Registration Dossier

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Description of key information

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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 Jun 2009 - 16 Jul 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study, tested with the source substance 1,2,4-Benzenetricarboxylic acid, decyl octyl ester (CAS 90218-76-1). 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:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
adopted October 3, 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy s.r.l.
- Age at study initiation: 27-29 days
- Weight at study initiation: 88-107 g
- Fasting period before study: no
- Housing: 5 of one sex in clear polycarbonate cages with a stainless steel mesh lid and floor
- Diet: rodent diet (4 RF 21, Mucedola S. r. l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) ad libitum
- Water: drinking water ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 15
- Air changes (per hr): 15-25
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: June 4 2009 To: July 16, 2009
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 20, 60, and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were diluted with 2-propanol and an internal standard was added. The dilutions were then analysed by GC/FID.

Analysis of samples of the formulations prepared on day 1 and week 4 to check the homogeneity and concentration
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
100, 300, and 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5

additional 5 animals per sex in high dose and control group as satellite groups for recovery assessment
Control animals:
yes, concurrent vehicle
Details on study design:
- Post-exposure recovery period in satellite groups: 2 weeks
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and once daily for clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before commencement of treatment and once a week thereafter
BODY WEIGHT: Yes
- Time schedule for examinations: on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption by each cage of rats was recorded at weekly intervals and mean daily diet consumption calculated as g food/animal/day.


OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of week 4 of treatment and at the end of week 2 of the recovery period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked: haematocrit, haemoglobin, red blood cell count, reticulocyte count, mean red blood cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, white blood cell count, differential leucocyte count (neutrophils, lymphocytes, eosinophils, basophils, monocytes, large unstained cells), platelets, prothrombin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of week 4 of treatment and at the end of week 2 of the recovery period
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, urea, creatinine, glucose, triglycerides, bile acids, phosphorus, total bilirubin, total cholesterol, total protein, albumin, globulin, A/G ratio, sodium, potassium, calcium, chloride


URINALYSIS: Yes
- Time schedule for collection of urine: at the end of week 4 of treatment and at the end of week 2 of the recovery period
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters checked: appearance, volume, specific gravity, pH, protein, total reducing substances, glucose, ketones, bilirubin, urobilinogen, blood; epithelial cells, leucocytes, erythrocytes, chrystals, spermatozoa and precursors, other abnormal components


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once before commencement of treatment and once a week thereafter
- Dose groups that were examined: all

OTHER:
- motor activity assessment, sensory reactivity to stimuli of different modalities and assessment of grip strength (once during week 4 of treatment and once during week 2 of recovery)
- hormone determination (T3, T4 and THS)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, post mortem examination including examination of the external surface and orifices; determination of organ weights (see table 1)

HISTOPATHOLOGY: Yes (see table 2)

Histopathological evaluation was performed on all males and females of the control group and of the high dose group (receiving the test item at 1000 mg/kg/day), sacrificed at the end of treatment. All abnormalities from all main phase animals were also examined.
Particular attention was paid to the gonads (ovaries and testes), accessory sex organs (uterus including cervix, epididymides, seminal vesicles with coagulation glands, dorsolateral and ventral prostate), vagina, pituitary, thyroid and adrenal glands for the evaluation of possible endocrine-related effects. In addition, as indicated by the international test program, synchronization of the oestrus cycle was also evaluated to better identify eventual disturbances on female sex hormone homeostasis.
The histopathological evaluation was performed following the suggestions provided by the OECD Test Guideline 407 (which refers to "Endocrine sisruption: A guidance document for histologic evaluation of endocrin and reproductive tests", Draft 3, May 16 2008).
On the basis of the pathological changes noted in the adrenals of high dose males and in the stomach of a few animals in the same treated group, the examination was extended to include the stomach, from males and females receiving the test item at dosages of 100 and 300 mg/kg/day, and the adrenals from the males dosed at 100 and 300 mg/kg and in the males killed after the recovery period.
Statistics:
For continued variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treatment group and the control group were assessed by Dunnet's test. The homogeneity of the data was verified by Bartlett's test before Dunnet's test.
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study. Clinical signs were limited to hair loss, observed in a total of 6/10 females dosed at 1000 mg/kg/day from Day 8 of treatment up to the end of the recovery period.

