Didodecyl fumarate

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:

experimental study

Adequacy of study:

key study

Study period:

2 May 2013 - 26 Nov 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline Study.

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Han-Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 328 to 395 g; Females: 217 to 255 g
- Fasting period before study: no
- Housing: In groups of three to five in Makrolon type-4 cages with wire mesh tops during acclimatization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (ISO-BLOX from Harlan Laboratories B.V. / Netherlands). During the prepairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet (e.g. ad libitum): Pelleted standard Harlan Teklad 2018C (batch nos. 43/12 and 56/12) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland), ad libitum.
- Water (e.g. ad libitum): Community tap-water from Füllinsdorf in water bottles, ad libitum
- Acclimation period: minimum 5 days; under test conditions after health examination; only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared daily using the test item as supplied by the Sponsor. Didodecyl fumarate was weighed into a glass beaker on a tared precision balance and 80 - 90% of the warmed-up (40 ± 5 °C) vehicle was added (w/v) with a syringe in small amounts under continuously stirring. The dose formulation was heated on approx. 50 °C for approx. 20 minutes and then the remaining warmed-up vehicle was added. Each dose formulation was homogenized with an Ultraturrax and stirred again for approx. 20 minutes at 37 - 40 °C. Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period by stirring at 37 - 40 °C temperature.
Dose Volume: 5 mL/kg bw
Dose Concentration: Group 1: 0 mg/mL; Group 2: 20 mg/mL; Group 3: 60 mg/mL; Group 4: 200 mg/mL

VEHICLE
- Source: Carl Roth GmbH
- Lot/batch no.: 103197718

Details on mating procedure:

During the pairing period, females were housed with sexually mature males from the same dose group (1:1) until evidence of copulation was observed. The females were removed and housed individually if:
- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.
The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.
If a female did not mate during the 14-day pairing period, a second pairing of this female with a male in the same group, which had already mated successfully, was considered. If mating was not recorded during this additional pairing period of a maximum of 14 days, the female was sacrificed and, if indicated, the reproductive organs examined histopathologically in order to ascertain the reason for the infertility.
All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

In the first week of dose formulation day a sample from the control group as well as three samples (top, middle and bottom) of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Due to high variation of the analytical homogeneity results, additional samples were taken in the first and second week from remaining formulations to evaluate optimized analytical conditions for sample workup and derivatization. These samples were analysed but results were not reported. Samples of each test item concentration were taken from the middle to confirm the stability (4 hours at room temperature 15 - 25 °C). Towards the end of the study, samples were taken from the middle to confirm concentration.
Since the dose formulation became not homogenous anymore at low temperature, samples of the exact amount of dose formulation were drawn and the entire sample was analyzed:
Groups 1 and 2: 500 mg dose formulation
Group 3: 250 mg dose formulation
Group 4: 100 mg dose formulation
The samples were analyzed by GC-FID. The test item was used as the analytical standard.

Duration of treatment / exposure:

Males: 48 days
Females: approx. 6 weeks

Frequency of treatment:

daily

Details on study schedule:

The study schedule can be found in "Any other information on materials and methods incl. tables".

Remarks:

Doses / Concentrations:
100, 300, 1000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

Termination of the Study
Males were sacrificed after treatment of at least 48 days, when no longer needed for the assessment of reproductive effects. Pups were sacrificed on day 4 post partum. Dams were sacrificed on day 5 post partum.
If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
The following observations were recorded:
Viability / Mortality: Twice daily
Clinical Signs: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

DETAILED CLINICAL OBSERVATIONS: Yes
Once prior to the first administration of the test item and weekly thereafter (in the gestation period on day 0, 6, 13 and 20 post coitum), detailed clinical observations were performed outside the home cage in a standard arena. Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Any changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.

BODY WEIGHT: Yes
- Time schedule for examinations: Body Weights: Recorded daily from treatment start to day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food Consumption: Males: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; after pairing period weekly. Females: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; gestation days 0 - 7, 7 - 14 and 14 - 21 and days 1 - 4 of the lactation. No food consumption was recorded during the pairing period.

WATER CONSUMPTION AND COMPOUND INTAKE: No

Litter observations:

PARAMETERS EXAMINED
The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.

