Registration Dossier

Administrative data

Description of key information

A dose range finding study- reproduction/ developmental toxicity screening test with 2,6-Di-tert-butyl-4-nonylphenol in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg body weight/ day was performed by oral gavage for a treatment period of 54 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed. Animals of an additional control group were handled identically as the dose groups but received corn oil, the vehicle used in this study.

No adverse effects of 2,6-Di-tert-butyl-4-nonylphenol were found up to dose level of 1000 mg/kg body weight. However, the fertility index was markedly lower in HD group (33%) compared to control (100%) and the test-item related effect cannot be excluded.

Based on the generated data the dose levels 100, 300 and 1000 mg/kg body weight/ day were selected for the main study.

A combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with 2,6-Di-tert-butyl-4-nonylphenol in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg body weight day was performed by oral gavage. Female animals were treated up to 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed. Animals of an additional control group were handled identically as the dose groups but received corn oil, the vehicle used in this study.

Test item related effects on body weight development, food consumption in male and female high-dose group, on haematology, coagulation and clinical biochemistry in male and female mid-dose and high-dose group and organ weights effect in all groups of males and females were observed. Histopathologically, in liver, hepatocellular vacuolation was observed in both surviving and decedent animals. Hepatocyte vacuolation (fatty changes) was observed in all dose groups and in both males and females. The observed hepatic change indicates hepatocyte functional alteration / disturbances related to the administration of 2,6-Di-tert-butyl-4-nonylphenol and the observed hepatic changes were considered to be adverse.

The LOAEL (Low Observed Adverse Effect Level) of 2,6-Di-tert-butyl-4-nonylphenol in this study for general toxicity is considered to be 100 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: oral, other
Remarks:
Combined Repeated Dose Oral Toxicity Study with the Reproduction / Developmental Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted on 28 July 2015
Qualifier:
according to
Guideline:
other: Health Effects guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test.
Version / remarks:
EPA 712-C-00-368, July 2000
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No. 440/2008, L 142, Appendix Part B,
Version / remarks:
May 30, 2008
GLP compliance:
yes (incl. certificate)
Limit test:
no
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Batch: 222375101
- Expiration date of the lot/batch: 02 May 2018
- Purity test date: 02 September 2016


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, closed bottle
- Stability under test conditions:
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Crl: WI(Han) (Full Barrier)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to the German Act on Animal Welfare the animals were bred for experimental purposes.
This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities. Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at the start of the treatment period: approx. 13-14 weeks old
- Body weight at the allocation of the animals to the experimental groups:
males: 327 – 362 g (mean: 344.50 g, ± 20% = 275.60 – 413.40 g)
females: 209 – 236 g (mean: 222.45 g, ± 20% = 177.96 – 266.94 g)
- Fasting period before study:
- Housing: Animals were housed in groups of 5 animals / sex / cage in type IV polysulphone cages) during the premating period for both males and females and during postmating period for males depending on the mating status. During mating period, males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages and males were returned to their original cage. In each cage Altromin saw fibre was used as bedding. Nesting material was provided latest on GD 18 for all mated females.
- Diet (e.g. ad libitum): Free access to Altromin 1324 maintenance diet for rats and mice
- Water (e.g. ad libitum): Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: Adequate acclimatisation period (at least 5 days) under laboratory conditions

DETAILS OF FOOD AND WATER QUALITY:
- 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)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

