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Diss Factsheets

Administrative data

Description of key information

In conclusion, no human hazard for systemic toxicity after repeated oral exposure was identified for the PFAE fumarate category members.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
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: Pelleted standard Harlan Teklad 2018C (batch nos. 43/12 and 56/12) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland), ad libitum.
- Water: 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 oral 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


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

Mating, Gestation and Lactation
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.

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
Observations and examinations performed and frequency:
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.
Pup Data: 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.

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

OPHTHALMOSCOPIC EXAMINATION: No

HEMATOLOGY: Yes
Blood samples were obtained on the day before or on the day of the scheduled necropsy from 5 males randomly selected from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum. Blood samples were drawn sublingually from all animals under light isoflurane anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.
The following hematology parameters were determined:
- Complete Blood Cell Count: Erythrocyte count, Hemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular hemoglobin, total Leukocyte count, Differential leukocyte count: Platelet count and Reticulocytes
- Coagulation: Prothrombin time (= Thromboplastin time), Activated partial Thromboplastin time

CLINICAL CHEMISTRY: Yes
The following clinical biochemistry parameters were determined: Glucose, Urea, Creatinine, total Bilirubin, total Cholesterol, Triglycerides, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Gamma-glutamyl-transferase, Bile acids, Sodium, Potassium, Chloride, Calcium, Phosphorus, total Protein, Albumin, Globulin, Albumin/Globulin ratio.
Furthermore, an additional blood sample (0.5 mL) was collected into serum tubes for possible future measurement of thyroid releasing hormone (TSH), and the thyroid hormones triiodothyronine (T3) and thyroxine (T4). Serum samples were stored at -80 ± 10 °C. Any samples remaining at finalization of the study report were discarded. These measurements were not necessary, since there were no changes at microscopic examination of the thyroid glands or thyroid gland weights.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
At one time during the study (males shortly before the scheduled sacrifice and females on day 3 or 4 post partum) relevant parameters were performed with five P generation males and five P generation females randomly selected from each group. This FOB assessment was conducted following the daily dose administration. Animals were observed for the following:
• Cage-side observations: faeces-balls, urine and posture as well as resistance to removal.
• Hand-held observations: muscle tone, constitution, skin, pupil size, palpebral closure lacrimation, salivation, reaction to handling and general abnormalities.
• Open field observations: level of ambulatory activity including rearing (one minute evaluation), unusual body movements (e.g. spasms, convulsions), gait evaluation, behavior, hair coat, respiration, quantity of faeces-balls and urine.
• Reflexes: blinking, pinna reflex, extensor thrust response, paw pinch, responsiveness to sharp noise, righting reflex and hearing ability (Preyer’s reflex).
• Measurements / Counts: hind limb / fore limb grip strength, rectal temperature.
Any abnormal findings were recorded and, where appropriate, graded in severity. Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported.
Sacrifice and pathology:
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. Dead pups, except those excessively cannibalized, were examined macroscopically. All parent animals and pups 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.
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
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Description (incidence and severity):
Since the differences were minor, they were considered not to be toxicologically relevant.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
The statistically significant differences from controls occurred in one gender only and the values were within the range of historical control data or showed no dose-dependency.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
ALAT levels in males were in the range of the historical control data. Nevertheless, the increase may be test item related but not adverse. Further statistically significant differences are considered not to be toxicologically relevant.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Due to the observed morphological alterations in the liver of males (1000 mg/kg bw/d), the increase was considered to be test item related but not adverse.
Gross pathological findings:
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.
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
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 bw/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.

HEMATOLOGY
The assessment of the hematology data did not reveal any test item-related effects in males and females at any dose level. The statistically significant differences from controls (higher numbers of neutrophils and reticulocytes in males at 1000 mg/kg bw/day; higher mean corpuscular hemoglobin concentration in females at 100 and 1000 mg/kg bw/day; lower MCV values in females at 100 mg/kg bw/day) occurred in one gender only and the values were within the range of historical control data or showed no dose-dependency.

CLINICAL CHEMISTRY
In males, the alanine aminotransferase levels were slightly, but dose-dependently increased at 300 and 1000 mg/kg bw/day (up to 2.2-fold). These levels were in the range of the historical control data. Nevertheless, due to the observed morphological alterations in the liver, the increase may be test item related but not adverse. Further statistically significant differences (higher glucose values at 100 and 1000 mg/kg bw/day; lower creatinine values at 1000 mg/kg bw/day; higher potassium values at 1000 mg/kg bw/day; higher protein levels at 100 and 1000 mg/kg bw/day, with higher globulin values and lower albumin/globulin ratio at 1000 mg/kg bw/day) were minor and within the historical control range except protein at 1000 mg/kg bw/day, which was borderline to historical control data. In the absence of corroborating changes (especially regarding histopathology) these differences are therefore considered not to be toxicologically relevant. In females, the assessment of the clinical biochemistry data did not reveal any test item-related effects at any dose level. Statistically significantly higher chloride values occurred at 100 mg/kg bw/day, but without dose-dependency.