GROSS EXAMINATION OF DEAD PUPS:
Dead pups, except those excessively cannibalized, were examined macroscopically. All pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
All animals sacrificed were subjected to a detailed macroscopic examination. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. At the scheduled sacrifice, all animals were weighed and sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated. All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred. For the parent animals, special attention was directed at the organs of the reproductive system. The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

HISTOPATHOLOGY: Yes
Tissue Preservation
The following tissues from all parental males were preserved in neutral phosphate buffered 4% formaldehyde solution: Prostate, Seminal vesicles with coagulating gland, Testes (in modified Davidson Solution), Epididymides (in modified Davidson Solution).
The following tissues from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution: Ovaries (with oviduct) Uterus (with vagina).
In addition, from all males and females the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution: Gross lesions, Brain, Spinal chord (cervical, thoracic, lumbar), Small and large intestines1 (incl. Peyer’s patches), Stomach (forestomach and glandular stomach), Liver, Kidneys, Adrenals, Spleen, Lymph nodes (axillary and mesenteric), Aorta2, Eyes with optic nerve and harderian gland2, Lacrimal gland2, Larynx2, Nasal cavity2, Esophagus2, Heart, Thymus, Thyroids, and parathyroids if possible, Trachea and lungs (preserved by inflation with fixative and then immersion), Pituitary gland2, Urinary bladder, Peripheral nerve (sciatic), Bone marrow (femur), Femur with knee joint2, Mammary gland (male and female)2, Pancreas2, Salivary glands – mandibular, sublingual2, Skeletal muscle2, Sternum with bone marrow2, Pharynx2.
(1 = Duodenum, jejunum, ileum, colon, caecum, rectum; 2 = Only examined by histopathology in case of macroscopic findings indicative of potential toxicity)

Histotechnique
All organ and tissue samples to be examined by the study pathologist were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin.

Histopathology
Macroscopical findings, testes, epididymides, prostate, seminal vesicles, ovaries, oviduct, vagina and uterus from all animals of the control and high dose group were examined. The same applied to all occurring gross lesions and to all animals, which died spontaneously or had to be terminated in extremis. The remaining organs/tissues of 5 randomly selected males and females of the control and high dose group, respectively, were examined histopathologically. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure. Since test item-related morphologic changes were detected in the liver at the high dose, these organs from the mid- and low-dose group were examined to establish a no-effect level. Histological examination of ovaries was carried out on any females that did not give birth. A histopathology peer review was performed.

Postmortem examinations (offspring):

GROSS PATHOLOGY: Yes
Dead pups, except those excessively cannibalized, were examined macroscopically. All pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.

HISTOPATHOLOGY: No

Statistics:

The following statistical methods were used to analyze food consumption, body and organ weights, grip strength, rectal temperature, clinical laboratory and reproduction data, locomotor activity and macroscopical findings:
• Means and standard deviations of various data were calculated and included in the report.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were minor morphological alterations in the liver of males is considered as adaptive in nature, within physiological limits and not a manifestation of frank toxicity.

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no unplanned deaths. All animals survived until scheduled necropsy. No test item-related clinical signs were noted in males and in females treated at 100, 300 and 1000 mg/kg bw/day. The findings noted during the detailed weekly clinical observations of males and females did not indicate any test item-related effects.

BODY WEIGHT AND WEIGHT GAIN
Males: There were no effects on mean body weight gain and mean body weights at any dose level and in any study phase. Statistically significant differences in body weight gain occurred on several occasions in males, but were either not dose-dependent or represented slightly higher body weight gains. Therefore, these differences were considered to be fortuitous. The overall differences in mean body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were: +11%, +12%, +12% and +10% during the pre-pairing period, +6%, +7%, +6% and +7% during the pairing period and +3%, +3%, +4% and +3% during the after pairing period (percentages refer to the body weight gain within the period).
Females: There were no effects on mean body weight gain and mean body weights at any dose level and in any study phase. The overall differences in mean body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were: +6%, +6%, +7% and +7% during the pre-pairing period, +54%, +57%, +57% and +57% during the gestation period and +4%, +4%, +4% and +4% during the lactation period (percentages refer to the body weight gain within the period).