IN-LIFE DATES:
From: Delivery of Animals: 26 January 2017
Acclimatisation Period: 26 January 2017 – 08 February 2017
Experimental Starting Date: 08 February 2017
To: Necropsies: 15 March 2017, 16 March 2017
Experimental Completion Date: 16 March 2017
Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Batch No.: MKBW9504V, MKBQ9948V
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a tared plastic vial on a precision balance.
The test item was dissolved in a suitable vehicle (corn oil). The dose formulations were prepared by adding the required volume of vehicle and further vortexing it for 2-3 minutes.
The test item and control formulations were prepared once in 10 days before the administration procedure.
The vehicle was also be used as control item.
The test item and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 4 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected following solubility check and also in consultation with sponsor. The same vehicle was used during the dose range finding study.
- Concentration in vehicle: 0, 25 , 75 and 250 mg/mL for the 0 (control), 100, 300 and 1000 mg/kg bw dose groups respectively.
- Amount of vehicle (if gavage): The application volume (test item and vehicle) for all groups was 4 mL/kg body weight.
- Lot/batch no. (if required): MKBW9504V, MKBQ9948V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
During the study samples were collected for the investigation of substance concentration.
Samples were taken from the middle of prepared formulations from all dose groups and from the middle of the control group in study week 1 (pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation / lactation) from all groups (16 samples).
Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 5 mL). The A-samples were analysed at Eurofins Munich and until then stored under appropriate conditions based on available stability data (Eurofins Munich study phase no. 166600). The B-samples were retained at -15 to -35 °C at BSL Munich (test facility) and discarded after completion of the final study report.
Dose formulation analysis for nominal concentration revealed that nominal concentrations for all formulations were confirmed throughout the study period during each occasions (study week 1, 3, 5 and in the last week of the study) as measured concentrations were within acceptance criterion of 10%.
Duration of treatment / exposure:
The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females. Then in females, treatment was done during the gestation period and up to post-natal day 12. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.
Frequency of treatment:
a single dose daily on 7 days per week
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 in the main test.
100 animals (40 males and 60 females) were included in the study. All females were screened for regular estrous cycles for 14 days before the treatment initiation and only 40 females (10 females/group) showing regular estrous cycles were continued in the study.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: According to the results of a previous dose range finding study (BSL Munich Study No. 166596) and in consultation with the sponsor the doses 100, 300 and 1000 mg/kg bw were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose).
- Rationale for animal assignment (if not random): Prior to the start of the treatment period, a detailed clinical observation outside the home cage was made. None of the animal showed pathological signs before the first administration. Before initiation of dosing, all females were screened for two weeks for regular estrous cyclicity and females (10 females/ group) with regular estrous cycle (4-5 day cycle) were used in the study. Before the first administration, all animals to be used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively. Randomisation was performed with validated IDBS Workbook 10.1.2 software.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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 are made once daily.
- Cage side observations were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure, and at least once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Clinical observations include spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4 and PND 13 along with the pups. All animals were weighed directly before termination. Any animals prematurely sacrificed were weighed prior to the sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE (gavage study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / Not specified
Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

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: Yes / No / Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (gavage study): Yes / No / Not specified
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Haematological parameters were examined in 5 randomly selected males and all females (only lactating females were evaluated) from each group at the end of the treatment prior to or as part of the sacrifice of the animals. Blood from the abdominal aorta of the animals was collected in EDTA-coated tubes.
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Yes / No / Not specified
- How many animals: 5 randomly selected males and all females (only lactating females were evaluated) from each group
- Parameters examined:
haematocrit value (Hct)
haemoglobin content (Hb)
red blood cell count (RBC)
mean corpuscular volume (MCV)
mean corpuscular haemoglobin (MCH)
mean corpuscular haemoglobin concentration (MCHC)
reticulocytes (Re)
platelet count (PLT)
white blood cells (WBC)
neutrophils (Neu)
lymphocytes (Lym)
monocytes (Mono)
eosinophils (Eos)
basophils (Baso)
large unstained cells (Luc)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Parameters of clinical biochemistry from 5 randomly selected males and all females (only lactating females were evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals. Blood from the abdominal aorta of the animals was collected in serum separator tubes.
- Animals fasted: Yes / No / Not specified
- How many animals: 5 randomly selected males and all females (only lactating females were evaluated) from each group
- Parameters examined:
alanine aminotransferase (ALAT)
aspartate-aminotransferase (ASAT)
creatinine (Crea)
total protein (TP)
albumin (Alb)
urea
total bile acids (TBA)
total cholesterol (Chol)
glucose (Gluc)
sodium (Na)
potassium (K)
From 2 female (if possible) pups/litter on day 4 after birth, from all dams and 2 pups/litter (1 male and 1 female, if possible) at termination on day 13 and from all adult males at termination blood samples were collected from the defined site. All blood samples were stored under appropriate conditions. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4). Further assessment of T4 in blood samples from the main study dams and day 4 pups were not deemed necessary. Additionally, assessment of TSH in day 4 pups, adult females, day 13 pups, and adult males were not considered necessary based on the fact that no histopathological findings were observed in thyroid/parathyroid gland of selected male and female adult animals and T4 hormone levels of males and day 13 pups. The study monitor was consulted for these decisions. Pup blood was pooled by litter for thyroid hormone analysis.

URINALYSIS: Yes
- Time schedule for collection of urine: A urinalysis was performed with samples collected from 5 randomly selected males and females (only lactating females were evaluated) prior to or as part of the sacrifice of the animals. Additionally, urine colour/ appearance were recorded.
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters examined:
specific gravity
nitrite
pH-value (pH)
protein
ketone bodies (ketones)
urobilinogen (ubg)
bilirubin
blood
leukocytes

NEUROBEHAVIOURAL EXAMINATION: Yes, Functional observations were performed.
- Time schedule for examinations:
Multiple detailed behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 randomly selected males and during the last week of lactation of the lactation period in 5 randomly selected females (only lactating females were evaluated) of each group outside the home cage using a functional observational battery of tests.
- Dose groups that were examined:
- Battery of functions tested: Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

IMMUNOLOGY: No

OTHER:
BLOOD COAGULATION:
Coagulation parameters from 5 randomly selected males and all females (only lactating females were evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals.
Blood from the abdominal aorta of the animals was collected in citrate tubes.
The following coagulation parameters were examined :
prothrombin time (PT)
activated partial thromboplastin time (aPTT)

ESTROUS CYCLES:
Estrous cycles were monitored before treatment starts to select females with regular cyclicity (using vaginal smears) for the study. Further on, vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating.

LITTER OBSERVATIONS:
The duration of gestation was recorded and is calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after the delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of littering (PND 0) and on PND 4 and PND 13. Live pups were identified by tattooing. In addition to the observations of the parent animals, any abnormal behaviour of the offspring was recorded.
The anogenital distance (AGD) of each pup was measured on PND 0. Pup body weight measured on PND 0 was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD / Cube root of pup weight). The number of nipples/areolae in male pups was counted on PND 12.


Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Males were sacrificed any time after the completion of the mating period (after a minimum dosing period of 28 days) and females were sacrificed on the respective PND 13 by using anesthesia (ketamine/xylazine). All surviving pups were killed by cervical dislocation on PND 13.
Vaginal smears were examined on the day of necropsy to determine the stage of estrous cycle.
Dead pups and all surviving pups on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.
Non-pregnant females were sacrificed on day 26 from the day of sperm positive vaginal smear or from the last day of mating period.
All adult animals 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.
Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), the thyroid/parathyroid glands and all organs showing macroscopic lesions of all adult animals were preserved in 4% neutral-buffered formaldehyde, except for testes and epididymides which were preserved in modified Davidson’s Solution for 24 hours and then transferred to 70% ethanol.
The number of implantation sites and corpora lutea were recorded for each parental female at necropsy. The number of corpora lutea and implantation sites were recorded for females sacrificed 26 days after the end of the mating period with no evidence of mating and or on day 26 post-coitum due to non-delivery.

HISTOPATHOLOGY: Yes
The following tissues of 5 randomly selected male and female animals (only lactating females were evaluated) from each group were preserved in 4% neutral-buffered formaldehyde except for testes and epididymides that were fixed in Modified Davidson’s Fixative for approximately 24 hours before they were transferred to 70% ethanol:
adrenal glands
all gross lesions
aorta
brain (incl. medulla/pons, cerebellar and cerebral cortex)
caecum
colon
duodenum
epididymides
eyes with optic nerve and Harderian gland
femur with knee joint
heart
ileum (including Peyer´s patches)
jejunum
kidneys
liver
lungs
lymph nodes (mandibular)
lymph nodes (mesenteric and axillary)
mammary gland area (male and female)
oesophagus
ovaries
oviducts
pancreas
parathyroid glands
pituitary
prostate and seminal vesicles with coagulating glands as a whole
rectum
salivary glands (sublingual, submandibular)
sciatic nerve
skeletal muscle
skin
spinal cord (cervical, thoracic and lumbar segments)
spleen
sternum (with bone marrow)
stomach
testes
thymus
thyroid/parathyroid glands
tongue
trachea
ureters
urinary bladder
uterus with cervix and vagina
Histopathologival examination was not performed on aorta, eyes with optic nerve and Harderian gland, femur with knee joint, lymph nodes (mandibular), mammary gland area (male and female), oesophagus, pancreas, paarathyroid glands, pituitary, salivary glands (sublingual, submandibular), skeletal muscle, skin, tongue, ureters.
A full histopathology was carried out on the preserved organs and tissues of the selected animals of the control and high dose groups which were sacrificed at the end of the treatment period. Thyroid/parathyroid glands from pups and from the remaining non-selected adult animals was not deemed necessary as no test item related histopathological findings were observed in thyroid/parathyroid gland of selected animals and there was also no test item related effect observed on T4 hormone level in males and pups sacrificed on PND 13.
A full histopathology was carried out on the preserved organs and tissues of all animals which died during the study or which were euthanised due to morbidity.
Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were trimmed, embedded into paraffin, cut at an approximated thickness of 2-4 μm and stained with hematoxylin and eosin and examined in control and HD animals and in non-pregnant female animals of the LD and MD animals. Testes, epididymides and accessory sex organs (prostate, seminal vesicle with coagulating gland) were also examined in the mating partners of the non-pregnant female LD and MD animals.
In addition, from 5 randomly selected males and females from the LD and MD group liver was processed and evaluated. Further, from the 5 selected males from the LD and MD group lung, axillary lymph nodes and kidney were processed and evaluated.
Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.
For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
The 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). The histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, 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.

Other examinations:
-Organ weights at necropsy:
The wet weight of the organs of 5 randomly selected male and female animals (only lactating females were evaluated) from each group were recorded as soon as possible. Paired organs were weighed together. Organ weights of animals found dead or euthanised for animal welfare reasons were not recorded.
Reproductive organs were weighed from all animals.
Thyroid/parathyroid glands from 1 pup/sex/litter/group (sacrificed on PND 13) and from all adult males and females were preserved. Weight of thyroid/parathyroid glands were measured after fixation.
Tissues/organs weighed at necropsy:
testes (paired weight)
levator ani + bulbocavernosus muscle complex (complete weight)
epididymides (paired weight)
glans penis
prostate, seminal vesicles and coagulating glands (complete weight)
uterus with cervix
Cowper’s gland (paired weight)
ovaries (paired weight)
thyroid/parathyroid glands (from 1 pup/sex/litter/group and from all adult males and females) – weighed after fixation (complete weight)
thymus
liver
spleen
kidneys (paired weight)
brain
adrenal (paired weight)
heart
pituitary gland


Statistics:
A statistical assessment of the results of body weight, food consumption, parameters of haematology, blood coagulation, clinical biochemistry and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights were statistically analyzed 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 GraphPad Prism V.6.01 software or Ascentos 1.1.3 software (p<0.05 was considered as statistically significant).