BODY WEIGHT AND WEIGHT GAIN (see table 3)
A trend towards a decrease in mean body weight, compared to controls, (from 5% to 9%) was recorded in the males receiving 1000 mg/kg/day from Day 8 throughout the administration period, up to the end of the recovery period. No changes of note were seen in the other male groups and in treated females. The body weight reductions noted in all animals on Day 29, when compared to Day 22, were due to the overnight fast preceding the bleeding procedures for clinical pathology analyses.

FOOD CONSUMPTION
Slight reductions in food intake were observed in the males dosed at 1000 mg/kg/day from Day 8 (-17%) to Day 29 (-7%). No changes were seen in the other male groups and in treated females. No differences between treated animals and controls were observed at the end of recovery.
The above changes were not considered to be toxicologically relevant as they were slight and comparable to the historical control data.
The food consumption reductions, noted in all animals on Day 29 when compared to Day 22, were due to the overnight fast preceding the bleeding procedures for clinical pathology analyses.

HAEMATOLOGY (see table 4a and 4b)
Leucocytosis was recorded in animals treated with 1000 mg/kg/day and in females dosed with 300 mg/kg/day. The increase in white blood cells mainly involved the lymphocytes and was 25% to 60% above controls.
The other statistically significant changes recorded, such as an increase in erythrocytes, haemoglobin and haematocrit, observed in males treated with 300 and/or 1000 mg/kg/day, a decrease in reticulocytes, seen in those receiving 1000 mg/kg/day, and of prothrombin time seen in males treated with 100 mg/kg/day and in females dosed with 1000 mg/kg/day, were of low magnitude and inconsistent between sexes, therefore they were considered to be of no toxicological significance. In the recovery group leucocytosis was still present in females treated with 1000 mg/kg/day (increased by 27% compared with controls.

CLINICAL CHEMISTRY (see table 5)
A moderate to severe increase in alkaline phosphatase (+67% and 6-fold higher for males of the mid- and high dose groups, +5-fold for high-dose females) and bile acids (+9 and 3-fold in high dose males and females, respectively) and a decrease in globulin were observed in males treated with 300 and 1000 mg/kg/day and in females dosed with 1000 mg/kg/day. In addition, increments of alanine aminotransferase (2-fold), gamma-glutamyl transferase (3-fold) and decrements of bilirubin (-27%), protein and sodium were observed in males dosed with 1000 mg/kg/day.
Females from the same groups showed additional increments of alanine aminotransferase (2.8-fold), aspartate aminotransferase (+36%), gamma-glutamyltransferase (+29%) and urea (+33%) and decrements of bilirubin (-40%), protein, sodium and calcium. Some of the above mentioned changes could be related to the modifications seen in the adrenals recorded at the histopathological examination and to the related increase in corticosteroids. The other statistically significant changes were considered to be incidental as they were not dose-related and/or inconsistent between sexes .

Changes recorded during the dosing phase showed a complete reversibility in the recovery groups. Even though phosphorus, in males dosed with 1000 mg/kg/day, and urea, in females from the same group, did not decrease, their mean values were comparable with controls.
The statistically significant changes detected (triglycerides, chloride and sodium in males, aspartate aminotransferase in females) were not seen during the dosing phase, therefore they were considered to be incidental.

URINALYSIS
No changes of toxicological significance were recorded. The differences of specific gravity between control and treated females were of low severity. In the recovery phase no changes were observed.

NEUROBEHAVIOUR
No treatment-related changes were found at the weekly neurotoxicity evaluation.

ORGAN WEIGHTS (see table 6)
Slight but statistically significant increases in absolute (+16% and 33%) and relative (+29% and 34%) liver weights were recorded at the end of the treatment period in both males and females of the highest dose group in comparison with the respective controls. In addition, the absolute and relative adrenal weights were increased in the males dosed at 1000 mg/kg/day (+8% and 20%, respectively). These changes were found to be completely recovered after 2 treatment-free weeks. The toxicological significance of the above changes is supported by the correlation with the clinical and microscopic pathology findings. No other changes of toxicological significance were reported.

GROSS PATHOLOGY
The only relevant change noted in treated animals, when compared with controls, during the post mortem examination, was a minimal (on one occasion moderate) hair loss on the skin of the head, detected in 4/5 females dosed at 1000 mg/kg/day. In the recovery group hair loss was again noted on the forelimbs and head region of females dosed at 1000 mg/kg/day and sacrificed after the recovery period. The remaining changes observed as enlarged ovaries, distended with clear fluid content in the uterus, swollen spleen or red colour of the thymus are considered to be incidental in origin and characteristically seen in untreated Sprague Dawley rats of the same age, under our experimental conditions.

HISTOPATHOLOGY:

Treatment-related changes were seen in the adrenals of the high dose males. This change consisted of cortical cell hypertrophy associated with vacuolation, as fatty change, primarily involving the zona fascicolata.
No similar changes were noted in the males receiving the test item at 100 and 300 mg/kg/day, sacrificed at the end of the treatment period or in those sacrificed at the end of the recovery period.
The evaluation of the reproductive system of male (testis, epididymis, prostate, coagulating gland and seminal vesicles) and female (ovary, uterus and vagina) animals did not show any pathological alteration in the spermatogenesis as well no irregularity in the oestrus cycle.

Squamous hyperplasia of the forestomach mucosa associated with gastric inflammation was only detected in one male and one female, dosed at 1000 mg/kg/day. Males and females dosed at 100 and 300 mg/kg/day did not show any alteration in the stomach.
This sporadic pathological change could be considered incidental and probably related to an individual stress-related change.
The remaining lesions reported were considered as an expression of spontaneous pathology or as incidental findings, commonly observed in this species at this age and under our experimental conditions.


OTHER FINDINGS
No significant differences between treated animals and controls were observed at the evaluation of sensory reaction performed at the end of treatment. Motor activity measurements performed at the end of the treatment period did not show changes which could be ascribed to treatment.