FOOD CONSUMPTION AND COMPOUND INTAKE
Males: There were no test item-related effects on mean food consumption in males at any dose level and in any study phase. At 1000 mg/kg be/day, food consumption was slightly higher between day 8 and day 20 of the after pairing period (approximately 10%). Since the differences were minor, they were considered not to be toxicologically relevant.
Females: There were no effects on mean food consumption in females at any dose level and in any study phase.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
All females mated within the first pairing period. For one female in the dose group 1000 mg/kg bw/d, mating was not detected. The median and mean precoital times were unaffected by treatment with the test item. Mean precoital times were 2.9, 1.8, 2.9 and 2.6 days in groups 1, 2, 3 and 4, respectively. The median precoital time was 3, 2, 2 and 2 days in order of ascending dose level. Two, one and one female in the control group and at 300 and 1000 mg/kg bw/day, respectively, were not pregnant. As a result the fertility index was 83.3%, 100%, 91.7% and 91.7% in order of ascending dose level.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In males at 1000 mg/kg bw/day, absolute and relative liver weights were statistically significantly increased. The value relative to body weight was in the range of the historical control data (2.39-3.56 g). Due to the observed morphological alterations in the liver, the increase was considered to be test item related but not adverse. Higher kidney values occurred in males at 100 and 1000 mg/kg bw/day. These differences were considered to be incidental since there was no dose-dependency. Further statistically significant differences comprised only derived relative weights or organ to brain ratios and were not accompanied by histopathological findings. No effects were observed at other dose levels or in females at any dose level.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no test item-related findings noted at necropsy in males and females. All gross lesions recorded were considered to be within the range of normal background alterations and showed no dose dependency.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were minor morphological alterations in the liver of males. This consisted of a minimal degree of diffuse, midzonal/centrilobular hepatocellular hypertrophy recorded in 3/5 rats at 1000 mg/kg bw/day. This finding may be considered as adaptive in nature, within physiological limits and not a manifestation of frank toxicity.

OTHER FINDINGS (PARENTAL ANIMALS)
- Corpora Lutea Count: Mean number of corpora lutea per dam (determined at necropsy) was similar in all groups (17.7, 16.8, 15.3 and 16.7 in order of ascending dose level) and gave no indication of a test item-related effect.
- Duration of Gestation: The mean duration of gestation was unaffected by exposure to the test item. Mean duration of gestation was 21.4, 21.7, 21.5 and 21.4 days, in order of ascending dose level.
- Implantation Rate and Post-Implantation Loss: No effects on implantation rate or post-implantation loss were observed at any dose level. The mean number of implantations per dam was 14.7, 14.6, 14.0 and 15.0 in order of ascending dose levels. The mean incidence of post-implantation loss as a percentage of total implantations was 9.5%, 9.1%, 12.3% and 11.5%, in order of ascending dose level.

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects were observed up to the highest dose tested (1000 mg/kg bw/d).

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY AND CLINICAL SIGNS (OFFSPRING)
No test item-related abnormal findings were noted at first litter check or during the first 4 days post partum at any dose level.

BODY WEIGHT (OFFSPRING)
Mean pup weights on day 0 and day 1 post partum were unaffected by treatment with the test item.

GROSS PATHOLOGY (OFFSPRING)
No test item-related findings were noted at macroscopic examination of F1 pups.

OTHER FINDINGS (OFFSPRING)
- Sex Ratios: Sex ratios at first litter check and on day 4 post partum were unaffected by exposure to the test item. The proportion of males at first litter check was 51%, 47%, 47% and 53%, in order of ascending dose level.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects were observed up to the highest dose tested (1000 mg/kg bw/d).

Reproductive effects observed:

not specified

Table 2: Summary of performance

P animals breeding for F1 litters
[mg/kg bw/d]	0	100	300	1000
Number of females paired	12	12	12	12
Number of females mated	12	12	12	12
Number of pregnant females (A)	10	12	11	11
Number of females with implantation sites only (B)	0	0	0	1
Number of females which reared their pups until day 4 post partum	10	12	11	10
Group 1 female nos. 52 and 54, group 3 female no. 84, group 4 female no. 88 were not pregnant. Group 4 female no. 96 lost its litter before first litter check was recorded and therefore only implantation sites were recorded.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

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 grouping of substances and read-across

The PFAE fumarates (Polyfunctional Aliphatic Ester) category consists of six members, which are either well-defined mono-constituent substances or related UVCB substances, with varying fatty alcohol chain lengths. The distinguishing feature of this category of chemicals is that its members are diester derivatives of fumaric acid (CAS 110-17-8). The alcohol moiety of the dicarboxylic esters generally falls in the C8-C22 carbon number range, including linear, even numbered alcohols. 

In order to avoid the need to test every substance for every endpoint, the category concept is applied for the assessment of environmental fate, environmental toxicity and human health hazards. Thus where applicable, environmental and human health effects are predicted from adequate and reliable data for source substance(s) within the group by inter- or extrapolation to the target substances in the group (read-across approach) applying the group concept in accordance with Annex XI, Item 1.5, of Regulation (EC) No 1907/2006. Structural similarities and similarities in properties and/or activities of the source and target substances in the category are the basis of read-across.