Clinical signs:
no effects observed
Description (incidence and severity):
No adverse effects of 2,6-Di-tert-butyl-4-nonylphenol were found on male and female clinical observations up to dose levels of 1000 mg/kg bw/day.
Mortality:
not specified
Description (incidence):
There were 7 mortalities (6 from HD (high dose) and 1 from Control group) observed in the study. Histopathologically, the cause of death/morbidity of 3 animals was not evident. Predominant causes of mortality/morbidity of other animals were incidental intra-uterine hemorrhage (hemometra), inflammatory process involving the heart (myocardium and or pericardium) and peri-renal hemorrhage
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test item related effects on body weight development in male and female HD group were observed.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Test item related effects on food consumption in male and female HD group were observed.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item related effects on haematology and coagulation in male and female MD (mid-dose) and HD (high-dose) group were observed.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Test item related effects on clinical biochemistry in male and female MD and HD group were observed.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No adverse effects of 2,6-Di-tert-butyl-4-nonylphenol were found on male and female urinalysis up to dose levels of 1000 mg/kg bw/day.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No adverse effects of 2,6-Di-tert-butyl-4-nonylphenol were found on male and female functional observations up to dose levels of 1000 mg/kg bw/day.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item related effects organ weights effect in all groups of males and females were observed.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No adverse effects of 2,6-Di-tert-butyl-4-nonylphenol were found on male and female gross pathological findings at necropsy up to dose levels of 1000 mg/kg bw/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathologically, in liver, hepatocellular vacuolation was observed in both surviving and decedent animals. Hepatocyte vacuolation (fatty changes) was observed in all dose groups and in both males and females. The observed hepatic change indicates hepatocyte functional alteration / disturbances related to the administration of 2,6-Di-tert-butyl-4-nonylphenol and the observed hepatic changes were considered to be adverse.
Histopathological findings: neoplastic:
not examined
Details on results:
*Mortality
During the treatment period of this study, few mortalities/moribund sacrifice were observed as follows:
- Female no. 42 (C) was found dead on gestation day (GD) 9. Clinical signs observed before death on GD 8 were abnormal breathing and severe salivation.
- Male no. 35 (HD) was found dead on day 27. No specific clinical signs were observed before death except moving the bedding from day 17-26 and salivation from day 21-26.
- Male no. 38 (HD) was euthanised on day 29 in moribund condition for animal welfare reasons. Clinical signs observed in this animal were moving the bedding from day 18-28, salivation on day 28, spontaneous activity markedly reduced, marked piloerection, nasal discharge, anaemia and slightly reduced grip strength on day 29.
- Male no. 40 (HD) was found dead on day 14. No specific clinical signs were observed before death.
- Female no. 72 (HD) was found dead on gestation day (GD) 13. Clinical signs observed before death were diarrhoea from premating day 3-4, moving the bedding from GD 1-9 and 12, moderate abnormal breathing and moderate piloerection on GD 9, slightly reduced spontaneous activity on GD 9-12, slight piloerection on GD 11-12 and nasal discharge (red) on GD 12.
- Female no. 78 (HD) was found dead on gestation day (GD) 12. Clinical signs observed before death were moving the bedding on premating day 2, 4 and GD 1-11, slight to severe salivation on GD 5 and GD 7-9 and slight piloerection on GD 11.
- Female no. 80 (HD) was euthanised in moribund condition on post natal day (PND) 0 due to animal welfare reasons. Predominant clinical signs observed were moving the bedding on GD 0-21, slight to moderate salivation on GD 1-3 and diarrhoea on GD 13. On PND (post-natal day) 0, severely reduced spontaneous activity, sunken flanks, severe piloerection and bloody vagina was observed.
Histopathologically, the cause of death/morbidity of animal no. 42 (C- female), 38 (HD-male) and 72 (HD- female) was not evident. The cause of morbidity of animal no. 80 (HD- female) was considered to be incidental intra-uterine hemorrhage (hemometra) and most likely of traumatic nature. The cause of death of animal no. 35 (HD- male) was inflammatory process involving the heart (myocardium). The cause of death of animal no. 78 (HD- female) was inflammatory process involving the heart (myocardium and pericardium) and intra-uterine hemorrhage (hemometra) which was deemed to be incidental and most likely of traumatic nature. Death of animal no. 40 (HD- male) was attributed to peri-renal hemorrhage.
*Clinical Observations
