Registration Dossier

Administrative data

Description of key information

In a reliable 3-week feeding study in male rats given Santicizer 261, an NOAEL of 60 mg/kg bw/day was determined. Longer-term studies on the structurally related

compounds, DINP and BBP, provide NOAELs of about 17 and 151 mg/kg bw/day respectively.

In a key combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in the rat (OECD 422), dietary exposure of 1500 ppm Sanicizer 261a (males = 67 mg/Kg/day; females = 151.83 mg/Kg/day) was considered to be the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of the F0 generation.


No data are available on the repeated dose inhalation toxicity of S261a, but testing by this route is not considered necessary because exposure of humans via inhalation is unlikely (taking into account the low vapour pressure of other phthalates and the low likelihood of exposure to aerosols, particles or droplets of an inhalable size). Reassurance can be drawn from two BBP studies in rats, which provided NOAECs of 0.218 mg/l after 13 weeks and 1 mg/l after 4 weeks.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998-11-11 to 1998-12-10
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Not a guideline study. However, study carried out to GLP, and data appears well documented and scientifically reasonable.
Reason / purpose:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
In male rats, the systemic toxicity (including to the liver and reproductive organs) was assessed following three weeks dietary exposure to the US or EU versions of the phthalate ester, Santicizer(R) 261.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl: CD(SD)BR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Raleigh, NC,
- Age at study initiation: Probably about 80 days
- Weight at study initiation: 334-360 g
- Housing: Rats individually housed in solid-bottom polycarbonate cages with stainless steel wire lids with Sani-Chip(R) cage bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: A "one-week quarantine"


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23
- Humidity (%): 51.2-62.3
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Purina certified rodent chow(R)
- Storage temperature of food: "frozen"
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stored frozen feed was analysed on 5 January 1999 [no further details given in study report]
Duration of treatment / exposure:
21 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
75, 750 and 1500 mg/kg bw/day
Basis:
other: target dose levels
Remarks:
Doses / Concentrations:
about 60, 600 and 1200 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
6 males/dose
Control animals:
yes, plain diet
other: positive control
Details on study design:
- Rationale for animal assignment (if not random): by a randomization procedure, stratified by body weight
Positive control:
Di(2-ethylhexyl) phthalate (DEHP), a known testicular and liver toxin, was examined at 1500 mg/kg bw/day only
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: a.m. and p.m.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily
The cage-side observations included changes in skin and fur, eyes, mucous membranes, respiratory system, circulatory system, autonomic and central nervous sytem, somatomotor activity, and behaviour pattern.

BODY WEIGHT: Yes
- Time schedule for examinations: 0, 7, 14 and 21 days


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


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: No


CLINICAL CHEMISTRY: No


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No


OTHER: Peroxisomal Acyl-CoA oxidase activity, an indication of hepatic peroxisome proliferation, was measured in the livers of the controls and treated rats at sacrifice.
Sacrifice and pathology:
GROSS PATHOLOGY: Performed, but no details on extent of examination. Organ weights (liver, brain, testis and epididymis) also assessed
HISTOPATHOLOGY: Limited to one testis from each rat in the high-dose group and in the untreated and positive controls
Other examinations:
Acyl-CoA oxidase activity in the liver assessed
Statistics:
Krusal-Wallis one-way analysis of variance by ranks was used to test for differences among dose groups for each test material. Whenever the results of this test were significant (p<0.05), the Mann-Whitney U test was used to make individual comparisons between untreated controls and test material-exposed groups for each test material for that measure (a one-tailed test was used for all parameters except for body weight, organ weight and feed consumption which were examined in a two-tailed test). Jonckheere's test for k independent samples was used to identify significant dose-response trends for each test material. For these data a Chi-square test for independence was used to analyse for differences among treatment groups, and the Cochran-Armitage test for linear trend of proportions was used to determine dose-related linear trends for each test material. When Chi-square revealed significant (p<0.05) differences between groups, then a one- or two-tailed Fisher's exact probability test was used for pair-wise comparisons between each test material-exposed group and the controls.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
All rats survived to scheduled sacrifice. Piloerection was observed in one untreated control animal (on day 7), in 2 males given the EU Santicizer(R) 261 at the top dose on day 1 and in one rat on days 2, 5 and 7, in none of the animals exposed to the US Santicizer(R) 261, and in 2 males exposed to DEHP on day 5 and one rat on day 7. In addition, one rat given the high dose EU Santicizer(R) 261 exhibited a sore shoulder on days 14-21 (relevance to treatment unknown).

BODY WEIGHT AND WEIGHT GAIN
No statistically significant differences in body weight or body weight gain were seen in the rats receiving about 60 mg/kg bw/day (of either the EU or US versions) compared to the untreated controls, whereas both test materials produced a statistically significant and dose-related reduction in body weight gain from 600 mg/kg bw/day, particularly during the first week.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
For those rats exposed to the EU version, food consumption was significantly reduced (compared to the untreated controls) at the top dose during the first 2 weeks (and returned to normal thereafter) and also during the second week at 600 mg/kg bw/day. With the US version the reduction in food consumption was evident only at the top dose during the first week. Food consumption was unaffected at the lowest dose of about 60 mg/kg bw/day (for either version). Due to the initial decrease in food intake (which was likely due to palatability problems) in the two test groups (and also in those animals receiving DEHP), phthalate intake was less during the first exposure week. Overall, the mean intakes for the US Santicizer(R) 261 were 62.5, 601.5 and 1206 mg/kg bw/day and for the EU Santicizer(R) 261 were 63.3, 593.1 and 1259 mg/kg bw/day, at 0.1, 1, and 2% in the diet, respectively. The overall mean intake of DEHP at 2% in the diet was 1093.9 mg/kg bw/day.

ORGAN WEIGHTS
Compared with untreated controls, both test materials produced statistically significant increases in relative liver weight, this being evident from 600 mg/kg bw/day with the EU version and at 1200 mg/kg bw/day with the US version. Neither compound affected absolute weights of the epididymis, testis or brain. The positive control, DEHP, produced an increase in relative liver weight and a decrease in absolute testis weight.

GROSS PATHOLOGY
There were apparently no gross necropsy findings at sacrifice (extent of examination unclear from study report).

HISTOPATHOLOGY: NON-NEOPLASTIC
At 1200 mg/kg bw/day, minimal testicular degeneration was seen in 1/6 and 4/6 rats given the US and EU versions, respectively. All six of the rats given DEHP exhibited testicular lesions, while there were none in the six untreated controls.

OTHER FINDINGS:
There was a statistically significant increase in acyl-CoA oxidase activity (an indication of peroxisome proliferation) in the livers of rats given the US or EU versions of Santicizer(R) 261 at 600 and 1200 mg/kg bw/day, when compared with untreated controls. The same effect was also observed in the positive controls given DEHP.
Dose descriptor:
NOAEL
Effect level:
ca. 60 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
Conclusions:
In a 3-week feeding study in which male rats were given two different formulations of Santicizer(R) 261 (one EU and one US) at around 60, 600 and 1200 mg/kg bw/day, effects observed from 600 mg/kg bw/day included reduced body weight gain and increases in liver weight and acyl-CoA oxidase activity (an indication of peroxisome proliferation). The EU version of the test material had a slightly greater effect, but the difference between the two versions was minimal.
Executive summary:

Groups of six male rats were fed either the US or EU versions of Santicizer(R) 261 at 0 (untreated controls), 1 or 2% in the diet, or DEHP at 2% in the diet (positive control), for 3 weeks. Overall, these dietary concentrations corresponded to actual mean intakes for the US test material of 62.5, 601.5 and 1206 mg/kg bw/day and for the EU version of 63.3, 593.1 and 1259 mg/kg bw/day. The overall mean intake of DEHP at 2% in the diet was 1093.9 mg/kg bw/day.

 

There were no deaths during the 3-week exposure period. Piloerection (a non-specific indicator of stress) was observed in one untreated control rat, and in "one to two" males in the high-dose EU Santicizer(R) 261 group and the DEHP group. One rat given the high-dose of the EU version exhibited a sore shoulder in the third week of exposure, but the relevance to treatment is not discussed in the study report.

 

No statistically significant differences in body weight or body weight gain were seen in the rats receiving about 60 mg/kg bw/day (of either the EU or US versions) compared to the untreated controls, whereas both test materials produced a statistically significant and dose-related reduction in body weight gain from 600 mg/kg bw/day (probably related to the significant reduction in food consumption seen at these higher doses).

 

Compared with untreated controls, both test materials produced statistically significant increases in relative liver weight, this being evident from 600 mg/kg bw/day with the EU version and at 1200 mg/kg bw/day with the US version. Neither compound affected absolute weights of the epididymis, testis or brain. The positive control, DEHP, produced an increase in relative liver weight and a decrease in absolute testis weight.

 

There were apparently no gross necropsy findings at sacrifice, although the extent of examination is unclear from the study report. At 1200 mg/kg bw/day, minimal testicular degeneration was seen in 1/6 and 4/6 rats given the US and EU versions, respectively. All six of the rats given DEHP exhibited testicular lesions, while there were none in the six untreated controls.

 

There was a statistically significant increase in acyl-CoA oxidase activity (an indication of peroxisome proliferation) in the livers of rats given the US or EU versions of Santicizer(R) 261 at 600 and 1200 mg/kg bw/day, when compared with untreated controls. The same effect was also observed in the positive controls given DEHP.

 

In conclusion, no statistically significant adverse effects were seen in male rats ingesting about 60 mg/kg bw/day (considered the NOAEL for this study) of either the EU or US versions of Santicizer(R) 261 for 3 weeks, compared to the untreated control animals. At 600 mg/kg bw/day (considered the LOAEL for this study) the male rats consuming the EU version for 3 weeks gained significantly less body weight and had increased liver weights, compared to the untreated controls. In addition at this dose, increased acyl-CoA oxidase activity was seen in the livers of the animals ingesting either the EU or US versions of Santicizer(R) 261.
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-04-03 to 2018-08-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Modified according to ECHA Final Decision Letter dated 19 May 2017 (see background material attached to RSS in Section 7.8.1)
Deviations:
yes
Remarks:
Study deviations neither affected the overall interpretation of study findings nor compromised the integrity of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
The rat was selected because it is a readily available rodent species acceptable to the regulatory authorities and is recommended for reproduction studies due to its
reproductive characteristics and the historical control data available at Covance-Harrogate.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Margate, UK
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: (P): At start of dosing: 10 to 12 wks
- Weight at study initiation: (P) Males: 360.7 g and 487.9 g; Females: 229.9 g and 320.6 g
- Fasting period before study: Not specified
- Housing: in cages that conform to the Code of Practice for the Housing and Care of Animals Bred, Supplied, or Used for Scientific Purposes(Home Office, 2014). During the pre-pairing phase, animals were housed in groups of up to four by sex and dose group. During the pairing phase, one female was housed with one male from the same dose group until mating was confirmed. Following mating, females were housed individually during gestation and with their litter during the lactation phase. Males were returned to group housing after the pairing phase
- Diet (e.g. ad libitum): 5LF2/5KB3 EU Rodent Diet (International Product Supplies Ltd., London, United Kingdom), or LabDiet 5002 diet (Special Diets Services Ltd, Witham, United Kingdom) ad libitum
- Water (e.g. ad libitum): Water from the main tap supply was provided ad libitum via water bottles
- Acclimation period: at least 12 days of acclimation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): minimum of 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours of light and 12 hours of dark

IN-LIFE DATES: From: 2018-04-16 To: 2018-08-29
Route of administration:
oral: feed
Details on route of administration:
The oral (dietary) route of administration was requested in the ECHA’s final decision letter, dated 19 May 2017; based on test article uses, this is a possible route of human
exposure.
Vehicle:
unchanged (no vehicle)
Remarks:
The control article (vehicle) was 5LF2/5KB3 EU Rodent diet
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): formulations were prepared weekly.
- Mixing appropriate amounts with (Type of food): The test article was formulated as a diet mix in 5LF2/5KB3 EU Rodent diet or LabDiet 5002 following dispensary SOPs and the formulation method as maintained in the study data.
- Storage temperature of food: Formulations were stored at room temperature (15 to 25°C) in a sealed container.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The control article (vehicle) was 5LF2/5KB3 EU Rodent diet
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulations prepared for use at the beginning (5LF2 EU rodent diet and Lab diet 5002) and end of the dosing period (5LF2 EU rodent diet) were analyzed to determine the achieved concentration. The mean % nominal concentration should be between 85 to 115% and with a relative standard deviation (RSD) ≤10.0%. Results were within these criteria.Test article was not detected in the Group 1 control samples.The analytical procedure was validated Covance study 8381228.
Duration of treatment / exposure:
The test item was administered to animals for a maximum duration of approximately 98 or up to 65 days for males and females, respectively.
Frequency of treatment:
Continuous in diet
Dose / conc.:
0 ppm
Remarks:
Control
Dose / conc.:
375 ppm
Remarks:
Low Concentration
Dose / conc.:
1 500 ppm
Remarks:
Intermediate Concentration
Dose / conc.:
6 000 ppm
Remarks:
High Concentration
No. of animals per sex per dose:
20/sex/concentration
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale:
Dose levels were selected based on a previous 14-day dietary study in rats (Covance Study 8381227 (Covance Laboratories Ltd., 2018a)) using dose levels of 250, 2500
and 7500 ppm 1,2-Benzenedicarboxylic acid, benzyl C7-9-branched and linear alkyl esters.

A constant ratio of 4 was used to set doses after deciding the high dose as to be 6000 ppm (ie 6000, 1500, 375 ppm). The high dose level of 6000 ppm was expected to
produce evidence of toxicity based on the substantially lower mean body weight gain noted in females previously administered 7500 ppm. The low dose level of 375 ppm
was selected in the absence of significant findings at 250 ppm in the previous study, and was expected to be the minimum NOAEL. The intermediate dose level of 1500 ppm was designed to explore dose-relationship.

- Rationale for animal assignment (if not random):
Upon arrival, animals were assigned to dose groups using a total randomization procedure. Animals were individually identified by electronic implant. During the FOB assessments, applicable animals were identified by tail marks.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed at the beginning and end (nominal) of each working day for signs of ill health or overt toxicity

DETAILED CLINICAL OBSERVATIONS: Yes (Table 2)
- Time schedule: Each animal was given a detailed physical examination once during acclimation (Day 1 of the predose phase) and on the days of body weight recording.

BODY WEIGHT: Yes
- Time schedule for examinations: Male body weights were recorded once during acclimation (Day 1 of the predose phase), on the first day of dosing (Pre-Pairing Day 1) and at weekly intervals thereafter, and on the day prior to, and of, necropsy (Post-Pairing Day 13 and terminal sacrifice).

Female body weights were recorded during acclimation (Days 1 and 8 of the predose phase); on the first day of dosing (Pre-Pairing Day 1); weekly prior to pairing until the confirmation of mating; on Gestation Days (GD) 0, 7, 14, and 20; and on LD 1, 4, 7, 13, 21, and 22 (prior to necropsy).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

The amount of food consumed was determined twice weekly prior to pairing (both sexes) and during the post-pairing phase for males. Daily food consumptions were recorded for females from GD 0 to 20; and from LD 1 to 21. Consumption was calculated as g/animal/ day.

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: withdrawn from the abdominal aorta at necropsy on Study Day 99 (Post-Pairing Day 14 for males) or on LD 22 for females
- Anaesthetic used for blood collection: Yes, Females were sacrificed by isoflurane anesthesia on LD 22 (those that achieved pregnancy) or 26 days post-coitum(the female that did not litter) after an overnight
period without food.
- Animals fasted: Yes
- How many animals: all animals in each group
- Parameters checked in table [No.3] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: withdrawn from the abdominal aorta at necropsy on Study Day 99 (Post-Pairing Day 14 for males) or on LD 22 for females
- Animals fasted: Yes
- How many animals: all animals in each group
- Parameters checked in table [No.4] were examined.

URINALYSIS: No (Quantitative assessments, including urine pools & fecal boli assessments were conducted under the functional observational battery [FOB])

NEUROBEHAVIOURAL EXAMINATION: Yes (Table 2)
All adult animals were assessed by detailed clinical observations, quantitative assessments, and for elicited responses. Males were assessed once prior to dose initiation and once weekly thereafter. Females were assessed once prior to dose initiation; once weekly during the pre-pairing and pairing phases; on GD 0, 7, 14, and 20; and on LD 1, 7, and 21.

IMMUNOLOGY: No

Blood samples for hematology (1 x 0.5 mL [EDTA], nominal), coagulation (1 x 0.5 mL [trisodium citrate], nominal), clinical chemistry (1 x 0.6 mL [lithium heparin], nominal), and bile acids (1 x 0.5 mL [serum separator tube], nominal) were withdrawn from the abdominal aorta at necropsy on Study Day 99 (Post-Pairing Day 14 for males) or on LD 22 for females. Samples were collected after animals were fasted overnight.

Each sample for bile acids was gently inverted 10 times and was left to clot for 30 minutes stored at room temperature (15 to 25°C). Samples were analyzed at Covance.

Blood samples for thyroid hormone (2 x 0.6 mL [serum separator tubes], nominal) were withdrawn from the jugular vein of adult males prior to necropsy on Post-Pairing Day 14. Blood samples for thyroid hormone (1 x 1.2 mL [serum separator tubes], nominal) were withdrawn from the abdominal aorta of adult females at necropsy on LD 22. Samples were collected after animals were fasted overnight. Sampling was performed at a similar time on each occasion. Adult male samples, one sample was used for analysis; one retained as a spare. Adult female samples were split into two equal aliquots of 0.6 mL; one sample was used for analysis, one was retained as a spare. The spare sample was retained in storage until report finalization.

Thyroid hormone sampling was performed at a similar time on each occasion (between 09:00 and 13:00); samples were protected from light until frozen storage. Each blood sample for thyroid hormone analysis was gently inverted 10 times and was stored at room temperature (15 to 25°C) and centrifuged at 2300g for 10 minutes at approximately 4°C. The resultant serum was separated, transferred to uniquely labeled amber polypropylene tubes, and frozen at <-20°C. Samples were centrifuged and aliquoted within 2 hours of collection, and were analyzed at Covance.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

- Male animals: Males were sacrificed on Day 99 of study (Post-Pairing Day 14), by isoflurane anaesthesia and after an overnight period without food. Sacrifices were carried out in controlled randomization order (one cage of animals from Group 1, 4, 2 then 3, then repeated). Once a suitable deep plane of anaesthesia was established, the major blood vessels were severed to exsanguinate the animal.

- Maternal animals: Females were sacrificed by isoflurane anaesthesia on LD 22 (those that achieved pregnancy) or 26 days post-coitum(the female that did not litter) after an overnight period without food. Once a suitable deep plane of anaesthesia was established, the major blood vessels were severed to exsanguinate the animal. Littered females were sacrificed in a controlled randomization order (i.e., females with the same day of mating/littering were necropsied together). The uterus of the apparently non-pregnant female (R0404 [Group 1]) was immersed in a 10% ammonium sulfide solution to reveal any evidence of implantation.

- Gross necropsy consisted of:
Females: Upon sacrifice, macroscopic examinations were conducted, and all lesions were recorded
Males: Upon sacrifice, macroscopic examinations were conducted, and all lesions were recorded

HISTOPATHOLOGY: Yes (see table 6)
The tissues indicated in Table [6] were prepared for microscopic examination and weighed, respectively.

Organ weights, as indicated in Table 6 were recorded at each scheduled sacrifice, excluding the nonlittered female. Organs were dissected free from fat and other contiguous tissue and weighed before fixation. Left and right organs were weighed together. The tissues listed in Table 6 were obtained from all adult animals within each group. Tissues were retained in 10% neutral-buffered formalin, unless otherwise indicated.

All tissues denoted by ‘E’ in Table 6 from all adult animals were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, and stained with hematoxylin and eosin. Additional sections of testes and epididymides were also stained with periodic acid-Schiff (PAS) for spermatogenic staging for all males.
Statistics:
Please see 'Any other information on materials and methods incl. tables' for information on statistics.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The clinical observations noted were predominantly related to thinning or staining of the fur, sore or lesions on the skin, and raised hair or hair loss on various body parts of control or test article-treated animals.

Other observations were vocalization for one male each administered diets containing 375 (animal R0105 on pre-pairing days 15, 22, 29 and 36) or 6000 ppm (animal R0301 on pre-pairing Day 22); thin appearance for two males administered diets containing 6000 ppm (R0319, R0320), noted on a few occasions (pre-pairing Days 50 and 57); impaired mobility, transiently observed for one male (animal R0307) administered diet containing 6000 ppm commencing on Day 51 until Day 64 of pre-pairing; minimally to moderately damaged paw(s) for one male each administered diets containing 375 (animal R0110 on pairing days 1, 7 and 14 and post pairing Day 7) or 6000 ppm (animal R0302 on postpairing Day 14); and one obese male (animal R0202) administered diet containing 1500 ppm at the end of pairing and into post pairing (pairing day 14 and post pairing Day 7).

Two females administered diet containing 375 ppm (Animal R0512 on Lactation Day 21 and Animal R0515 on Gestation Days 0 and 7) had one or both eyes protruding, and one female (Animal R0706) administered diet containing 6000 ppm had minimal excretion/discharge from the urogenital area on Gestation Day 15 however littering data for this female did not indicate a loss of implantations.

The clinical observations noted in the study were considered incidental as they were either present in controls and/or comparable with observations routinely noted at this laboratory for rats, infrequent, or showed no relationship to dose or trend over the duration of dosing.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean male body weight gain when administered diet containing 6000 ppm throughout the dosing period was lower than the Controls (approximately 13% from pre-pairing day 1 to post pairing day 13, 154.8 g versus 177.1 g control value). However, statistical significance was not achieved, except pre-pairing days 1 to 36 where the body weight change was statistically significantly reduced by approximately 21%. These reduction in body weight gain by up to 21% compared to control was considered adverse effect of test article. Mean male body weight gain over the dosing period for males administered diets containing 375 or 1500 ppm was similar to the controls.

Body weight gain for females administered diet containing 6000 ppm was reduced by approx. 23% on pre-pairing day 1 to 15 and was considered adverse effect of test article due to lack of effects on food consumption. On GD 14 to 20 mean body weight gain was statistically increased for females (83.7g compared with 71.6 g of control) administered diet containing 6000 ppm. These transient changes had no effect on the clinical condition of the adult females or the litters and was considered incidental and unrelated to the test article. The overall weight gain from pre-pairing day 1 to lactation day 21 was approximately 15% higher in females administered 6000 ppm compared to control.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was not affected in the study.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Hemoglobin distribution width (HDW) was statistically significantly increased (3.06 g/dL) in males administered diet containing 6000 ppm, compared with controls (2.69 g/dL). This HDW increase occurred in the absence of effects on absolute reticulocyte counts and other RBC parameters, therefore, this is of uncertain mechanism and unlikely relationship to test article due to the absence of changes in other erythroid parameters.

Eosinophil counts were statistically significantly decreased in males administered diets containing 375, 1500, or 6000 ppm, compared with control. However, the group mean values were within the historical control data range (0.04 x 10^9/L to 0.30 x 10^9/L). The range of values were within or near the range of the controls and therefore considered most consistent with normal variability. The changes in eosinophil counts were therefore not test article-related.

Statistically significantly decreased hemoglobin (HB) and packed cell volume (PCV) were noted in females administered diet containing 6000 ppm. The mean and individual values were within the historical control range (hemoglobin 13.3 to 16.9 g/dL; PCV: 39.6 to 51.3%). These changes were considered not to be an adverse effect of the test article.

Statistically significantly increased WBC (10.3x10^9/L compared with 8.0x10^9/L of control) and large unstained cells (LUC) - (0.10 x 10^9/L compared with 0.05 x 10^9/L of control) were noted in females administered diet containing 6000 ppm. The mean and most individual values of WBC and LUC were within the historical control range (WBC = 4.4 to 14.6 10^9/L; LUC = 0.02 to 0.18 x 10^9/L). Test article-related statistically significantly increased neutrophil was noted in females administered diets containing 1500 or 6000 ppm (3.13 and 3.08 x 10^9/L compared with 2.16 x 10^9/L of control) and was outside the range of historical control data (0.36 to 2.59 x 10^9/L). In the absence of evidence of inflammation this finding was considered not to be adverse.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significantly increased cholesterol was noted in males and females administered diet containing 6000 ppm (males = 1.5 mmol/L compared with 1.2 mmol/L of control; females = 1.9 mmol/L compared with 1.5 mmol/L of control). Individual values of test article treated animals were near or within the range of concurrent control and also were within the historical control range (males = 1.2 to 2.6 mmol/L; females = 1.1 to 3.5 mmol/L). Therefore, the changes were considered not test article related.

Aspartate aminotransferase activity (AST) increased (110 IU/L compared with 94 IU/L of control) in females administered diet containing 6000 ppm. The mean and individual values were within the historical control range (58 to 160 IU/L). Additionally, the finding corroborated with microscopic liver changes, which were associated with an adaptive response. Thus, these findings were considered not adverse effects of the test article.

Individual females administered 1500 ppm (animals R0602, R0604, R0611, R0616) or 6000 ppm (animals R0702, R0706, R0711, R0713, R0716) had decreased globulin concentration with secondary increase in albumin:globulin ratio. Therefore, mean albumin:globulin ratio was statistically significantly increased (2.5 compared with 1.9 of control), and globulin was statistically significantly decreased (18 and 17 g/L, respectively compared with 21 g/L of control) in females administered 1500 or 6000 ppm. However, these changes were though uncertain relationship to test article were considered not adverse due to individual values were within the historical control range (albumin:globulin ratio: 1.1 to 3.4; globulin: 13 to 34 g/L).

Total protein decreased (57 g/L compared with 60 g/L of control) in females administered 6000 ppm. Individual values were within the concurrent control and the historical control range (total protein: 52 to 85 g/L). Thus, the changes were considered not adverse effects of the test article.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No specific clinical observations (FOB) were noted in test article-treated groups, compared with controls, and the findings were considered incidental as they were present in controls, infrequent, showed no relationship to dose, or were in single pups in a given group.

Quantitative assessments, including urine pools, fecal boli, latency, rears, foot splay, forelimb grip, and hindlimb grip, revealed no effects in males or females.

Locomotor activity, including total activity, mobility and rearing, was not affected in parent males (postpairing) and females (lactation).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test article-related changes in liver and kidney weights were recorded, compared with concurrent controls.

Group mean absolute liver weights and body weight ratios were higher for both sexes fed diets containing 6000 ppm, compared with concurrent controls. The differences achieved statistical significance for males and females and a magnitude of change of approximately 35% in females.

Group mean absolute kidney weights and body weight ratios were marginally higher for males fed diets containing 6000 ppm, compared with concurrent controls. The differences achieved statistical significance (p<0.001).

None of the other absolute or organ weight ratio changes were considered test article-related as they were small in magnitude, not dose-dependent, inconsistent between sexes, due to normal interanimal variability, and/or lacked a microscopic correlate.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Upon macroscopic examination, test article-related findings were noted in the liver. An increased incidence of large appearance was recorded for females fed diets containing 6000 ppm.

No other macroscopic findings considered related to the test article were recorded.

Other tissues were macroscopically unremarkable, or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain and age at this laboratory.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Upon microscopic examination, test article-related findings were recorded for the liver, thyroid, and kidney. In the liver, diffuse hepatocyte hypertrophy was noted in both sexes fed diets containing 6000 ppm.

This finding was characterized by the presence of enlarged hepatocytes without consistent zonal pattern and correlated with the large appearance noted macroscopically and the increased organ weight. Hepatocyte hypertrophy is commonly observed as an adaptive response associated with the metabolism of xenobiotics or their metabolites (Greaves, 2012, Hallet al., 2012).

In the thyroid, an increased incidence and severity of follicular cell hypertrophy was recorded for both sexes fed diets containing 6000 ppm. This diffuse finding was characterized by increased height of the follicular epithelium and reduced amounts of colloid leading to decreased follicular diameter.

In the kidney, an increased incidence and severity of pigment was recorded for males fed diets containing 1500 or 6000 ppm. This finding was characterized by the presence of yellow-brown cytoplasmic droplets in renal tubular epithelial cells. In addition, there was a marginal increase in incidence and severity of hyaline droplets in males fed diets containing 6000 ppm.

No other test article-related microscopic findings were recorded. Microscopic findings in other tissues were generally infrequent, of a minor nature, and consistent with the usual pattern of findings in rats of this strain and age at this laboratory.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid Hormone Assessment:
The thyroid stimulating hormone (TSH) and thyroxine (T4) concentrations were not statistically significantly different in test article-treated groups, compared with control. However, dose-related increased TSH and dose-related decreased T4 concentrations were noted in both sexes administered diet containing 375, 1500 or 6000 ppm. In animals administered diet containing 6000 ppm, these changes corroborated with a microscopically increased incidence and severity of follicular cell hypertrophy in the thyroid gland, characterized by increased height of the follicular epithelium and reduced amounts of colloid leading to decreased follicular size. These findings were considered not adverse as the microscopic changes in the thyroid were considered adaptive changes and also secondary to diffuse hepatocyte hypertrophy. In the rat, thyroid follicular cell hypertrophy is generally considered an adaptive change due to increased thyroid hormone metabolism in the liver and is commonly associated with liver cell hypertrophy (McClain, 1989; Hardisty and Boorman, 1990; Maronpot and Brix; Curran and DeGroot, 1991; Ennulat et al., 2010).
Key result
Dose descriptor:
NOAEL
Effect level:
1 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic Toxicity
Remarks on result:
other: equivalent to 67 mg/Kg/day (males) and 151.83 mg/Kg/day (females)
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
6 000 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Formulation Analysis:

Formulations prepared for use at the beginning and end of the dosing period were analyzed to determine the achieved concentration. The mean % nominal concentration of formulated diet were between accepted criteria of 85 to 115% and with a relative standard deviation (RSD) of ≤10.0%. Test article was not detected in Group 1 control samples.

Test Article Consumption:

During the pre-pairing phase, the average test article consumption for males was 18.2, 73.3, or 298.8 mg/kg/day for those administered 375, 1500, or 6000 ppm, respectively. During the post-pairing phase, the average test article consumption was 15.7, 60.7, or 260.2 mg/kg/day for those administered 375, 1500, or 6000 ppm, respectively.

 

During the pre-pairing phase, the average test article consumption for females was 23.6, 99.2, or 367.3 mg/kg/day for those administered 375, 1500, or 6000 ppm, respectively. During the gestation phase, the average test article consumption was 25.3, 106.2, or 410.7 mg/kg/day for those administered 375, 1500, or 6000 ppm, respectively, and during the lactation phase, the average test article consumption was 65.5, 250.1, or 974.7 mg/kg/day for those administered 375, 1500, or 6000 ppm, respectively.

Table 7. Summary of Body Weight Gain per Interval (Males)

Group

(Dose Level ppm)

 

Data Presented in "g" Interval X through X

Phase

Pre-pairing

Pre-pairing 1 -

Day

1-8

1-15

1-22

1-29

1-36

1-43

1-50

1-57

1-64

1-71

Pairing 7

Pairing 14

Post Pairing 7

Post Pairing 13

1

Control (0)

Mean

32.8

52.5

70.4

81.9

102.9

122.5

128.0

135.4

150.3

157.8

163.2

159.7

178.9

177.1

SD

15.26

20.96

20.95

26.08

26.90

30.01

34.26

31.42

37.13

42.57

39.33

39.35

41.73

38.23

N

20

20

20

20

20

20

20

20

20

20

20

20

20 

 20

 

2

(375)

Mean

29.0

46.5

64.9

77.1

96.8

104.8

119.0

129.7

144.2

155.4

156.7

163.5

176.5

173.4

SD

16.22

19.78

21.52

24.06

28.28

29.85

33.96

31.56

34.09

36.21

41.05

39.29

44.84

40.95

N

20

20

20

20

20

20

20

20

20

20

20

20

 20

 20

 

3

(1500)

Mean

33.7

54.8

71.7

86.4

103.9

116.5

126.7

142.7

154.0

166.5

165.7

163.7

183.6

180.7

SD

21.75

25.35

28.83

30.52

33.42

37.74

40.66

40.31

43.96

45.45

47.47

52.32

51.29

48.66

N

20

20

20

20

20

20

20

20

20

20

20

20

 20

 20

 

4

(6000)

Mean

26.4

44.0

59.0

70.7

81.7*

94.1

99.1

111.6

125.9

137.8

135.9

137.9

156.1

154.8

SD

11.32

15.00

14.85

14.18

16.20

20.23

40.12

27.45

28.70

33.36

33.44

40.24

32.89

31.96

N

20

20

20

20

20

20

20

20

20

20

20

20

20

20

 

Statistics

AT

AT

AT

AT

AT

AT

AT

AT

AT

AT

AT

AT

AT

AT

* P<=0.05

** P<=0.01

*** P<=0.001

A = ANOVA and Dunnett's

T = Rank-transformed data

 

Table 8. Summary of Body Weight Gain per Interval (Females)

Group

(Dose Level ppm)

 

PA: 1-

PA: 8

PA: 8-

PA: 15

GD: 0- GD: 7

GD: 7- GD: 14

GD: 14- GD: 20

GD: 20- LA: 1

LA: 1- LA: 4

LA: 1- LA: 7

LA: 7- LA: 13

LA: 13- LA: 21

LA: 21- LA: 22

1

Control (0)

Mean

-1.7

7.9

23.8

27.9

71.6

-100.6

9.1

12.7

15.7

0.9

-32.0

SD

11.92

9.59

8.65

9.21

17.60

27.29

12.11

14.28

13.61

22.23

11.49

N

20

20

16

16

16

16

19

19

19

19

17

 

2

(375)

Mean

-0.2

5.9

21.2

28.3

76.8

-102.3

11.1

10.0

13.7

-0.7

-34.1

SD

5.65

6.43

7.21

6.64

12.26

17.42

14.79

13.77

15.81

12.17

9.02

N

20

20

20

20

20

20

20

20

20

20

14

 

3

(1500)

Mean

1.2

7.8

19.7

29.5

79.8

-101.2

7.3

15.8

12.5

-1.9

-35.9

SD

7.68

11.21

8.52

6.03

9.85

13.52

18.37

13.85

14.07

14.51

12.12

N

20

20

20

20

20

20

20

20

20

20

18

 

4

(6000)

Mean

0.9

3.8

18.9

33.3

83.7*H

-109.9

12.3

8.1

20.1

0.6

-38.5

SD

6.33

4.86

8.43

6.25

11.49

16.32

8.53

7.19

11.77

14.46

6.21

N

20

20

20

20

20

20

20

20

20

20

11

PA - Pre-pairing

GD - Gestation

LA - Lactation

*H = Dunnett Exact Homogeneous Test Significant: 0.05 level

Table 9. Summary of Hematology Results

Group

(Dose Level ppm)

Sex

Males

 

Females

Parameter

HDW

g/dL

E

109/L

MPV fL

HB

g/dL

PCV

%

WBC

109/L

N

109/L

LUC. 109/L

N%

%

PT

sec

Phase

Post Pairing

Post Pairing

Post Pairing

Lactation

Lactation

Lactation

Lactation

Lactation

Lactation

Lactation

Day

14

14

14

22

22

22

22

22

22

22

1

Control

(0)

Mean

2.69

0.15

7.4

15.4

47.6

8.0

2.16

0.05

27

24.0

SD

0.130

0.053

0.42

0.67

2.16

2.13

0.811

0.023

7.4

1.59

N

17

17

17

19

19

19

19

19

19

18

 

 

2

(375)

Mean

2.79

0.11*

7.7

15.7

48.5

8.6

2.51

0.06

29

23.5

SD

0.226

0.040

0.54

0.57

1.81

1.89

0.689

0.037

7.2

1.38

N

20

20

20

20

20

20

20

20

20

20

 

 

3

(1500)

Mean

2.82

0.11**

7.8*

15.6

48.4

9.2

3.13**

0.06

34*

23.0

SD

0.182

0.041

0.36

0.50

1.65

3.01

1.475

0.081

7.5

1.09

N

19

19

19

20

20

20

20

20

20

20

 

 

4

(6000)

Mean

3.06***

0.11*

7.4

14.9**

45.5**

10.3*

3.08*

0.10*

31

22.7**

SD

0.267

0.030

0.45

0.50

1.79

2.76

0.864

0.073

6.2

0.74

N

18

18

18

20

20

20

20

20

20

20

 

Statistics

AT

A

A

A

A

A

A

A

A

A

* P<=0.05

** P<=0.01

*** P<=0.001

A = ANOVA and Dunnett's

T = Rank-transformed data

HDW = Haemoglobin Distribution Width

E = Eosinophils

MPV = Mean Platelet Volume

HB = Haemoglobin

PCV = Packed Cell Volume

WBC = White Blood Cells

N = Neutrophils

LUC = Large Unstained Cells

N% = Neutrophils %

PT = Tox Prothrombin time

Table 10. Summary of Clinical Chemistry Results

Group

(Dose Level ppm)

Sex

Males

 

Females

Parameter

CHOL mmol/L

HCRE umol/L

ASTP

IU/L

CHOL mmol/L

TP

g/L

ALB

g/L

GLOB

g/L

A\G RATIO

GLUC mmol/L

Phase

Post Pairing

Post Pairing

Lactation

Lactation

Lactation

Lactation

Lactation

Lactation

Lactation

Day

14

14

22

22

22

22

22

22

22

1

Control

(0)

Mean

1.2

29

94

1.5

60

39

21

1.9

7.6

SD

0.23

3.0

14.3

0.36

3.6

2.9

2.9

0.38

1.75

N

20

20

19

19

19

19

19

19

19

 

2

(375)

Mean

1.3

26*

97

1.5

61

41

20

2.2

9.0*

SD

0.26

3.9

15.6

0.26

2.4

2.9

2.8

0.34

2.09

N

20

20

20

20

20

20

20

20

20

 

3

(1500)

Mean

1.4

28

104

1.7

60

42**

18**

2.5***

9.8***

SD

0.27

2.2

15.3

0.28

3.0

3.5

2.9

0.54

1.76

N

20

20

20

20

20

20

20

20

20

 

4

(6000)

Mean

1.5*

25**

110**

1.9***

57*

41

17***

2.5***

8.7

SD

0.28

3.0

15.2

0.28

2.5

2.0

2.6

0.46

1.38

N

20

20

20

20

20

20

20

20

20

 

Statistics

A

AT

A

A

A

A

A

AT

A

* P≤0.05

** P≤0.01

*** P≤0.001

A = ANOVA and Dunnett's

T = Rank-transformed data

CHOL: Total Cholesterol

HCRE: Enzymatic Creatinine

ASTP: Aspartate Aminotransferase

TP: Total Protein

ALB: Albumin

GLOB: Globulin

A / G: Albumin/Globulin Ratio

GLUC: Glucose

Table 11. Summary of Thyroid Hormone Analysis (Males)

Group

(Dose Level ppm)

Parameter

TSHI µIU/mL

IMT4 nmol/L

Phase

Post Pairing

Post Pairing

Day

14

14

1

Control

(0)

Mean

0.75

64

SD

0.561

11.5

N

20

11

 

2

(375)

Mean

0.76

63

SD

0.575

8.7

N

20

16

 

3

(1500)

Mean

1.04

61

SD

1.200

12.7

N

20

11

 

4

(6000)

Mean

1.46

54

SD

1.694

10.1

N

20

14

 

Statistics

AT

A

A = ANOVA and Dunnett's

T = Rank-transformed data

 

Table 12. Summary of Thyroid Hormone Analysis (Females)

Group

(Dose Level ppm)

Parameter

TSHI µIU/mL

IMT4 nmol/L

Phase

Lactation

Lactation

Day

22

22

1

Control

(0)

Mean

0.77

44

SD

0.466

10.3

N

12

12

 

2

(375)

Mean

0.81

48

SD

0.602

11.5

N

19

19

 

3

(1500)

Mean

1.16

43

SD

0.692

13.2

N

14

14

 

4

(6000)

Mean

1.47

37

SD

1.016

6.4

N

16

16

 

Statistics

AT

A

A = ANOVA and Dunnett's

T = Rank-transformed data

Conclusions:
Daily oral dietary administration of 375, 1500, or 6000 ppm of 1,2-Benzenedicarboxylic acid, benzyl C7-9-branched and linear alkyl esters to groups of male and female Crl:CD (Sprague Dawley [SD]) rats for 98 days (males during pre-pairing, pairing, and post pairing phases) or up to 65 days (females during pre-pairing, pairing, gestation, and lactation phases) resulted in reduction in body weight gain for males and females administered 6000 ppm; an increased incidence of large appearance of liver for females administered 6000 ppm and increased group mean absolute and relative (to body weight) liver weights for animals administered 6000 ppm, compared with concurrent controls.
 
Increased haemoglobin distribution width (HDW) concentrations in males administered 6000 ppm was considered uncertain mechanism and unlikely to be related to the test material. Treatment-related microscopic findings were observed in the liver and thyroid of animals administered 6000 ppm and were considered to be adaptive rather than treatment-related. The increased pigment in the kidneys for males administered 1500 or 6000 ppm was likely to be non-adverse in the absence of any tubular degeneration, necrosis or inflammation.
 
Based on the results of this study, dietary exposure of 1500 ppm (males = 67 mg/Kg/day; females = 151.83 mg/Kg/day) is considered to be the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of the F0 generation.
Executive summary:

A key combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in the rat was conducted. Repeated dose administration of the test material (1,2-Benzenedicarboxylic acid, benzyl C7-9-branched and linear alkyl esters; CAS# 68515-40-2) was evaluated for systemic effects and to screen for effects on male and female reproductive performance (i.e. gonadal function, mating behavior, conception, development of the conceptus and parturition), and offspring growth until Lactation Day 21.

 

Four groups of Crl:CD(SD) rats (20/sex/concentration) were administered 0 (control article), 375, 1500, or 6000 ppm of test material in diet, for a duration of 98 days for males or up to 65 days for females. The control group was provided access to basal diet ad libitum.

 

Adults were observed for clinical effects, body weight, food consumption, motor activity, grip strength and foot splay, estrous cycling, mating, fertility and pregnancy indices, and offspring parameters. Pup clinical observations, litter size, sex, and body weights were recorded. Anogenital distance was recorded on Postnatal Day (PND) 4, and nipple retention was also recorded for male pups on PND 13. Complete necropsies were performed on all animals, and any macroscopic abnormalities were noted. Blood samples for hematology, clinical chemistry, bile acids and thyroid hormone assessments were collected at necropsy from all adults. Additionally, samples were collected at necropsy from selected PND 21 pups for clinical chemistry, and PND 4 and 21 pups for thyroid hormone assessments, thyroid weight recording and macroscopic examination. Organ weights were recorded and microscopic examinations were performed.

 

No test material-related mortality or clinical signs of toxicity were observed. Reduction in body weight gain by up to 21% and 23% was observed in males and females, respectively administered diet containing 6000 ppm. This was considered to be adverse and treatment-related. Food consumption was not affected by oral exposure to the test material.

 

During the pre-pairing phase, the average test material consumption for males was 18.2, 73.3, or 298.8.26 mg/Kg/day for those administered 375, 1500, or 6000 ppm, respectively. During the pre-pairing phase, the average test material consumption for females was 23.6, 99.2, or 367.3 mg/Kg/day for those administered 375, 1500, or 6000 ppm, respectively. During the post-pairing phase, the average test material consumption for males was 15.7, 60.7, or 260.2 mg/Kg/day for rats administered 375, 1500, or 6000 ppm, respectively. During the gestation phase, the average test material consumption was 25.3, 106.2, or 410.7 mg/Kg/day for rats administered 375, 1500, or 6000 ppm, respectively, and during the lactation phase, the average test material consumption was 65.5, 250.1, or 974.7 mg/Kg/day for rats administered 375, 1500, or 6000 ppm, respectively.

 

There were no adverse findings in either sex during the functional observational battery and locomotor activity assessments.

 

No effects on clinical chemistry parameters were observed in parental animals. Haematology resultsrevealed statistically significantly increased haemoglobin distribution width (HDW) in males administered 6000 ppm. In the absence of changes in red blood cell count (RBC), hemoglobulin (HB), mean corpuscular volume (MCV), or red cell distribution width (RDW), compared with control, the change in HDW was not considered not to be an adverse treatment-related effect. Statistically significantly increased neutrophil counts were observed in females administered 1500 or 6000 ppm and was considered treatment-related. However, in the absence of evidence of inflammation, this finding was not considered to be adverse.

 

At necropsy, treatment-related findings were observed in the liver. An increased incidence of large appearance was recorded for females fed diets containing 6000 ppm. No other macroscopic findings considered related to the test material were recorded. Other tissues were macroscopically unremarkable, or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain and age at the laboratory.

 

Absolute and relative liver weights were higher for both sexes receiving 6000 ppm, attaining statistical significance (p<0.01 to p<0.001) with an increased magnitude of change observed in the females (approximately 35%), which also correlated with an increased incidence of large appearance macroscopically in females and microscopically in both sexes with diffuse hepatocyte hypertrophy, characterized by the presence of enlarged hepatocytes without consistent zonal pattern in the liver. This was associated with an increased incidence and severity of follicular cell hypertrophy in the thyroid gland, characterized by increased height of the follicular epithelium and reduced amounts of colloid leading to decreased follicular size, accompanied with decreased T4 levels (approximately 16% of control), and increased TSH levels (approximately 90% of control), respectively. The liver changes were also accompanied with increased aspartate aminotransferase activity in female rats administered 6000 ppm. Dose-related increased TSH and dose-related decreased T4 concentrations were observed in both sexes administered diet containing 375, 1500 or 6000 ppm and considered to be the result of adaptive changes in the thyroid gland and secondary to hepatocyte hypertrophy. Kidney weights (both absolute and relative) were increased for males given 6000 ppm, attaining statistical significance (p<0.001 to p<0.05, respectively), which also correlated microscopically with an increased incidence and severity of pigment for males given 1500 ppm or 6000 ppm. However, the increased weight and pigment in the kidneys for males administered 1500 or 6000 ppm was considered to be non-adverse in the absence of any associated tubular degeneration, necrosis or inflammation.

 

In conclusion, daily oral dietary administration of 375, 1500, or 6000 ppm of 1,2-Benzenedicarboxylic acid, benzyl C7-9-branched and linear alkyl esters to groups of male and female Crl:CD (Sprague Dawley [SD]) rats for 98 days (males during pre-pairing, pairing, and post pairing phases) or up to 65 days (females during pre-pairing, pairing, gestation, and lactation phases) resulted in reduction in body weight gain for males and females administered 6000 ppm; an increased incidence of largeappearance of liver for females administered 6000 ppm and increased group mean absolute and relative (to body weight) liver weights for animals administered 6000 ppm, compared with concurrent controls.

 

Increased haemoglobin distribution width (HDW) concentrations in males administered 6000 ppm was considered uncertain mechanism and unlikely to be related to the test material. Treatment-related microscopic findings were observed in the liver and thyroid of animals administered 6000 ppm and were considered to be adaptive rather than treatment-related. The increased pigment in the kidneys for males administered 1500 or 6000 ppm was likely to be non-adverse in the absence of any tubular degeneration, necrosis or inflammation.

 

Based on the results of this study, dietary exposure of 1500 ppm (males = 67 mg/Kg/day; females = 151.83 mg/Kg/day) is considered to be the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of the F0 generation.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
60 mg/kg bw/day
Study duration:
subacute
Species:
rat

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

In a reliable 3-week feeding study, groups of 6 male rats were given two different formulations of Santicizer 261 (one EU and one US) at around 60, 600 or 1200 mg/kg bw/day. A control group received normal diet. There were no effects at 60 mg/kg bw/day (the NOAEL), while at 600 mg/kg bw/day and above body weight gain was reduced (associated with reduced food consumption) and there were increases in liver weight and acyl-CoA oxidase activity (an indication of peroxisome proliferation). The EU version of the test material had a slightly greater effect, but the difference between the two versions was minimal (Center for Life Sciences and Toxicology, 1999).

 

As this oral toxicity study was of short duration and used only male rats, additional insights can be gained from longer-term studies on the structurally related compounds, di-isononyl phthalate (DINP) and butyl benzyl phthalate (BBP). In a reliable chronic toxicity and carcinogenicity study on DINP, no treatment-related effects were seen in male and female rats given a dietary level of 0.03% (about 17 mg/kg bw/day) for up to 2 years, compared to controls. Systemic toxicity (including effects on the liver, kidney and blood) and mononuclear cell leukemia were seen at 0.3% (about 175 mg/kg bw/day) and above (Lington et al. 1997). A 90-day dietary study on BBP in male and female rats reported an NOAEL of 151 mg/kg bw/day in the males. The higher doses of 381 and 960 mg/kg bw/day resulted in histopathological changes in the pancreas and increased relative kidney weight, while high-dose males also showed gross pathological changes in the liver. This reliable study was performed to a standard equivalent to GLP but prior to its introduction (British Industrial Biological Research Association, 1981).

In addition to this 90-day study, BBP has been tested in a number of other repeated dose dietary studies in rodents (mainly rats) with durations of between 14 days and 2 years. An NOAEL of 160 mg/kg bw/day, was obtained in a 14-day rat study which reported testicular damage from 480 mg/kg bw/day, while in 2-year studies the NOAELs were 240-300 mg/kg bw/day in rats and 780 mg/kg bw/day in mice (European Chemicals Bureau, 2007).

A key combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in the rat was conducted (Covance Laboratories Ltd., 2019; Klimisch score = 1). Repeated dose administration of the test material (1,2-Benzenedicarboxylic acid, benzyl C7-9-branched and linear alkyl esters; CAS# 68515-40-2) was evaluated for systemic effects and to screen for effects on male and female reproductive performance (i.e. gonadal function, mating behavior, conception, development of the conceptus and parturition), and offspring growth until Lactation Day 21.

 

Four groups of Crl:CD(SD) rats (20/sex/concentration) were administered 0 (control article), 375, 1500, or 6000 ppm of test material in diet, for a duration of 98 days for males or up to 65 days for females. The control group was provided access to basal diet ad libitum.

 

Adults were observed for clinical effects, body weight, food consumption, motor activity, grip strength and foot splay, estrous cycling, mating, fertility and pregnancy indices, and offspring parameters. Pup clinical observations, litter size, sex, and body weights were recorded. Anogenital distance was recorded on Postnatal Day (PND) 4, and nipple retention was also recorded for male pups on PND 13. Complete necropsies were performed on all animals, and any macroscopic abnormalities were noted. Blood samples for hematology, clinical chemistry, bile acids and thyroid hormone assessments were collected at necropsy from all adults. Additionally, samples were collected at necropsy from selected PND 21 pups for clinical chemistry, and PND 4 and 21 pups for thyroid hormone assessments, thyroid weight recording and macroscopic examination. Organ weights were recorded and microscopic examinations were performed.

 

No test material-related mortality or clinical signs of toxicity were observed. Reduction in body weight gain by up to 21% and 23% was observed in males and females, respectively administered diet containing 6000 ppm. This was considered to be adverse and treatment-related. Food consumption was not affected by oral exposure to the test material.

 

During the pre-pairing phase, the average test material consumption for males was 18.2, 73.3, or 298.8.26 mg/Kg/day for those administered 375, 1500, or 6000 ppm, respectively. During the pre-pairing phase, the average test material consumption for females was 23.6, 99.2, or 367.3 mg/Kg/day for those administered 375, 1500, or 6000 ppm, respectively. During the post-pairing phase, the average test material consumption for males was 15.7, 60.7, or 260.2 mg/Kg/day for rats administered 375, 1500, or 6000 ppm, respectively. During the gestation phase, the average test material consumption was 25.3, 106.2, or 410.7 mg/Kg/day for rats administered 375, 1500, or 6000 ppm, respectively, and during the lactation phase, the average test material consumption was 65.5, 250.1, or 974.7 mg/Kg/day for rats administered 375, 1500, or 6000 ppm, respectively.

 

There were no adverse findings in either sex during the functional observational battery and locomotor activity assessments.

 

No effects on clinical chemistry parameters were observed in parental animals. Haematology resultsrevealed statistically significantly increased haemoglobin distribution width (HDW) in males administered 6000 ppm. In the absence of changes in red blood cell count (RBC), hemoglobulin (HB), mean corpuscular volume (MCV), or red cell distribution width (RDW), compared with control, the change in HDW was not considered not to be an adverse treatment-related effect. Statistically significantly increased neutrophil counts were observed in females administered 1500 or 6000 ppm and was considered treatment-related. However, in the absence of evidence of inflammation, this finding was not considered to be adverse.

 

At necropsy, treatment-related findings were observed in the liver. An increased incidence of large appearance was recorded for females fed diets containing 6000 ppm. No other macroscopic findings considered related to the test material were recorded. Other tissues were macroscopically unremarkable, or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain and age at the laboratory.

 

Absolute and relative liver weights were higher for both sexes receiving 6000 ppm, attaining statistical significance (p<0.01 to p<0.001) with an increased magnitude of change observed in the females (approximately 35%), which also correlated with an increased incidence of large appearance macroscopically in females and microscopically in both sexes with diffuse hepatocyte hypertrophy, characterized by the presence of enlarged hepatocytes without consistent zonal pattern in the liver. This was associated with an increased incidence and severity of follicular cell hypertrophy in the thyroid gland, characterized by increased height of the follicular epithelium and reduced amounts of colloid leading to decreased follicular size, accompanied with decreased T4 levels (approximately 16% of control), and increased TSH levels (approximately 90% of control), respectively. The liver changes were also accompanied with increased aspartate aminotransferase activity in female rats administered 6000 ppm. Dose-related increased TSH and dose-related decreased T4 concentrations were observed in both sexes administered diet containing 375, 1500 or 6000 ppm and considered to be the result of adaptive changes in the thyroid gland and secondary to hepatocyte hypertrophy. Kidney weights (both absolute and relative) were increased for males given 6000 ppm, attaining statistical significance (p<0.001 to p<0.05, respectively), which also correlated microscopically with an increased incidence and severity of pigment for males given 1500 ppm or 6000 ppm. However, the increased weight and pigment in the kidneys for males administered 1500 or 6000 ppm was considered to be non-adverse in the absence of any associated tubular degeneration, necrosis or inflammation.

 

In conclusion, daily oral dietary administration of 375, 1500, or 6000 ppm of 1,2-Benzenedicarboxylic acid, benzyl C7-9-branched and linear alkyl esters to groups of male and female Crl:CD (Sprague Dawley [SD]) rats for 98 days (males during pre-pairing, pairing, and post pairing phases) or up to 65 days (females during pre-pairing, pairing, gestation, and lactation phases) resulted in reduction in body weight gain for males and females administered 6000 ppm; an increased incidence of largeappearance of liver for females administered 6000 ppm and increased group mean absolute and relative (to body weight) liver weights for animals administered 6000 ppm, compared with concurrent controls.

 

Increased haemoglobin distribution width (HDW) concentrations in males administered 6000 ppm was considered uncertain mechanism and unlikely to be related to the test material. Treatment-related microscopic findings were observed in the liver and thyroid of animals administered 6000 ppm and were considered to be adaptive rather than treatment-related. The increased pigment in the kidneys for males administered 1500 or 6000 ppm was likely to be non-adverse in the absence of any tubular degeneration, necrosis or inflammation.

 

Based on the results of this study, dietary exposure of 1500 ppm (males = 67 mg/Kg/day; females = 151.83 mg/Kg/day) is considered to be the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of the F0 generation.

 

No data are available on the repeated dose inhalation toxicity of S261a, but testing by this route is not considered necessary because exposure of humans via inhalation is unlikely (taking into account the low vapour pressure of other phthalates and the low likelihood of exposure to aerosols, particles or droplets of an inhalable size). Reassurance can be drawn from two reliable GLP-compliant studies on BBP, in which Sprague-Dawley rats were exposed by inhalation (6 hours/day on 5 days/week) for 13 weeks or 4 weeks (Environmental Health Laboratory, 1982a,b). The NOAECs were 0.218 and 1 mg/l, respectively, and the only effect observed after 13 weeks’ exposure at 0.789 mg/l was increased relative liver and kidney weights (but with no accompanying histopathological findings).


Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver

Justification for classification or non-classification