The available studies providing information on the human health hazard assessment within the PFAE fumarates category were conducted with the category member Didodecyl fumarate (CAS 2402-58-6). This substance was selected for testing, because it represents the category member with the shortest fatty alcohol side chain, and consequently with the lowest molecular weight, which is regarded as worst-case approach in terms of hazard assessment of the PFAE fumarates for the local as well as for systemic effects.

Furthermore, the category is supported by another polyfunctional aliphatic ester, namely Bis(2-ethylhexyl) adipate (CAS 103-23-1). This supporting chemical is used to cover toxicological endpoints, exclusively. The read across of Bis(2-ethylhexyl) adipate (CAS 103-23-1) to the PFAE fumarate category is justified due to the similar structural and physico-chemical properties, as well as their toxicological, and ecotoxicological profiles.

A detailed justification for the grouping of chemicals and read-across is provided in the technical dossier (see IUCLID Sections 7.1 and 13) and within Chapter 5.1 of the CSR.

Endpoint specific data matrix:

ID #	CAS	Toxicity to reproduction – Fertility
1	2402-58-6	Experimental result: NOAEL P (males/females) ≥ 1000 mg/kg bw/day RA: CAS 103-23-1
2	10341-03-4	RA: CAS 2402-58-6 RA: CAS 103-23-1
3	68610-90-2	RA: CAS 2402-58-6 RA: CAS 103-23-1
4	68921-51-7	RA: CAS 2402-58-6 RA: CAS 103-23-1
5	68921-52-8	RA: CAS 2402-58-6 RA: CAS 103-23-1
6	68921-53-9	RA: CAS 2402-58-6 RA: CAS 103-23-1
7	103-23-1 (a)	Experimental result: NOAEL P (males/females) ≥ 2102/2399 mg/kg bw/day

(a) Analogue substances are either chemicals forming part of a related category of structurally similar fatty acid esters or precursors/breakdown products of category members (i.e. alcohol and fatty acid moieties). Available data on these substances are used for assessment of toxicological properties by read-across on the same basis of structural similarity and/or mechanistic reasoning as described below for the present category. These substances are not subject to the REACh Phase-in registration deadline of 31 May 2013 and are indicated in normal font.

Toxicity to reproduction

Within the PFAE fumarate category a study is available assessing toxicity to reproduction which was conducted with Didodecyl fumarate (CAS 2402-58-6). Furthermore, there is a reliable study for the structurally related substance Bis(2-ethylhexyl) adipate (CAS 103-23-1)available.

CAS 2402-58-6

A Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test in the Han Wistar Rat is available (Senn, 2013). The study was conducted according to OECD test guideline 422 and under GLP conditions to investigate the toxicological effects resulting from repeated oral-gavage administration of the test item Didodecyl fumarate (CAS 2402-58-6). 12 animals per sex and dose were administered the test material in corn oil as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day. The controls received the vehicle only. Didodecyl fumarate was administered to male rats for 48 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post-partum. Neither clinical signs nor effects on mean food consumption, mean body weight, reproduction or breeding were noted at any dose level. Based on these results, the NOAEL (P) for reproduction was considered to be ≥ 1000 mg/kg body weight/day.

CAS 103-23-1

Toxicity to reproduction of Bis(2-ethylhexyl) adipate (CAS 103-23-1) has been investigated in a one-generation reproduction toxicity study similar to OECD 415.

The effect of Bis(2-ethylhexyl) adipate on the fertility of Alpk:APfSD (Wistar-derived) rats was investigated in a GLP-conform study similar to OECD guideline 415 (Tinston, 1988). Groups of 15 male and 30 female parental animals were exposed daily to the test substance at dietary concentrations of 300, 1800 or 12000 ppm, corresponding to mean achieved dose levels of 52, 178 and 2102 mg/kg bw/day for males and 61, 203 and 2399 mg/kg bw/day for females, respectively. A similar constituted group of animals received the plain diet and served as controls. After 10 weeks of treatment the animals were mated to produce a single litter (F1), which were reared until Day 36 post-partum. Male parent animals were killed after completion of mating and female parent animals were killed after weaning their litter. There was no evidence for any clear effects on bodyweight or food consumption during the premating phase of the study apart a marginal reduction in bodyweight gain for female rats in the 12000 ppm test group. This decrease in body weight continued through gestation in the female animals of the highest dose group compared to controls. There were no treatment-related effects on pre-coital interval, length of gestation, or on male and female fertility. Offspring weight gain, total litter weight and litter size in the 12000 ppm test group were reduced compared to controls, but there was no effect on the number of pups born live or on their survival at any dose level. An increase in absolute and relative liver weights was observed in both male and female parent animals receiving dietary levels of 12000 ppm. No treatment-related findings were observed at gross pathology, except for accentuated lobular pattern in the liver of two female rats fed diets containing 12000 ppm of the test substance. No histological changes were noted in the reproductive organs of those males and females which failed to breed and were thus suspected of being infertile. Based on the results of this study the NOAEL for fertility was set at ≥ 12000 ppm in the diet, which corresponds to mean achieved dose levels of 2102 and 2399 mg/kg bw/day in males and females, respectively. The NOAEL for systemic toxicity for parental animals (P) and offspring (F1) was considered to be at 1800 ppm, equivalent to dose levels of 178 and 203 mg/kg bw/day in males and females, respectively.

In summary, the NOAEL for fertility from the one-generation reproduction toxicity study of Bis(2-ethylhexyl) adipate was set at ≥ 12000 ppm in the diet, which corresponds to mean achieved dose levels of ≥ 2102 and ≥ 2399 mg/kg bw/day in males and females, respectively.

Conclusion for toxicity to reproduction

There is one study available which was conducted with Didodecyl fumarate (CAS 2402-58-6) addressing the endpoint toxicity to reproduction of dicarboxylic esters with fumaric acid. Furthermore, toxicity to reproduction of the analogue substance Bis(2-ethylhexyl) adipate (DEHA; CAS 103-23-1) was assessed in various studies.

In an OECD 422 study with Didodecyl fumarate (CAS 2402-58-6) in rats, a NOAEL of ≥ 1000 mg/kg bw/day was identified for the parental generation. No adverse effects on fertility were observed up to the highest dose tested in either males or females.

Adverse effects on fertility in females (prolonged estrous stage) have been observed with the analogue substance Bis(2-ethylhexyl) adipate (CAS 103-23-1) in a subacute repeated dose toxicity study according to OECD 407 (dose levels: 40, 200 and 1000 mg/kg bw/day) where findings at histopathology and in vaginal smears examinations revealed increased ovarian follicle atresia and prolongation of the estrous stage in 4/10 and 2/10 females at 1000 mg/kg bw/day, respectively. No substance-related effects were observed on sperm parameters and histopathology of reproductive organs in males (Miyata et al., 2006). In contrast, a one-generation toxicity study with the same test substance (Bis(2-ethylhexyl) adipate, CAS 103-23-1) did not result in any adverse effects on fertility up to a maximum dietary dose level of 12000 ppm, which corresponds to 2102 and 2399 mg/kg bw/day in males and females, respectively. 

In rats Bis(2-ethylhexyl) adipate (DEHA) is hydrolysed to adipic acid and 2-ethylhexanol, the latter of which is oxidized to ethylhexanoic acid (EHA) (Takahashi et al., 1981). As EHA is a known reproductive toxicant, read-across from Bis(2-ethylhexyl) adipate (DEHA) can be considered as a worst case approach for the assessment of reproductive/ developmental toxicity within the category. In female rats delayed estrous cycle has been observed when treated with EHA (Pennanen et al., 1993), an effect also observed in the subacute study with Bis(2-ethylhexyl) adipate. However, fertility was not affected in a one-generation study with Bis(2-ethylhexyl) adipate, the study with the longest treatment duration.

The overall NOAEL for reproduction toxicity was set at ≥ 1000 mg/kg bw/day, indicating no hazard for reproduction toxicity within the PFAE fumarate category.

A detailed reference list is provided in the technical dossier (see IUCLID, section 13) and within CSR.

Short description of key information:
No hazard for reproductive toxicity was identified for the members of the PFAE fumarate category.

Justification for selection of Effect on fertility via oral route:
GLP study supported by other studies in read across

Effects on developmental toxicity

Description of key information

No hazard for developmental toxicity was identified for the members of the PFAE fumarate category. 

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Study period:

15 Sep - 16 Oct 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - guideline study. In accordance to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Alpk:APfSD (Wistar derived)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF colony maintained at the Animal Breeding Unit, ICI Pharmaceuticals, Alderley Park, Cheshire, UK.
- Age at study initiation: 12 weeks
- Weight at study initiation: 218-278 g
- Housing: individually in stainless steel cages with absorbent paper over collecting trays.
- Diet (e.g. ad libitum): CTI diet, Special Diets Services Ltd., Essex, UK.
- Water (e.g. ad libitum): yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24
- Humidity (%): 44-70
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: From: 15 Sept - 16 Oct 1987.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The experimental diets were prepared in 30 kg batches from premixes.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical stability was observed at 300 ppm up to at least 32 days. This interval was in excess of the maximum period of use of the first batch of diet (21 days from preparation). The achieved concentration was within 8% of target and the doses received by the test groups were approximately 28, 170 and 1080 mg/kg bw/day.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: not reported
- Length of cohabitation: overnight
- Proof of pregnancy: sperm in vaginal smear referred to as day 1 of pregnancy

Successfully mated females were delivered to the experimental unit.
A total of 96 mated females was supplied over a two week period.

Duration of treatment / exposure:

days 1-22 of gestation.

Frequency of treatment:

Continuous feeding

Duration of test:

22 days

Remarks:

Doses / Concentrations:
0, 28, 170 and 1080 mg/kg bw/day
Basis:
other: approximately received doses (as given in study report)

Remarks:

Doses / Concentrations:
0, 300, 1800 and 12000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

24

Control animals:

yes, plain diet

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on arrival and daily

BODY WEIGHT: Yes
- Time schedule for examinations: daily during days 1-22 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 22
- Organs examined: uterus, ovaries, liver, spleen, kidney, stomach, rectum, abdominal cavity, pelvic cavity,

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, in each ovary.
- Number of implantations: Yes
- Number of early resorptions: Yes (early intra-uterine deaths)
- Number of late resorptions: Yes (late intra-uterine deaths)
- Other: Each foetus was weighed and individually identified within the litter by means of a cardboard tag. After weighing the foetuses were killed with an intra-cardiac injection of pentobarbitone.

Fetal examinations:

- External examinations and cleft palate: Yes, each foetus
- Soft tissue examinations: Yes, all
- Skeletal examinations: Yes, all
- Head examinations: Yes, the head of each foetus was cut along the fronto-parietal suture line and the brain was examined for macroscopic abnormalities.

Statistics:

Analysis of variance, Student's t-test, Fisher's Exact Test

Indices:

- Pre-implantation loss (No. of corpora lutea / No. of implantations)
- Post implantation loss (No. of implantations / No. of live foetuses)

Historical control data:

Yes, data on variants and frequency of occurence in rats of this strain were given.
Defects like bipartite 5th sternebrae, slightly dilated ureters and kinked ureters were seen in historical controls of this strain.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
At 12000 ppm a small but statistically significant reduction in maternal bodyweight gain was observed.
There was also a small but statistically significant reduction in food consumption at this dose level from days 2-18 inclusive of gestation.
There was no evidence of maternal toxicity at 300 or 1800 ppm.

Dose descriptor:

NOAEL

Effect level:

ca. 170 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOAEL

Effect level:

ca. 1 080 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

>= 1 080 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOEL

Effect level:

ca. 28 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

LOEL

Effect level:

ca. 170 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes. Remark: (non-adverse)

Details on embryotoxic / teratogenic effects:
There was no effect at any dose on foetal weight, litter weight, gravid uterus weight, numbers of intra-uterine deaths or numbers of external abnormalities. At 12000 ppm there was a minimal increase of pre-implantation loss with an associated decrease in litter size.
Six major abnormalities (in five foetuses) were seen in the treated groups and eight in the control group (of which seven consisted of multiple minor skull defects in one litter). There was no evidence that the type or distribution of these abnormalities was related to treatment with the test substance.
Overall, minor skeletal defects were increased in a dose-related manner at 1800 and 12000 ppm of the test substance, while skeletal variants (as a percentage of foetuses affected) were increased at the top dose only. These findings indicated slightly poorer ossification at dose levels of 1800 and 12000 ppm, which were considered to be the result of slight fetotoxicity. However, the slightly poorer ossification is not considered as adverse effect.

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1: Foetal defects and variants - Intergroup comparison of foetal defects and variants

	Dietary conc. of DEHA (ppm)
	0	300	1800	12000
No. of litters examined	24	23	24	23
External and visceral defects
No. of foetuses examined	282	263	278	243
No. of foetuses showing major defects	1	2	0	2
No. of foetuses showing minor defects only	7	8	9	3
Variants
No. of foetuses showing variants	69	69	81	78*
Skeletal defects
No. of foetuses examined	282	263	278	243
No. of foetuses showing major defects	7	0	1	1
No. of foetuses showing minor defects only	70	78	97**	120**
Variants
No. of foetuses showing variants	270	257	268	243**

 

Table 2: Summary of the type and incidence of major defects

	Dietary conc. of DEHA (ppm)
	0	300	1800	12000
External/Visceral
Situs inversus totalis	0	0	0	1
Left adrenal, kidney and ureter absent	1	0	0	0
Cysts attached to liver	0	1	0	0
Small right kidney	0	1	0	0
Umbilical hernia	0	0	0	1
Skeletal
Skull: Multiple minor defects	7	0	0	0
3^rdand 7^thribs (left) not ossified0	0	0	0	1
1^strib (right) partially ossified	0	0	1	0

 There was no evidence that the test substance is teratogenic to the rat at any of the dose levels tested (up to 12000 ppm -approximately 1000 mg/kg/day). Administration of 12000 ppm DEHA resulted in slight maternal toxicity and slight foetotoxicity.