NEUROBEHAVIOUR
None of the parameters under investigation during the functional observational battery gave an indication of a test item-related effect. A slightly but statistically significantly lower body temperature (-1.3%) was noted in males at 300 mg/kg bw/day. However, due to the lack of dose-dependency, this finding was deemed to be incidental.
Locomotor activity was not affected by the treatment with the test item at any dose level.

ORGAN WEIGHTS
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
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: NON-NEOPLASTIC
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.
Dose descriptor:
NOAEL
Remarks:
general toxicity
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).
Critical effects observed:
not specified

Table 2: Alanine Aminotransferase (ALAT) in males (on the day of the scheduled necropsy) and in females (on day 5 post partum)

Males

Females

 

ALAT [U/L]

ALAT [U/L]

Control

20.4

38.6

100 mg/kg bw/d

24.3-

32.5-

300 mg/kg bw/d

32.7*

46.3-

1000 mg/kg bw/d

44.1**

42.0-

*/**/- : Significant at 5% (*), 1% (**) or not significant (-)

Table 3: Liver weights and liver/body weight ratios in males

 

Control

100 mg/kg bw/d

300 mg/kg bw/d

1000 mg/kg bw/d

Liver weights

MEAN

11.33

11.57-

11.30-

13.56**

ST.DEV.

0.63

0.86

0.47

0.63

MINIMUM

10.63

10.85

10.82

12.67

MAXIMUM

12.08

12.94

11.86

14.22

N

5

5

5

5

Liver/body weight ratio (%)

MEAN

2.48

2.57-

2.54-

3.12**

ST.DEV.

0.12

0.11

0.08

0.14

MINIMUM

2.37

2.48

2.43

2.95

MAXIMUM

2.67

2.76

2.64

3.28

N

5

5

5

5

*/**/- : DUNNETT-Test based on pooled variance significant at 5% (*), 1% (**) or not significant (-)

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
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

Repeated dose toxicity oral

Repeated dose toxicity inhalation

Repeated dose toxicity dermal

1

2402-58-6

Experimental result: NOAEL >/= 1000 mg/kg bw/day (subchronic)

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 = 200 mg/kg bw/day (subchronic)

--

--

(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. Lack of data for a given endpoint is indicated by “--“.

Repeated dose toxicity - oral

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

Subacute

CAS 103-23-1

In a study according to OECD 407, Bis(2-ethylhexyl) adipate (CAS 103-23-1) in corn oil was administered daily via oral gavage to 10 Crj: CD(SD) rats per sex and group at dose levels of 40, 200 and 1000 mg/kg bw/day (Miyata et al., 2006).

After 28-day treatment with the test substance, no treatment-related mortalities were observed in the animals. The relative organ weight of kidneys was significantly increased in males and females at 1000 mg/kg bw/day. At the same dose level, increased relative adrenal weights in females and increased relative liver weights in both sexes were observed. The changes in kidney weights at 1000 mg/kg bw/day were accompanied by clear spotty pattern in kidneys of 2 male rats as well as increased eosinophilic bodies (7/10 males) and hyaline droplets (8/10 males) at microscopic examination. These kidney effects are specific to male rats (alpha-2 micro globulin nephropathy syndrome) and of no concern to man. Further findings at histopathology involved increased ovarian follicle atresia in 4/10 females at 1000 mg/kg bw/day. Examination of vaginal smears revealed prolongation of the estrous stage in 2/10 females of the 1000 mg/kg bw/day dose group. No substance-related effects were observed on sperm parameters and histopathology of reproductive organs in males.

Based on the results of this study, the NOAEL for male and female Crj:CD(SD) rats was established at 200 mg/kg bw/day.

Subchronic

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. In males at 1000 mg/kg bw/day, slightly higher levels of alanine aminotransferase were observed. Although a relationship to treatment cannot be excluded, the values were well within the normal range of the rat strain used. Therefore, these differences were not considered as adverse. Furthermore, increased liver weights were observed and minor morphological alterations in the liver of males. This consisted of a minimal degree of diffuse, midzonal/centrilobular hepatocellular hypertrophy. This finding may be considered as adaptive in nature, within physiological limits and not a manifestation of frank toxicity. None of these observations were recorded at females. In males at 300 mg/kg bw/day, the alanine aminotransferase levels were also slightly higher but no other liver alterations were recorded at this dose level. No test item-related effects were observed in female rats at any dose level. Neither clinical signs nor effects on mean food consumption or mean body weight were noted at any dose level. Based on these results, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 1000 mg/kg body weight/day.

