. et al (1983). Toxicity of azelaic acid. Drugs Exptl. Clin. Res. 9(6): 447-455.

Scott, W.J. Jr et al. (1994). Pharmacokinetic Determinants of Embryotoxicity in Rats Associated with Organic Acids. Environ Health Perspect, 102, Suppl 11: 97-101.

Seidenberg, J.M. and Becker, R.A. (1987) A Summary of the Results of 55 Chemicals Screened for Developmental Toxicity in Mice. Teratogenesis, carcinogenesis, and mutagenesis 7(1): 17-28.

Poulos, S.P. et al. (2001). Pre- and Postnatal Dietary Conjugated Linoleic Acid Alters Adipose Development, Body Weight Gain and Body Composition in Sprague-Dawley Rats. J. Nutr. 131(10): 2722-2731.

 

Justification for selection of Effect on developmental toxicity: via oral route:
Hazard assessment is conducted by means of read-across based on a category approach. The selected study is the most adequate and reliable study based on overall assessment of duration and dose-descriptor level (refer to the endpoint discussion for further details).

Justification for classification or non-classification

All available data on toxicity on reproduction of the members of the fatty acids category do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.

No information is available on effects via lactation.