In terminally sacrificed males, predominant clinical sign observed during the treatment period (premating day 1 to mating day 14) was slightly to moderately increased salivation in few animals of LD and almost all animals of MD and HD group. Isolated incidences of diarrhoea in 2 males (no. 24 and 25) of MD group was considered to be incidental.
In terminally sacrificed females, predominant clinical signs observed during the treatment period (PD 2 to PND 12) were moving the bedding, slightly to moderately increased salivation in few animals of LD, MD and HD group.
Isolated incidences of alopecia on various body parts, moderate to severe piloerection, sunken flanks, hunched posture, anemia and bloody vagina were observed on few occasions in very few female animals in all group including control group and considered to be incidental in nature.
Moving the bedding was observed transiently in few males and all females of the LD and almost all male and female animals of MD and HD group. The clinical signs salivation and moving the bedding were observed immediately after the dose administration and therefore were considered to be a sign of discomfort due to a local reaction to the test item rather than a systemic adverse effect and has no toxicological relevance.
None of the females showed signs of abortion or premature delivery.
During the weekly detailed clinical observation, no relevant differences between the groups were found.
*Functional Observations
In males, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. There were no biologically relevant differences in body temperature between the groups.
In females, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period except statistically significantly higher supported rearing count was observed in last week of the treatment in HD group when compared with the controls. As this type of difference was marginal, it has no toxicological relevance and could be considered as biological variation. There were no biologically relevant differences in body temperature between the groups.
*Body Weight Development
In males, there was no statistically significant difference observed on body weight between the LD, MD and HD dose groups and the control group during the entire study period.
Group mean body weight gain in HD group was statistically significantly lower during premating day 1-7, premating day 14 to mating and post mating day 7 and premating day 1 to terminal sacrifice when compared with the control. There was also a lower body weight gain observed in HD group during premating day 1 and post mating day 14 without achieving the statistical significance when compared with the control.
In females, statistically significantly higher group mean body weight was observed during premating day 14, lactation day 4 and 13 in LD group when compared with the controls.
There was statistically significantly lower group mean body weight gain observed during gestation day 14-20 and during overall gestation period i.e. gestation day 0-20 in HD group when compared with the controls.
This statistically and biologically significant effect on body weight gain in HD group male and female was considered as test item related and toxicologically relevant. However, due to lack of dose dependency and consistency, statistically significant effect on female group mean body weight in LD group was not considered to be toxicologically relevant.
*Food Consumption
In males, in correlation to the body weight and body weight gain, the food consumption during treatment period tended to increase with the progress of the study in the control, the LD, the MD and the HD group. However, In HD group, during premating period, food consumption was lower without achieving statistical significance compared to the control group and this effect on food consumption in males was considered to be test item related.
In females, no statistically significant effect on food consumption was observed during premating period, gestation and lactation period in treatment groups when compared with the controls. However, in correlation to body weight development, marginally lower food consumption was observed in MD and HD animals during gestation day 7-14 and 14-20 when compared with the controls.
*Haematology and Coagulation
In males sacrificed at the end of treatment period, no test item related adverse effects were observed for haematological parameters. However, there was a statistically significantly higher platelets (PLT) and reticulocytes (RE) count observed in HD group compared to the control group. There was also statistically significantly higher prothrombin time (PT) observed in MD group when compared to the controls. In HD group, due to clotted samples and few invalid values, no group mean data was available.
All mean and most of the individual values were within the historical control data range. As group mean value was within historical control data limit, statistically significant effect on PLT and RE in HD group was considered to be incidental and not related to the treatment with test item. However, increase in prothrombin time (PT) in MD, could be attributed to hepatocellular injury of the liver revealed after histopathology evaluation and inability of liver to synthesise clotting factors.