At 1800 ppm, there was no evidence of maternal toxicity although minimal foetotoxicity was observed. A dietary level of 300 ppm DEHA was a clear no-effect level for embryonic development. 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

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 grouping of substances and read-across

The PFAE fumarates (Polyfunctional Aliphatic Ester) category consists of six members, which are either well-defined mono-constituent substances or related UVCB substances, with varying fatty alcohol chain lengths. The distinguishing feature of this category of chemicals is that its members are diester derivatives of fumaric acid (CAS 110-17-8). The alcohol moiety of the dicarboxylic esters generally falls in the C8-C22 carbon number range, including linear, even numbered alcohols. 

In order to avoid the need to test every substance for every endpoint, the category concept is applied for the assessment of environmental fate, environmental toxicity and human health hazards. Thus where applicable, environmental and human health effects are predicted from adequate and reliable data for source substance(s) within the group by inter- or extrapolation to the target substances in the group (read-across approach) applying the group concept in accordance with Annex XI, Item 1.5, of Regulation (EC) No 1907/2006. Structural similarities and similarities in properties and/or activities of the source and target substances in the category are the basis of read-across.

The available studies providing information on the human health hazard assessment within the PFAE fumarates category were conducted with the category member Didodecyl fumarate (CAS 2402-58-6). This substance was selected for testing, because it represents the category member with the shortest fatty alcohol side chain, and consequently with the lowest molecular weight, which is regarded as worst-case approach in terms of hazard assessment of the PFAE fumarates for the local as well as for systemic effects.

Furthermore, the category is supported by another polyfunctional aliphatic ester, namely Bis(2-ethylhexyl) adipate (CAS 103-23-1). This supporting chemical is used to cover toxicological endpoints, exclusively. The read across of Bis(2-ethylhexyl) adipate (CAS 103-23-1) to the PFAE fumarate category is justified due to the similar structural andphysico-chemical properties, as well as their toxicological, ecotoxicological profiles.

A detailed justification for the grouping of chemicals and read-across is provided in the technical dossier (see IUCLID Sections 7.1 and 13) and within Chapter 5.1 of the CSR.

Endpoint specific data matrix:

ID #	CAS	Toxicity to reproduction – Developmental Toxicity oral
1	2402-58-6	Experimental result: NOAEL developmental ≥1000 mg/kg bw/day NOAEL maternal ≥1000 mg/kg bw/day RA: CAS 103-23-1
2	10341-03-4	RA: CAS 2402-58-6 RA: CAS 103-23-1
3	68610-90-2	RA: CAS 2402-58-6 RA: CAS 103-23-1
4	68921-51-7	RA: CAS 2402-58-6 RA: CAS 103-23-1
5	68921-52-8	RA: CAS 2402-58-6 RA: CAS 103-23-1
6	68921-53-9	RA: CAS 2402-58-6 RA: CAS 103-23-1
7	103-23-1 (a)	Experimental result: NOAEL developmental ≥ 1080 mg/kg bw/day NOAEL maternal = 170 mg/kg bw/day

(a) Analogue substances are either chemicals forming part of a related category of structurally similar fatty acid esters or precursors/breakdown products of category members (i.e. alcohol and fatty acid moieties). Available data on these substances are used for assessment of toxicological properties by read-across on the same basis of structural similarity and/or mechanistic reasoning as described below for the present category. These substances are not subject to the REACh Phase-in registration deadline of 31 May 2013 and are indicated in normal font.

Developmental toxicity/teratogenicity

Within the PFAE fumarate category a study is available assessing developmental toxicity which was conducted with Didodecyl fumarate (CAS 2402-58-6). Furthermore, there is one reliable study on the structurally related substance Bis(2-ethylhexyl) adipate (CAS 103-23-1), which is used for read-across based on the analogue approach.

CAS 2402-58-6

A Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test in the Han Wistar Rat is available (Senn, 2013). The study was conducted according to OECD test guideline 422 and under GLP conditions to investigate the toxicological effects resulting from repeated oral-gavage administration of the test item Didodecyl fumarate (CAS 2402-58-6). 12 animals per sex and dose were administered the test material in corn oil as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day. The controls received the vehicle only. Didodecyl fumarate was administered to male rats for 48 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post-partum. Neither clinical signs nor effects on mean food consumption, mean body weight orpup developmentwere noted at any dose level. Based on these results, the NOAEL (P) for development was considered to be ≥ 1000 mg/kg body weight/day.

CAS 103-23-1

In a prenatal developmental toxicity study according to OECD guideline 414, the effects of Bis(2-ethylhexyl) adipate (CAS 103-23 -1) on mated female Alpk:APfSD (Wistar derived) rats were investigated during Days 1 to 22 of gestation (Hodge, 1988). Groups of 24 females received the test substance at dietary concentrations of 300, 1800 and 12000 ppm, which approximately corresponded to dose levels of 28, 170 and 1080 mg/kg bw/day. A further group of 24 mated females received the plain diet and served as controls. On Day 22 of gestation, dams were sacrificed and maternal as well as foetal examinations were performed. Maternal toxicity occurred at 12000 ppm and involved a small, but statistically significant decrease in body weight gain compared to controls. This effect was accompanied by slight, statistically significant reduction in food consumption between Days 2 to 18 of gestation. No treatment-related clinical signs were observed during the study and no adverse findings were noted at macroscopic examination of dams. There was no effect at any dose level on fetal weight, litter weight, gravid uterus weight, numbers of intra-uterine deaths or numbers of external abnormalities. At 12000 ppm, a minimal increase of pre-implantation loss associated with a decrease in litter size was observed. Six major abnormalities (in five fetuses) were seen in the treated groups and eight in the control group (of which seven consisted of multiple minor skull defects in one litter). There was no evidence that the type or distribution of these abnormalities was related to test substance treatment. Overall, minor skeletal defects were increased in a dose-related manner at 1800 and 12000 ppm, while skeletal variants (as a percentage of fetuses affected) were increased at 12000 ppm only. These findings indicated slightly poorer ossification at dose levels of 1800 and 12000 ppm. However, the slightly poorer ossification is not considered as an adverse effect. There was no evidence at any dose level, that the test substance was teratogenic in rats. 

Based on the results of the study, the NOEL for developmental toxicity in Alpk:APfSD (Wistar derived) rats was set at 300 ppm, which corresponded to approximately 28 mg/kg bw/day. The NOAEL for developmental toxicity in Alpk:APfSD (Wistar derived) rats was ≥ 12000 ppm, which is equivalent to ca. ≥ 1080 mg/kg bw/day. The NOAEL for maternal toxicity in Alpk:APfSD (Wistar derived) rats was 1800 ppm, which is equivalent to ca. 170 mg/kg bw/day.

Conclusion for developmental toxicity/teratogenicity

Members of the PFAE fumarate category are hydrolysed to fumaric acid and fatty alcohols (C8-C22). The alcohol and the dicarboxylic acids are expected to feed into metabolic pathways. There is one study available which was conducted with Didodecyl fumarate (CAS 2402-58-6) addressing the endpoint toxicity to reproduction of dicarboxylic esters with fumaric acid. No effects onpup developmentwere noted at any dose level. Based on these results, the NOAEL (P) for development was considered to be ≥ 1000 mg/kg body weight/day. Furthermore, one study investigating the developmental toxicity via the oral route is available for the analogue substance Bis(2-ethylhexyl) adipate (CAS 103-23-1, DEHA). Like fumaric acid diesters, DEHA is hydrolyzed to give the corresponding fatty acid and the alcohol, i.e. adipic acid and 2-ethylhexanol. The latter of which is oxidized to ethylhexanoic acid (EHA) which is a known to be a potent developmental toxicant. Therefore the read-across from Bis(2-ethylhexyl) adipate based on an analogue approach should be considered a worst case approach for the assessment of reproductive/ developmental toxicity within the PFAE fumarate category. Due to the absence of adverse effects, the NOAEL for developmental toxicity of Bis(2-ethylhexyl) adipate was set at ≥ 1080 mg/kg bw/day.

Therefore, no hazard for developmental toxicity was identified for the members of the PFAE fumarate category.

A detailed reference list is provided in the technical dossier (see IUCLID, section 13) and within CSR.

Justification for selection of Effect on developmental toxicity: via oral route:
Well performed study, used in read across

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the group concept is applied to the members of the PFAE fumarate category, data will be generated from representative reference substance(s) within the category to avoid unnecessary animal testing. Additionally, once the group concept is applied, substances will be classified and labelled on this basis.

Therefore, based on the group concept, all available data on toxicity to reproduction/developmental toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.