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: at 1000 mg/kg bw: reduced body weight (males), hair loss (females), increased leucocytes (females), effects on adrenals and liver (reversible)
Critical effects observed:
not specified
Table 3: Body weights of males (mean values and standard deviations)
Group Control 100 mg/kg/d 300 mg/kg/d 1000 mg/kg/d
day 1 212.17 ± 8.62 207.60 ± 7.77 201.87 ± 6.58 206.30 ± 7.82
day 8 261.78 ± 12.72 254.79 ± 8.80 247.26 ± 9.25 247.53 ± 110.39*
day 15 309.27 ± 15.05 298.20 ± 11.60 288.67 ± 12.22* 284.52 ± 11.81**
day 22 339.44 ± 17.19 327.37 ± 9.40 320.53 ± 16.65 311.11 ± 15.12**
day 29 340.78 ± 19.25 329.45 ± 12.26 317.14 ± 19.98 309.66 ± 10.99**
Recovery phase
Day 1 365.39 ± 21.24 326.39 ± 17.49*
Day 8 382.19 ± 23.58 340.30 ± 19.49*
Day 15 377.09 ± 24.86 337.03 ± 22.06*
*p < 0.05; ** p < 0.01
Table 4a: Haematology findings in males (mean values and standard deviations)
Group Control 100 mg/kg/d 300 mg/kg/d 1000 mg/kg/d
Red blood cells (*106/µl) 7.316 ± 0.133 7.574 ± 0.248 7.635 ± 0.162 7.798 ± 0.258**
Haemaglobin (g/dl) 14.06 ± 0.29 14.44 ± 0.44 14.83 ± 0.43* 15.04 ± 0.29**
Haematocrit (%) 40.24 ± 0.62 41.30 ± 1.42 42.25 ± 1.08* 43.44 ± 1.06**
Reticulocytes 137.04 ± 26.46 123.04 ± 6.83 1325.25 ± 18.78 104.14 ± 12.13*
White cell blood count (*103/µl) 11.148 ± 2.220 9.3486 ± 0.805 10.458 ± 2.149 13.282 ± 1.959
Lymphocytes (*103/µl) 8.712 ± 2.307 7.332 ± 0.880 8.425 ± 1.941 10.866 ± 2.217
*p < 0.05; ** p < 0.01
Table 4b: Haematology findings in females (mean values and standard deviations)
Group Control 100 mg/kg/d 300 mg/kg/d 1000 mg/kg/d Recovery control group Recovery group 1000 mg/kg/d
White cell blood count (*103/µl) 6.208 ± 1.028 5.684 ± 1.362 9.174 ± 1.010** 9.930 ± 1.261** 7.222 ± 0.704 9.198 ± 1.196
Lymphocytes (*103/µl) 5.196 ± 0.756 4.752 ± 1.196 7.838 ± 0.838** 8.410 ± 1.090** 6.016 ± 0.616 7.808 ± 1.190*
*p < 0.05; ** p < 0.01
Table 5: Clinical chemistry findings (mean values and standard deviations)
Group Control 100 mg/kg/d 300 mg/kg/d 1000 mg/kg/d Recovery control group Recovery group 1000 mg/kg/d
Alkaline phosphatase (U/l) males 357.96 ± 28.25 398.90 ± 97.75 597.06 ± 106.54** 2212.06 ± 429.06** 276.30 ± 29.77 301.44 ± 15.90
females 287.54 ± 26.85 336.94 ± 93.45 361.86 ± 61.66 1421.66 ± 631.98* 184.30 ± 20.87 189.12 ± 26.00
Alanine Amino-transferase (U/l) males  51.52 ± 17.46 35.70 ± 5.23 43.38 ± 5.97 103.24 ± 17.68** 40.56 ± 2.93 41.74 ± 5.05
females 30.68 ± 12.08 26.42 ± 3.94 25.32 ± 4.21 86.06 ± 26.53* 31.40 ± 1.57 30.66 ± 3.45
Aspartate Amino-transferase (U/l) females 57.70 ± 6.29 57.26 ± 4.39 60.26 ± 4.94 78.40 ± 4.99** 84.10 ± 5.43 73.94 ± 3.59**
¿-Glutamyl transferase (U/l) males 1.34 ± 1.02 0.88 ± 0.28 1.98 ± 0.56 4.12 ± 1.45* 2.08 ± 2.00 2.56 ± 1.03
females 0.70 ± 0.23 0.78 ± 0.13 0.60 ± 0.19 0.90 ± 0.16 2.50 ± 1.31 1.86 ± 1.02
Bile acids (µmol/l) males 10.08 ± 5.72 9.10 ± 5.25 16.12 ± 3.88 89.96 ± 29.39** 20.46 ± 11.16 8.54 ± 5.26
females 15.46 ± 8.78 16.14 ± 10.07 5.06 ± 2.75 43042 ± 32.32 9.44 ± 8.02 7.68 ± 3.80
Bilirubin (mg/dl) males 0.188 ± 0.018 0.192 ± 0.033 0.188 ± 0.026 0.138 ± 0.026* 0.160 ± 0.034 0.170± 0.035
females 0.226 ± 0.024 0.186 ± 0.026 0.232 ± 0.022 0.136 ± 0.027** 0.216 ± 0.038 0.230 ± 0.014
Protein (g/dl) males 5.96 ± 0.05 5.90 ± 0.14 5.88 ± 0.30 5.24 ± 0.18** 6.48 ± 0.26 6.60 ± 0.55
females 5.42 ± 0.11 5.42 ± 0.23 5.58 ± 0.27 4.78 ± 0.26** 6.34 ± 0.15 6.24 ± 0.11
Globulin males 2.30 ± 0.12 2.20 ± 0.07 2.08 ± 0.16* 1.64 ± 0.09** 2.38 ± 0.52 2.66 ± 0.53
females 1.94 ± 0.15 1.92 ± 0.16 1.96 ± 0.15 1.50 ± 0.14** 2.32 ± 0.08 2.38 ± 0.08
Albumin/Globulin ratio males 1.60 ± 0.13 1.68 ± 0.08 1.83 ± 0.14* 2.20 ± 0.12** 1.82 ± 0.57 1.53 ± 0.30
females 1.80 ± 0.18 1.83 ± 0.15 1.85 ± 0.13 2.20 ± 0.16** 1.74 ± 0.11 1.62 ± 0.09
Urea (mg/dl) females 37.54 ± 5.56 43.48 ± 3.56 39.34 ± 5.30 49.88 ± 3.81** 66.14 ± 16.19 66.00 ± 1.97
Sodium (mmol/l) males 149.76 ± 1.20 152.00 ± 1.12 149.50 ± 1.34 145.92 ± 1.76** 148.22 ± 1.53 151.22 ± 1.05**
females 150.28 ± 1.64 150.62 ± 1.68 148.92 ± 2.27 146.62 ± 0.52** 150.16 ± 1.25 148.18 ± 1.54
Calcium (mmol/l) females 2.322 ± 0.077 2.310 ± 0.022 2.342 ± 0.047 2.200 ± 0.063** 2.582 ± 0.058 2.578 ± 0.047
*p < 0.05; ** p < 0.01
Table 6: Organ weights (mean values and standard deviations)
Group Control 100 mg/kg/d 300 mg/kg/d 1000 mg/kg/d Recovery control group Recovery group 1000 mg/kg/d
Liver (absolute) males 10.279 ± 1.304 9.713 ± 0.500 9.874 ± 0.957 11.960 ± 1.125* 9.981 ± 0.826 9.013 ± 0.997
females 6.201 ± 0.371 6.646 ± 0.693 6.395 ± 0.413 8.229 ± 0.401** 5.907 ± 0.526 6.299 ± 0.430
Liver (relative) males 3.009 ± 0.217 2.954 ± 0.106 3.118 ± 0.165 3.868 ± 0.246** 2.671 ± 0.103 2.688 ± 0.135
females 2.765 ± 0.080 2.960 ± 0.201 2.934 ± 0.052 3.697 ± 0.194** 2.627 ± 0.200 2.727 ± 0.100
Adrenal (absolute) males 0.0536 ± 0.0080 0.0510 ± 0.0022 0.0480 ± 0.0031 0.0580 ± 0.0056 0.0552 ± 0.0053 0.0506 ± 0.0059
females 0.0674 ± 0.0084 0.0664 ± 0.0060 0.0656 ± 0.0051 0.0662 ± 0.0056 0.0584 ± 0.0047 0.0688 ± 0.0066*
Adrenal (relative) males 0.0157 ± 0.0022 0.0155 ± 0.0005 0.0152 ± 0.0016 0.0188 ± 0.0013* 0.0148 ± 0.0018 0.0152 ± 0.0019
females 0.0301 ± 0.0038 0.0297 ± 0.0027 0.0301 ± 0.0024 0.0297 ± 0.0018 0.0261 ± 0.0028 0.0298 ± 0.0019*
*p < 0.05; ** < 0.01
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The endpoint conclusion "No adverse effect observed" was selected based on the result of the 28-d study. As the effects observed in this oral subacute repeated dose study were seen only at the highest dose of 1000 mg/kg bw/day and therefore the NOAEL was determined to be 300 mg/kg bw/day, classification for specific target organ toxicity after repeated exposure is not warranted. However, DNELs were derived based on the NOAEL of 300 mg/kg bw/day of this study.