CAS 103-23-1

A subchronic oral toxicity study similar to OECD 408 was performed with Bis(2-ethylhexyl) adipate (CAS 103-23-1) in Fischer 344 rats and B6C3F1 mice at dose levels of 1600, 3100, 6300, 12500 and 25000 ppm for a period of 90 days (NTP, 1982). Ten animals per sex and dose received the test substance daily via diet, whereas a similar constituted control group was administered the plain diet. No signs of toxic effects and no mortality were observed in any of the animals during the study period. In mice, an adverse decrease in body weight gain compared to controls was noted starting at 3100 ppm in males and at 6300 or 25000 ppm in females, respectively. In rats, body weight gain was adversely reduced in males at 12500 and 25000 ppm. Average food consumption was not altered between treated and control groups of both genders and species. No adverse effects were noted at histopathological examination in rats and mice. Clinical chemistry and haematological parameters were not reported in this study. Based on these results, a NOAEL of 1600 ppm was derived for male B6C3F1 mice, corresponding to an actual ingested dose of 200 mg/kg bw/day. In male rats, the NOAEL was set at 6300 ppm, which is equivalent to a dose of 630 mg/kg bw/day. In female rats, the NOAEL was set at 25000 ppm, which was equivalent to a dose of 2187 mg/kg bw/day.

The effect of Bis(2-ethylhexyl) adipate (CAS 103-23-1)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. Male and female rats were continuously treated 10 weeks prior to mating and throughout mating. Male rats were sacrificed after mating. Treatment of female rats was continued until Day 36 post-partum when the animals were sacrificed. A similar constituted group of animals received the plain diet and served as controls. There was no evidence for any clear effect on bodyweight or food consumption. An increase in absolute and relative liver weights was observed in both male and female rats receiving dietary levels of 12000 ppm. No treatment-related findings were observed at gross pathology, except for accentuated lobular pattern in the livers 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 suspected of being infertile. Based on the results of this study the NOAEL for systemic toxicity was considered to be 1800 ppm, equivalent to dose levels of 178 and 203 mg/kg bw/day in males and females, respectively.

Based on the study results, a NOAEL of 178 mg/kg bw/day was derived for Bis(2-ethylhexyl) adipate based on the effects seen in males in the fertility study.

Conclusion for repeated dose toxicity

There is one study available which was conducted with Didodecyl fumarate (CAS 2402-58-6) addressing the repeated dose toxicity of dicarboxylic esters with fumaric acid. Furthermore, oral toxicity after repeated exposure with the analogue substance Bis(2-ethylhexyl) adipate (CAS 103-23-1) was investigated in four studies.

In a subchronic oral toxicity studie with Didodecyl fumarate (CAS 2402-58-6) in rats, a systemic NOAEL of 1000 mg/kg bw/day was identified. No adverse effects were observed up to the highest dose tested.

In subacute to subchronic oral toxicity studies with Bis(2-ethylhexyl) adipate in rats and mice systemic NOAELs of 178 and 200 mg/kg bw/day were identified.

Reduced body weight gain and increased liver weight were main findings after repeated oral exposure to Bis(2-ethylhexyl) adipate. These effects are attributed to the strong rodent specific activation of the peroxisome proliferation, predominantly via the peroxisome proliferator activated receptor (PPAR) alpha pathway (Reddy et al., 1986; Keith et al., 1992). Marked species differences have been observed for PPAR alpha activation. While rodents are very susceptible to hepatic peroxisome proliferation, guinea pigs and marmosets did not respond to Bis(2-ethylhexyl) adipate exposure with a marked increase in hepatic PPAR alpha activity (Cornu et al., 1992). This is supported by the finding that Bis(2-ethylhexyl) adipate metabolism in marmosets in contrast to rats does not lead to the formation of significant amounts of 2-Ethylhexanoic acid, a known PPAR alpha agonist (Elcombe, 1986). Thus, the observed effects after oral application are considered to be not relevant for humans.

In conclusion, no human hazard for systemic toxicity after repeated oral exposure was identified for the PFAE fumarate category members.

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


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
GLP study, supported by certified sub-chronic studies 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 repeated dose 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.