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Toxicological information

Repeated dose toxicity: oral

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1,2,4-Benzenetricarboxylic acid, decyl octyl ester (Label name: [trade name])
- Substance type: pure active substance
- Physical state: liquid
- Lot/batch No.: 03319
- Expiration date of the lot/batch: July 31, 2010
- Storage condition of test material: ambient temperature

Test animals

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

Administration / exposure

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
Doses / concentrations
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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

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)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion