Bis(2-butoxyethyl) adipate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In the assessment and evaluation of the toxic characteristics of chemicals, the determination of toxicity using repeated doses by oral gavage is carried out after initial information on toxicity has been obtained from previous studies.
The test is performed in the rat. The rat is a widely used rodent species in toxicological studies and acceptable to regular authorities.
This study provides information on the possible health hazards which could arise from repeated exposure over a certain period of time.
The test item was orally administered daily in graduated doses to several groups of test animals, one dose level per group, for a period of 90 days. During the period of administration, the animals were observed precisely each day for signs of toxicity. At the end of the test the animals were euthanised and then examined macroscopically and histopathologically.
A battery of functional observation tests was performed to detect possible neurotoxic effects of the test item
No validated in vitro method is available for assessing systemic toxicity after repeated exposure.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-butoxyethyl) adipate

EC Number:

205-466-0

EC Name:

Bis(2-butoxyethyl) adipate

Cas Number:

141-18-4

Molecular formula:

C18H34O6

IUPAC Name:

1,6-bis(2-butoxyethyl) hexanedioate

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Proviplast 0142 (bis(2-butoxyethyl)adipate)
- Chemical Name: Bis(2-butoxyethyl)adipate
- Substance type: liquid
- Physical state: clear liquid with a very faint odour
- Analytical purity:98,83%
- Composition of test material, percentage of components: see Condifential details
- Purity test date: 2011-07-11
- Lot/batch No.:11701913 SA
- Expiration date of the lot/batch: 2012-12-14
- Storage condition of test material: store in tightly closed containers between -20 and +40°C

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Species/strain:healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source :Charles River, 97633 Sulzfeld, Germany
Sex:male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: approx. 7-8 weeks ol

Sex:

male/female

Details on test animals or test system and environmental conditions:

Housing and Feeding Conditions
-Full barrier in an air-conditioned room
-Temperature: 22  3°C
-Relative humidity: 55  10%
-Artificial light, sequence being 12 hours light, 12 hours dark
-Air change: 10 x / hour
-Free access to Altromin 1324 maintenance diet for rats and mice
-Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
-The animals were kept in groups of 2 animals / sex / group / cage in IVC cages (type III H, polysulphone cages) on Altromin saw fibre bedding
-Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
-Adequate acclimatisation period (at least 5 days)

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

An evaluation of the toxic characteristics of DBEA, using repeated doses by oral gavage is carried out .

Vehicle:

corn oil

Details on oral exposure:

The test item formulation was prepared at least once every ten days based on available stability data (Eurofins Munich study no. 156144) and stored at room temperature. The test item formulation was prepared with corn oil and administered daily during a 90-day treatment period to male and female animals. Dose volumes were adjusted individually based on weekly body weight measurements

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration analysis of formulation samples was determined in study weeks 1, 5, 9 and last for all dose groups. The mean recoveries observed in the LD, MD and HD groups were 102.1 %, 101.2 % and 103.9 % of the nominal concentration, respectively. Nominal concentrations were confirmed for all dose groups, as measured concentrations did not differ from nominal concentration by more than 10 %.
Homogeneity of formulation samples was investigated in study week 1, 5 and in last study week for the LD and HD group. The coefficients of variation of the different sampling locations (top, middle, bottom) were between 1.4% and 3.8% in the LD group and between 0.3% and 3.1% in the HD group. All samples were homogenous, as COV was below or equal 10%.

Duration of treatment / exposure:

The aim of this study was to assess the possible health hazards which could arise from repeated exposure of Bis(2-butoxyethyl) adipate via oral administration to rats over a period of 90 days.

Frequency of treatment:

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 90 days. Animals of an additional control group were handled identically as the dose groups but received corn oil, the vehicle used in this study. The 4 groups comprised of each 10 male and 10 female Wistar rats.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

350 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).

Control animals:

yes

Details on study design:

The test item formulation or vehicle was administered at a single dose to the animals by oral gavage. The application volume for all groups was 4 mL/kg body weight (Table 2).
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.
Table 2: Dose, Application Volume and Concentration
Group No. Group
Name Dose
[mg/kg bw] Application volume
[mL/kg bw] Concentration
[mg/mL]
1 C 0 4 0
2 LD 50 4 12.5
3 MD 200 4 50
4 HD 350 4 87.5
C = control, LD = low dose, MD = medium dose, HD = high dose

Positive control:

no

Examinations

Observations and examinations performed and frequency:

Body Weight and Food Consumption

The body weight was recorded once before the assignment to the experimental groups, on the first day of administration, weekly during the treatment period and on the day of necropsy.
Food consumption was measured weekly during the treatment period.

Clinical Observations

All animals were observed for clinical signs during the entire treatment period of 90 days.
General clinical observations were made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Detailed cage side observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were made outside the home cage in a standard arena once before the first administration and at least once a week thereafter.
Ophthalmological examinations using an ophthalmoscope were made on all animals before the first administration and in the last week of the treatment period.

Functional Observations

Once before the first exposure and once in the last week of exposure multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests . These tests were conducted in all animals

Haematology

Haematological parameters were examined at the end of the treatment period prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in EDTA-coated tubes.
The following haematological parameters were examined (Table 3).
Table 3: Haematology

Parameter Instrument Units
haematocrit value (Hct) ADVIA®120 (Siemens) %
haemoglobin content (Hb) ADVIA®120 (Siemens) g/dL
red blood cell count (RBC) ADVIA®120 (Siemens) 1012/L
mean corpuscular volume (MCV) ADVIA®120 (Siemens) fL
mean corpuscular haemoglobin (MCH) ADVIA®120 (Siemens) pg/erythrocyte
mean corpuscular haemoglobin concentration (MCHC) ADVIA®120 (Siemens) g/dL
reticulocytes (Re) ADVIA®120 (Siemens) %
platelet count (PLT) ADVIA®120 (Siemens) 109/L
white blood cells (WBC) ADVIA®120 (Siemens) 109/L
neutrophils (Neu) ADVIA®120 (Siemens) %
lymphocytes (Lym) ADVIA®120 (Siemens) %
monocytes (Mono) ADVIA®120 (Siemens) %
eosinophils (Eos) ADVIA®120 (Siemens) %
basophils (Baso) ADVIA®120 (Siemens) %
large unstained cells (Luc) ADVIA®120 (Siemens) %

Blood Coagulation

Coagulation parameters were examined at the end of the treatment period prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in citrate tubes.
The following coagulation parameters were examined (Table 4):

Table 4: Coagulation Parameters

Parameter Instrument Units
prothrombin time (PT) ACL 7000 (IL Instrumental) or Kugelkoagulometer (ADW) sec
activated partial thromboplastin time (aPTT) ACL 7000 (IL Instrumental) or Kugelkoagulometer (ADW) sec

Clinical Biochemistry

Parameters of clinical biochemistry were examined at the end of the treatment period prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in serum separator tubes.
The following parameters of clinical biochemistry were examined (Table 5):

Table 5: Clinical Biochemistry

Parameter Instrument Units
alanine aminotransferase (ALAT) Olympus AU 480 Beckman Coulter U/L
aspartate-aminotransferase (ASAT) Olympus AU 480 Beckman Coulter U/L
alkaline phosphatase (AP) Olympus AU 480 Beckman Coulter U/L
creatinine (Crea) Olympus AU 480 Beckman Coulter µmol/L
total protein (TP) Olympus AU 480 Beckman Coulter g/L
albumin (Alb) Olympus AU 480 Beckman Coulter g/L
urea Olympus AU 480 Beckman Coulter mmol/L
total bilirubin (TBIL) Olympus AU 480 Beckman Coulter µmol/L
total bile acids (TBA) Olympus AU 480 Beckman Coulter µmol/L
total cholesterol (Chol) Olympus AU 480 Beckman Coulter mmol/L
glucose (Gluc) Olympus AU 480 Beckman Coulter mmol/L
sodium (Na) Olympus AU 480 Beckman Coulter mmol/L
potassium (K) Olympus AU 480 Beckman Coulter mmol/L

Urinalysis

A urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals. Additionally, urine colour/ appearance was recorded.
The following parameters (Table 6) were measured using qualitative indicators (Heiland Urine Stripes URI 10SL).

Table 6: Urinalysis

Parameter Method/Device
specific gravity URI 10SL
nitrite URI 10SL
pH-value (pH) URI 10SL
protein URI 10SL
glucose URI 10SL
ketone bodies (Ket) URI 10SL
urobilinogen (UBG) URI 10SL
bilirubin (BIL) URI 10SL
erythroctes (Ery) URI 10SL
leukocytes (Leu) URI 10SL

Sacrifice and pathology:

Gross necropsy

One day after the last administration (study day 91) all animals were sacrificed using anesthesia (ketamine and xylazin) and were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

Organ Weight

The wet weight of the organs (Table 7) of all sacrificed animals was recorded as soon as possible. Paired organs were weighed together.

Table 7: Organs Weighed at Necropsy

Tissue/Organ Tissue/Organ
liver uterus with cervix
kidneys thymus
adrenals thyroid/ parathyroid glands
testes spleen
epididymides brain
prostate, seminal vesicles and
coagulating glands pituitary gland
ovaries heart

The following tissues (Table 8) from all animals were preserved in 4% neutral-buffered formaldehyde except eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70% ethanol.
Table 8: Preserved and Examined Tissues
Tissue/Organ Preserved at Necropsy Histopatho-logical Examination
adrenal glands x x
all gross lesions x x
aorta x x
brain (incl. medulla/pons, cerebellar and
cerebral cortex) x x
caecum x x
colon x x
duodenum x x
epididymides x x
eyes with optic nerve and Harderian gland x x
femur with knee joint x --
heart x x
ileum (including Peyer´s patches) x x
jejunum x x
kidneys x x
liver x x
lungs x x
lymph nodes (mandibular) x --
lymph nodes (mesenteric and axillary) x x
mammary gland area (male and female) x x
oesophagus x x
ovaries x x
oviducts x --
pancreas x x
pituitary x x
prostate and seminal vesicles with
coagulating glands as a whole x x
rectum x x
salivary glands (sublingual, submandibular) x x
sciatic nerve x x
skeletal muscle x x
skin x x
spinal cord (cervical, thoracic and lumbar segments) x x
spleen x x
sternum (with bone marrow) x x
stomach x x
testes x x
thymus x x
thyroid gland including parathyroid glands x x
tongue x --
trachea x x
ureters x --
urinary bladder x x
uterus with cervix and vagina x x


Histopathology
The afore-listed organs (Table 8) were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining for the animals of the groups 1 and 4 sacrificed at the end of the treatment period.
These examinations were extended to animals of all other dosage groups for treatment-related changes that were observed in the liver, spleen and bone marrow (sternum) of the HD group.
Any gross lesion macroscopically identified was examined microscopically in all animals.
Histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). Histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten, Switzerland (test site for histopathology). The study phases from test site 1 and 2 were performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted 18 May 2005 [SR 813.112.1] (Status as of 01 December 2012). Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites.
The principal investigator for histopathological tissue processing sent all raw data (including blocks, slides, paper raw data, statement of compliance and quality assurance statement) to the study director.
The principal investigator for histopathological evaluation provided the histopathology results to the study director by e-mail and sent a pathology phase report to the study director upon the completion of the study.

Statistics:

Parameters like body weight gain and food consumption were calculated as the difference in weight measured from one week to the next. Mean body weights are also presented as figures.
The relative organ weights were calculated in relation to the brain weight and in relation to the body weight (measured at necropsy) and are presented as percentage.
All results are reported in a tabular form (summarised in mean or summary tables and/or listed in individual data tables).
Analytical results and histopathological findings are presented in separate phase-reports attached to this report (Annex 1 and Annex 2).
With few exceptions, toxicology and pathology data was captured using the validated computerised system Ascentos® System (version 1.1.3, Pathology Data Systems Ltd.). If not possible raw data will be recorded on paper according to appropriate SOPs.
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights was performed for each gender by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with Ascentos 1.1.3 software (p<0.05 was considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

Salivation was noted dose dependently in 10/10 males and 9/10 females of the HD group on several days of treatment and in 2/10 females of the MD group on some days of treatment. Furthermore, moving the bedding was observed in all males and females of the HD group and in 4/10 females of the MD group. As the symptoms of salivation and moving the bedding were noted transiently after dose administration, these signs were considered to be a sign of discomfort due to a local reaction to the test item and not as an adverse systemic effect.
Haematuria was observed in one single female of the HD group on the second day of treatment and in 2 males (cage mates) of the HD group on day 37. A relation to the treatment with the test item cannot be excluded.
Low incidences of slight clinical signs like alopecia, crust or scratch on various body parts and transient nasal discharge and piloerection were noted in few males and/or females of the dose groups and/or the control group. These signs were considered as incidental.
During the weekly detailed clinical observation, slightly to moderately increased salivation was noted from week 3 onwards in the male HD group and from week 5 onwards in the female HD group which was related to the earlier daily dose administration of the animals. After approximately half of the study period, slight habituation of the animals to the open field and handling was noticed throughout all groups including control with slightly reduced activity and reactivity e.g. concerning response to handling, arousal, fear and/or head touch and occasionally upright lying body position instead of standing in the open field. Slight differences apart from salivation between the dose groups and the respective controls in some weeks of treatment and single deviating values in individual animals were considered as incidental. In few weeks, spontaneous activity was noted to be slightly more reduced in the male HD group (week 7, 11), the female HD group (week 8, week 10), the male MD group (week 8) and the male LD group (week 10) when compared to the respective controls. However, as this was seen without consistency and without relevant impact on other parameters like food consumption, it was not assumed to be toxicologically relevant.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred in the control or any of the dose groups during the treatment period of this study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In males and females, the mean body weight overall increased with the progress of the study in the control, the LD, the MD and the HD group. However, mean body weight gain was noted to be marginally lower in the first week after initiation of treatment in the male MD (21 % below controls, p < 0.05) and HD group (22 % below controls, p < 0.05) when compared to the control group. A tendency towards lower body weight gain in the first week of treatment was also observed in the female HD group (40 % below controls) but without achieving statistical significance. Furthermore, slightly to moderately and statistically significantly lower body weight gain was noted in the male HD group in week 7 (47 % below controls, p < 0.01) and week 10 (107 % below controls, p < 0.001) whereas a moderately higher body weight gain was seen in one single week 11 (117 % above controls, p < 0.05). Statistically significantly higher body weight gain in week 2 in the male LD group was seen without dose dependency and considered as incidental. For females, a slight but statistically significant mean body weight loss was noted in week 9 (-2.80 g compared to a slight gain of 3.80 g in controls, p < 0.01). No statistically or biologically significant effect was noted in the remaining study weeks or when related to the entire treatment period from day 1 to day 90 in the male and female dose groups. Furthermore, there was no effect on mean body weight in any of the dose groups. Thus, slight transient differences to body weight gain in single weeks of treatment in the dose groups were not considered as toxicologically relevant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

During the treatment period, there was no effect of toxicological relevance on food consumption in any of the dose groups. However, in correlation to the lower mean body weight change in the first week of treatment of the male MD and HD group, also slightly lower food consumption was observed when compared to the control group (12 % and 11 % below controls, respectively, each p < 0.01). Similarly, slightly lower food consumption was observed in the female HD group in the first week of treatment (26 % below controls, p < 0.001). As no such effect was noticed for the following weeks and total food consumption was comparable to controls, this was not considered as adverse.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmologic findings were observed in any of the animals of this study.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In males and females, slightly to moderately and statistically significantly lower red blood cell count (RBC) was noted in the MD and the HD group when compared to the respective controls with following a dose dependent pattern (males: 17 % and 28 %. below controls, each p < 0.001; females: 15 % and 20 % below controls, each p < 0.001). The same effect was noted for haemoglobin (HGB) (males: 10 % and 17 % below controls, p < 0.01 and p < 0.001, respectively; females: each 8 % below controls, each p < 0.01). Mean values for RBC of the male MD (7.511 x 1012/L) and HD group (6.568 x 1012/L) were below two standard deviations of the mean of historical control data (mean 9.3 1012/L, - 2 fold of SD: 8.2 x 1012/L). The same was noted for the female HD group (6.361 1012/L compared to historical control data mean of 8.3 x 1012/L, - 2 fold of SD: 6.7 x 1012/L).
The observed reduced red blood cell mass was accompanied by a marked increase of reticulocytes in the male and female MD and HD group. Mean percentage of reticulocytes was dose dependently and statistically significantly higher in the male MD and HD group (176 % above controls, p < 0.01, and 470 % above controls, p < 0.001, respectively) and the female MD and HD group (179 % above controls, p < 0.01, and 566 % above controls, p < 0.001, respectively) when compared to controls. Mean values of the male MD (3.797 %) and HD group (7.829 %) and the female MD (4.812 %) and HD group (11.483 %) were above maximum value of historical control data (males 2.8 %, females 4.2 %).
This marked regenerative response was correlated to an increase in cell volume. Thus, mean corpuscular volume (MCV) was noted to be slightly to moderately and statistically significantly higher in the male MD and HD group (11 % and 24 % above controls, each p < 0.001) and the female MD and HD group (15 % and 31 % above controls, each p < 0.001). Accordingly, the mean corpuscular haemoglobin concentration (MCHC) was noted to be slightly lower in the male HD group (7 % below controls, p < 0.001) and the female HD group (12 % below controls, p < 0.001). A marginal effect was also seen in the female MD group (6 % below controls, p < 0.001) but not the male MD group.
Furthermore, mean corpuscular haemoglobin (MCH) was slightly higher in the male MD and HD group (9 % and 15 % above controls, each p < 0.001) and the female MD and HD group (8 % above controls, p < 0.05, and 15 % above controls, p < 0.001).
Further slight but statistically significant difference to controls for haematological parameters was noted for haematocrit (HCT) in males but not females (male MD group 8 % below controls, p < 0.05, male HD group 10 % below controls, p < 0.01).
All other haematological parameters were within the normal range of variation. Thus, slight differences to controls for percentage of the different kind of leukocytes were not assumed to be biologically relevant.
Blood coagulation was also affected by the test item. Prothrombin time (PT) was noted to be marginally but statistically significantly higher in males and females of the HD group (11 % above controls, p < 0.001, and 7 % above controls, p < 0.05). As values were within historical control data, this marginal difference was not considered as adverse. No effect was seen for the other dose groups and for activated partial thromboplastin time (aPTT).

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant effects on clinical biochemistry parameters were found at the end of the treatment period of this study. All mean values were within the range of historical control data. However, statistically significantly higher values for total bile acids (TBA) were observed in the female MD and HD group (200 % above controls, p < 0.01, and 129 % above controls, p < 0.05) but did not follow a dose dependent pattern. Slightly higher mean value of TBA in the male MD and HD group compared to controls were seen without statistical significance and were based on deviating values of individual animals. Furthermore, mean values of TBA for males and females were within the normal range of variation so that no biological relevance was assumed.
The observation of statistically significantly higher urea in the female HD group (27 % above controls, p < 0.05) and statistically significantly lower total protein in the female HD group (8 % below controls, p < 0.05) was not assumed to be toxicologically relevant, as differences to the control group were slight, values were within the normal range of variation and no such effect was noted for males.

Urinalysis findings:

no effects observed

Description (incidence and severity):

All parameters of urinalysis of the dose groups at the end of the treatment period were not considerably different to the corresponding control group and were within the normal range of variation. Slightly deviating values for individual animals throughout all groups were considered as incidental, isolated findings without relation to the treatment with the test item

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No toxicologically relevant effects were observed in any of the parameters of the functional observation battery

Immunological findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant effects were observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period, a test item related effect was noted for liver weight in the dose groups of both genders. Slightly to moderately higher liver weight was recorded in the male LD group (absolute weight 13 % above controls, p < 0.05), the male MD group (absolute weight 11 % above controls without achieving statistical significance, relative weight (to body weight) 18 % above controls, p < 0.001) and the male HD group (absolute weight 15 % above controls, p < 0.01). In females, absolute liver weight was dose dependently higher in the LD (14 % above controls, p < 0.01), the MD (25 % above controls, p < 0.001) and the HD group (26 % above controls, p < 0.001).
Spleen weight was observed to be markedly higher in the male and female HD group when compared to the respective controls (absolute weight 71 % and 86 % above controls, p < 0.001, respectively). Furthermore, slightly higher spleen weight was also observed in the male MD group (absolute weight 23 % above controls, p < 0.05). A slight tendency towards higher spleen weight was noted in the female MD group (18 % above controls) but did not achieve statistical significance.
Marginally higher relative (to body weight) but not absolute kidney weight was seen in the male HD group (15 % above controls, p < 0.05). In females, a marginal tendency towards higher absolute kidney weight was noted in all dose groups, the LD, the MD and the HD group but without clear dose dependency (11 % above controls, p < 0.05; 8 % above controls without statistical significance; 11 % above controls, p < 0.05, respectively). Relative (to body weight) kidney weight was 17 % above controls (p < 0.001) in the female HD group compared to controls. Slightly higher relative (to body weight) but not absolute adrenals weight was seen in the male MD group (21 % above controls, p < 0.01). Without correlating microscopic lesions and without clear dose dependency, marginal to slight weight differences to controls in kidneys and adrenals were not assumed to be biologically relevant.
There were no further statistically or biologically significant differences between the dose groups and the control group for the remaining organ weight data.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Predominant macroscopic finding observed in the dose groups at necropsy was dose dependently enlarged spleen in one male of the MD group, 2 males of the HD group and 2 females of the HD group. This finding correlated with the microscopic finding of a test item induced dose dependent centrilobular to diffuse hepatocellular hypertrophy in the liver.
Few more gross pathological findings like discoloured (red) thymus, large sized adrenals, discoloured (red) pancreas, ovary cysts and malpositioned ovary were observed in single animals without dose dependency. Furthermore, dilated uterus was noted in several females at the end of the treatment period. The distributions among the groups were considered to be incidental, reflecting the usual individual variability. After histopathological evaluation, these findings were considered as normal background lesions that may be recorded in animals of this strain and age.

Neuropathological findings:

not examined

Description (incidence and severity):

No toxicologically relevant effects were observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period, a dose-dependent centrilobular to diffuse hepatocellular hypertrophy was recorded in the liver of all dosed groups (except males of the LD group).
Further liver findings consisted of hemosiderin deposits in females of the MD and HD group and males of the HD group. This finding was characterized by an accumulation of brownish pigment mainly in Kupffer cells. In the male and female HD group the hemosiderin deposits were also found in histiocytes along with slightly increased inflammatory cell foci.
Furthermore, there was an increase in extramedullary hematopoiesis and hemosiderin deposits in the spleen of the male and female MD and HD group.
A slight decrease in fatty replacement was recorded in the sternal bone marrow of the male and female MD and HD group.

Histopathological findings: neoplastic:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day (actual dose received)

System:

haematopoietic

Organ:

blood

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

On the basis of the present study, the 90-Day Repeated Dose Oral Toxicity study with Bis(2-butoxyethyl) adipate in male and female Wistar rats with dose levels of 50, 200, and 350 mg/kg body weight/day the following conclusions can be made:
No adverse effects of Bis(2-butoxyethyl) adipate were found at a dose level of 50 mg/kg body weight/day.
At dose levels of 200 and 350 mg/kg body weight/day dose dependent, adverse effects on parameters of haematology were noted. In males and females, red blood cell count and haemoglobin were slightly to moderately lower when compared to controls. This was associated with a marked compensatory increase in reticulocytes. This regenerative response was further correlated to an increase in cell volume (mean corpuscular volume) in the respective dose groups. Accordingly, the mean corpuscular haemoglobin concentration was noted to be slightly lower in males at 350 mg/kg body weight/day and females at 200 and 350 mg/kg body weight/day. Furthermore, mean corpuscular haemoglobin was slightly higher at 200 and 350 mg/kg body weight/day.
The microscopic findings at 200 and 350 mg/kg body weight/day of increased extramedullary hematopoiesis in the spleen, increased hemosiderin deposits in the spleen and liver (except for males at 200 mg/kg body weight/day) and a slight decrease in fatty replacement in the sterna bone marrow were considered secondary to the changes in the red blood parameters. The latter represented an activation of the bone marrow. Observed hemosiderin deposits in the liver were seen along with slightly increased inflammatory cell foci at 350 mg/kg body weight/day. Dose dependent centrilobular to diffuse hepatocellular hypertrophy was observed in the liver of all test item dosed groups (except for males at 50 mg/kg body weight/day) that correlated with statistically significantly increased absolute and/or relative liver weight. This finding was considered to represent an adaptive and non-adverse change. Observed microscopic findings in the spleen (increased extramedullary hematopoiesis and hemosiderin deposits) correlated with statistically significantly increased spleen weight and macroscopically observed enlarged spleen in some animals.
Though no clinical signs of anaemia became apparent in the animals during the study period, marked changes to haematological parameters beyond historical control data in at 200 and 350 mg/kg body weight/day in terms of reduced blood cell mass, regenerative processes and other associated changes to red blood cell parameters were considered as test item related, adverse effects.
Thus, the NOAEL of this study was considered to be 50 mg/kg body weight/day.

Executive summary:

No mortality occurred in the control or any of the dose groups during the treatment period of this study. Salivation and/or moving the bedding were observed occasionally in the MD and HD group after dose administration. This was considered as a slight sign of discomfort attributed to a local reaction to the test item and not as an adverse systemic effect. Likewise, during the weekly detailed clinical observation, slightly to moderately increased salivation was noted from week 3 onwards in the male HD group and from week 5 onwards in the female HD group which was related to the earlier daily dose administration of the animals. No toxicologically relevant effects were observed in any of the parameters of the functional observation battery. At the end of the treatment period, there were no biologically relevant differences in body temperature between the dose groups and the respective controls. No ophthalmologic findings were observed in any of the animals of this study. Haematuria was observed transiently in one single female of the HD group on the second day of treatment and in 2 males of the HD group on day 37. A relation to the treatment with the test item cannot be excluded. In both males and females, the mean body weight increased with the progress of the study in the control, the LD, the MD and the HD group. However, mean body weight gain was noted to be marginally but statistically significantly lower in the first week after initiation of treatment in the male MD (21 % below controls) and HD group (22 % below controls) when compared to the control group. A tendency towards lower body weight gain in the first week of treatment was also observed in the female HD group (40 % below controls) but without achieving statistical significance. Differences of the male MD and HD group and the female HD group to the respective controls in the first week of treatment were considered as slight and transient but not adverse effect of the treatment with the test item. There was no effect on mean body weight in any of the dose groups. Thus, further slight transient differences in body weight gain in single weeks of treatment in the dose groups were not considered as toxicologically relevant. There was no effect of toxicological relevance on food consumption during the treatment period of any group. However, in correlation to the lower mean body weight gain in the first week of treatment also slightly lower food consumption was observed in the male MD and HD group and the female HD group when compared to the control group. All parameters of urinalysis of the dose groups at the end of the treatment period were not considerably different to the corresponding control group and were within the normal range of variation. No toxicologically relevant effects on clinical biochemistry parameters were found at the end of the treatment period of this study. All mean values were within the range of historical control data. At the end of the treatment period, dose dependently, slightly to moderately lower red blood cell count (RBC) and haemoglobin (HGB) were noted in the male and female MD and HD group with achieving statistical significance when compared to the respective controls. Values of RBC in the MD and HD group were 17 % and 28 % below controls in males and 15 % and 20 % below controls in females, respectively. The observed reduced red blood cell mass was accompanied by a marked increase of reticulocytes in the male and female MD and HD group. Mean percentage of reticulocytes was dose dependently and statistically significantly higher in the male MD and HD group (approx. 2.8-fold and approx. 5.7-fold of control values) and the female MD and HD group (approx. 2.8-fold and approx. 6.7-fold of control values) when compared to controls. This marked regenerative response was correlated to an increase in cell volume. Thus, mean corpuscular volume (MCV) was noted to be slightly to moderately and statistically significantly higher in the male MD and HD group (11 % and 24 % above controls) and the female MD and HD group (15 % and 31 % above controls). Accordingly, the mean corpuscular haemoglobin concentration (MCHC) was noted to be slightly lower in the male HD group (7 % below controls) and the female HD group (12 % below controls). A marginal effect was also seen in the female MD group (6 % below controls) but not the male MD group. Furthermore, mean corpuscular haemoglobin (MCH) was slightly and statistically significantly higher in the male MD and HD group (9 % and 15 % above controls) and the female MD and HD group (8 % and 15 % above controls). In males but not females a slight but statistically significant difference to controls was also noted for haematocrit (HCT) (MD group 8 % below controls, HD group 10 % below controls). Furthermore, blood coagulation was affected by the test item. Prothrombin time (PT) was noted to be marginally but statistically significantly higher in males and females of the HD group (11 % and 7 % above controls). As values were within historical control data, this marginal difference was not considered as adverse. Though no clinical signs of anaemia became apparent in the animals during the study period, marked changes to haematological parameters beyond historical control data in the MD and HD group in terms of reduced blood cell mass, regenerative processes and other associated changes were considered as test item related, adverse effects of the treatment with the test item. The microscopic findings in the MD and the HD group of increased extramedullary hematopoiesis in the spleen, increased hemosiderin deposits in the spleen and liver (except for males of the MD group) and a slight decrease in fatty replacement in the sterna bone marrow were considered secondary to the changes in the red blood parameters. They possibly reflect a saturation of the haematopoietic capacity of the bone marrow. Observed hemosiderin deposits in the liver were seen along with slightly increased inflammatory cell foci in the HD group. Furthermore, the test item induced a dose dependent centrilobular to diffuse hepatocellular hypertrophy in the liver of all test item dosed groups (except for males of the LD group) that correlated with statistically significantly increased absolute and/or relative liver weight. Absolute liver weight was noted to be 13 %, 11 % and 15 % higher in the male LD, MD and HD group and 14 %, 25 % and 26 % higher in the female LD, MD and HD group, respectively, when compared to the respective controls. This finding was considered to represent an adaptive and non-adverse change. Findings in the spleen (increased extramedullary hematopoiesis and hemosiderin deposits in the MD and HD group) correlated with increased spleen weight and macroscopically observed enlarged spleen in some animals of the HD group and one male of the MD group. Mean spleen weight was observed to be markedly and statistically significantly higher in the male and female HD group when compared to the respective controls (absolute weight 71 % and 86 % above controls, respectively). Furthermore, slightly but statistically significantly higher spleen weight was also observed in the male MD group (absolute weight 23 % above controls) and without statistical significance in the female MD group (absolute weight 18 % above controls). Few other observed gross macroscopic lesions at necropsy were considered as normal background lesions that may be recorded in animals of this strain and age. Based on the findings, a histopathological NOEL was established at the low dose for males and a histopathological NOAEL at the high dose for males and females. Formulation analysis for concentration verification and homogeneity was performed on collected samples at various intervals during the study. Nominal concentrations were confirmed for all dose groups as measured concentration did not differ from nominal concentration by more than 10 %. All samples were homogenous as COV was below or equal to 10 %.