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 Polyfunctional acid ester (PFAE) aromatic category covers fatty alcohol esters of Benzene-1,2,4-tricarboxylic acid. The category contains both mono constituent and UVCB substances with fatty alcohol carbon chain lengths from C8-C13 (linear and iso-alcohols) building tri-esters with Benzene-1,2,4-tricarboxylic acid in variable proportions. A further surrogate substance of similar structure is included, namely a triester of Benzene-1,2,4-tricarboxylic acid with a C8 alcohol, but the alcohol moiety is branched (2-ethylhexyl).

The available data allows for an accurate hazard and risk assessment of the category and the category concept is applied for the assessment of environmental fate, environmental 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 interpolation 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. In particular, for each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across.

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:

Discussion

Repeated dose toxicity

Table: Repeated dose toxicity.

ID No.

CAS

Repeated dose toxicity oral

Repeated dose toxicity inhalation

Repeated dose toxicity dermal

#1

3319-31-1 (c)

Subacute

NOAEL >/=1000 mg/kg bw

--

--

#2

90218-76-1 (b) 

(former CAS: 67989-23-5)

Subacute

NOAEL 300 mg/kg bw

--

--

#4

94279-36-4 (a)

RA: CAS 90218-76-1
RA: CAS 3319-31-1

--

--

#5

72361-35-4

RA: CAS 90218-76-1
RA: CAS 3319-31-1

--

--

(a) Category members subject to the REACh Phase-in registration deadline of 31 May 2013 are indicated in bold font.

(b) Substances that are either already registered under REACh or not subject to the REACh Phase-in registration deadline of 31 May 2013 are indicated in normal font. Lack of data for a given endpoint is indicated by “--“.

(c) Surrogate 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.

 

Repeated dose toxicity - oral

Subacute

CAS 90218-76-1

The Subacute oral toxicity of 1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters was investigated in a study performed according to OECD guideline 407 under GLP conditions (Longobardi, 2010). The test substance (> 97.5% pure) was administered once daily via gavage to groups of 5 Sprague-Dawley rats per sex at doses of 100, 300, and 1000 mg/kg bw in corn oil for a period of 28 days. A control group received the vehicle alone. Additionally, satellite groups of 5 animals per sex (control and high dose) were used to investigate the reversibility of effects during a 14-day post-exposure recovery period.

No mortality occurred during the study and clinical signs were limited to hair loss, observed in a total of 6/10 high dose females from day 8 of treatment up to the end of the recovery period. A trend towards a decrease in mean body weight, compared to controls, (from 5% to 9%) was recorded in high dose males. Slight reductions in food intake were observed in high dose males from day 8 (-17%) to Day 29 (-7%). The above changes were not considered to be toxicologically relevant as they were slight and comparable to the historical control data.

Effects in haematological parameters were seen and included leucocytosis high dose males and in mid and high dose females. The increase in white blood cells mainly involved the lymphocytes and was 25% to 60% above controls. Leucocytosis was still present in high dose females after the recovery period (increased by 27% compared with controls). The other statistically significant changes recorded (increase in erythrocytes, haemoglobin and haematocrit, observed in mid and high dose males; decrease in reticulocytes in high dose males, decrease in prothrombin time low dose males and high dose females) were of low magnitude and inconsistent between sexes, therefore, they were considered to be of no toxicological significance.

Changes in clinical chemistry included a moderate to severe increase in alkaline phosphatase (+67% and 6-fold higher for males of the mid- and high dose groups, +5-fold for high-dose females) and bile acids (+9 and 3-fold in high dose males and females, respectively) and a decrease in globulin in mid and high dose males and high dose females. In addition, increments of alanine aminotransferase (2-fold), gamma-glutamyl transferase (3-fold) and decrements of bilirubin (-27%), protein and sodium were observed in high dose males. Females from the same groups showed additional increments of alanine aminotransferase (2.8-fold), aspartate aminotransferase (AST) (+36%), gamma-glutamyltransferase (+29%) and urea (+33%) and decrements of bilirubin (-40%), protein, sodium and calcium. Some of the above mentioned changes could be related to the modifications seen in the adrenals recorded at the histopathological examination and to the related increase in corticosteroids. The other statistically significant changes were considered to be incidental as they were not dose-related and/or inconsistent between sexes. Changes recorded during the dosing phase showed a complete reversibility in the recovery groups.

Slight but statistically significant increases in absolute (+16% and 33%) and relative (+29% and 34%) liver weights were recorded at the end of the treatment period in both males and females of the highest dose group in comparison with the respective controls. In addition, the absolute and relative adrenal weights were increased in the males dosed at 1000 mg/kg/day (+8% and 20%, respectively). All changes were found to be completely recovered after the recovery period. However, the toxicological significance of the above changes is supported by the correlation with the clinical and microscopic pathology findings. No other changes of toxicological significance were reported.

The changes observed at gross pathology included enlarged ovaries, distended with clear fluid content in the uterus, swollen spleen or red colour of the thymus and were considered to be incidental in origin and characteristically seen in untreated Sprague Dawley rats of the same age, under our experimental conditions.

Treatment-related changes in the adrenals were seen at histological examination of the high dose males. This change consisted of cortical cell hypertrophy associated with vacuolation, as fatty change, primarily involving the zona fascicolata. No similar changes were noted in low and mid dose males or in those of the recovery group. The evaluation of the reproductive system of male (testis, epididymis, prostate, coagulating gland and seminal vesicles) and female (ovary, uterus and vagina) animals did not show any pathological alteration in the spermatogenesis as well as no irregularity in the oestrus cycle. Squamous hyperplasia of the forestomach mucosa associated with gastric inflammation was only detected in one male and one female, dosed at 1000 mg/kg/day.

This sporadic pathological change could be considered incidental and probably related to an individual stress-related change.

Based on the observed reduction in body weight (males), hair loss (females), the leucocytosis (females), as well as the effects on liver in males and females (clinical chemistry, organ weight) and adrenals in males (organ weight, histopathological changes) in the high dose groups (1000 mg/kg bw), the NOAEL was established at 300 mg/kg bw and this effect level was used for the derivation of the long-term systemic effect DNELs.

CAS 3319-31-1

The subacute oral toxicity of Tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate was investigated in a study performed according to OECD guideline 407 under GLP conditions (Inoue, 1996). Dilutions of the test substance (> 99% pure) in corn oil were administered once daily via oral gavage to groups of 5 Crj: CD(SD) rats per sex at doses of 100, 300 and 1000 mg/kg bw for a period of 28 days. A similar constituted group received the vehicle and acted as a control. In addition, satellite groups of 5 animals per sex, each for the control and high dose group, were used to investigate the reversibility of effects during a 14-day post-exposure recovery period. No substance-related mortalities occurred during the whole study period. No clinical signs were observed and body weight, body weight gain and food consumption was similar between treated and control animals. Slight effects were seen in haematological parameters (prolonged prothrombin time in high dose males; increased haemoglobin in high dose recovery females), clinical chemistry (decreased chloride in mid and high dose females; increased potassium and AST in high dose recovery males and females), and organ weights (increased absolute and relative kidney and adrenal weight in high dose females; decreased relative kidney weight in recovery high dose males). These effects were considered normal (physiological) variations and not compound-related. Coloured patch was observed in lung of 1/5 male, 2/5 males and 3/5 males of the low, mid, and high dose group, respectively. This was not regarded as a test substance related change. Eosinophilic body was observed in the kidneys of 1/5 male, 2/5 males and 3/5 males of the low, mid and high dose group, respectively. According to historical data, the frequency was 22.2% and this change was not observed in all males of 1000 mg/kg bw/day group. Therefore, this change was not regarded as compound related effect, either.

Based on the results of this subacute toxicity study, the NOAEL of Tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate was established at 1000 mg/kg bw/day.

No data on repeated exposure via inhalation or dermal treatment and no oral study with prolonged exposure period is available for any category member. Since the group concept is applied to the members of the PFAE aromatic Category, additional data will be generated from representative reference substance(s) within the category to avoid unnecessary animal testing. The selected reference substance for additional testing within the PFAE aromatic category to cover the endpoint repeated dose toxicity is 1,2,4-benzenetricarboxylic acid, mixed decyl and octyl triesters (CAS 90218-76-1). To evaluate the subchronic toxicity of 1,2,4-benzenetricarboxylic acid, mixed decyl and octyl triesters (CAS 90218-76-1), a testing proposal for the conduct of a 90-day subchronic study was already made in 2010, to be performed according to OECD 408. The re-assessment of the repeated dose toxicity and the classification and labelling of all the substances within the PFAE aromatic category will based on the outcome of this test once it will be available.

Conclusion for repeated dose toxicity

Toxicity after repeated exposure was investigated in two oral subacute studies. Adverse effects have been reported in an oral studies (28 days) with the category member 1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters (CAS 90218-76-1) at doses of 1000 mg/kg bw/d, while no adverse effects have been observed for the category member Tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate (CAS 3319-31-1) up to the limit dose of 1000 mg/kg bw/d. The NOAEL for subacute oral toxicity of the category member Tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate (CAS 3319-31-1) in rats was established at 1000 mg/kg bw/day. In the subacute oral toxicity study with 1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters (CAS 90218-76-1) a NOAEL of 300 mg/kg bw/day was identified.

To evaluate the subchronic toxicity of 1,2,4-benzenetricarboxylic acid, mixed decyl and octyl triesters (CAS 90218-76-1), a testing proposal for the conduct of a 90-day subchronic study was already made in 2010, to be performed according to OECD 408. The re-assessment of the repeated dose toxicity and the classification and labelling of all the substances within the PFAE aromatic category will based on the outcome of this test once it will be available.

In conclusion, no human hazard for systemic toxicity after repeated oral, dermal, or inhalation exposure was identified for the PFAE aromatic category members for the time being. An update of this dossier will be submitted as soon as possible.

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 aromatic 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.

To evaluate the subchronic toxicity of 1,2,4-benzenetricarboxylic acid, mixed decyl and octyl triesters (CAS 90218-76-1), a testing proposal for the conduct of a 90-day subchronic study was already made in 2010, to be performed according to OECD 408. The re-assessment of the repeated dose toxicity and the classification and labelling of all the substances within the PFAE aromatic category will based on the outcome of this test once it will be available. An update of this dossier will be submitted as soon as possible.