In females sacrificed at the end of treatment period, higher but statistically insignificant PLT count was observed in HD group when compared with the controls. There was also statistically significantly lower prothrombin time (PT) was observed in LD, MD and HD group when compared to the controls.
All mean and most of the individual values were within the historical control data range. As group mean value was within historical control data limit, statistically significant effect on PLT in HD group was considered to be incidental and not related to the treatment with test item. Lover prothrombin time has no clinical significance and therefore, this effect on prothrombin time in female was not considered to be adverse
*Clinical Biochemistry
In males sacrificed at the end of treatment period, significantly lower total bile acids (TBA) was observed in MD and HD group although statistical significance was only achieved in HD group when compared with the controls. There was also marginally higher alanine aminotransferase (ALAT) and alkaline phosphatase (AP) observed in MD and HD group without achieving statistical significance when compared with the controls.
In females sacrificed at the end of treatment period, higher aspartate aminotransferase (ASAT) ALAT and AP were observed in HD group without achieving statistical significance when compared with the controls. There was also statistically significantly higher total protein (TP), cholesterol, Potassium in HD group and statistically significantly lower creatinine and TBA in MD and glucose in MD and HD group when compared with the controls.
This effect on clinical biochemistry parameters (TBA, ASAT, ALAT, AP) in male and female was considered to be test item related and could be attributed to hepatocellular injury of liver as correlated with histopathology evaluation.
Furthermore, there was data variation observed in individual values for sodium and potassium in few males (Control – 2/5 and HD- 1/5) and females (control- 3/9, LD- 4/10, MD- 6/10) of all groups including controls.
It is common to have data variation in electrolyte measurement without effect on animal health for either individual or group of animals which do not appear to be dose related or correlate with histopathological findings in kidney. Excess water intake occurs when excessive thirst is induced by the test item or where renal excretion does not act as a compensatory mechanism, this result in lowering of the sodium. However, no indication of excess water intake was observed during clinical examination of the animals in this study. In the light of fact that no major test item related clinical signs (except moving the bedding and occasional salivation in few LD and MD females), absence of relevant histopathological lesions in kidney, animals with lower sodium values in control group and no dose dependency in in the dose groups, this effect was considered to be incidental and not related to treatment with test item.
Generally, lower potassium is observed in animals with diarrhoea, renal tubular defects and thrombocytopenia. As no such clinical signs, neither relevant kidney lesions nor effect on platelet count was observed in these animals, this effect on potassium in few male and female animals was not considered to be of toxicological relevance.
*Urinalysis
The urinalysis performed in selected male and female animals sacrificed at the end of treatment period revealed no test item treatment related effect and all urinary parameters were in the normal range of variation. High protein levels were found in the urine of few male and females of all groups including control group. Therefore, this effect on urine parameters was not considered to be test item related.
*Pathology
Few specific macroscopic changes were recorded for the male and female animals, which based on microscopic examination were not considered to be of test item treatment relevance.
The macroscopic changes observed were fluid filled- thoracic cavity (male no. 34 of HD group, female no. 78 of HD group), oesophagus – red fluid filled (male no. 34 of HD group), heart – hard (female no. 76 of HD group), pericardium- oily fluid filled and enlarged (female no. 78 of HD group ), lung – abnormal surface (female no. 76 of HD group), stomach – filled with dark fluid or (male no. 38 of HD group, female no. 80 of HD group), Jejunum- mass 5-8 cm in diameter and adhesion with right kidney and left testes (male no. 40 of HD group), payer’s patches- dark discolouration (female no. 78 of HD group), caecum- gas filled (female no. 42 of control group), caecum- filled with dark fluid (male no. 38 of HD group), liver- abnormal colour and spotted (female no. 42 of control group), kidneys- dark abnormal colour (male no. 40 of HD group), uterus- filled with blood (female no. 72 of HD group), spleen- abnormal colour – black (male no. 40 of HD group, female no. 42 of control group), spleen- small (male no. 38 of HD group), thymus- dark abnormal colour and enlarged on both sides (male no. 35 of HD group) testes (male no. 40 of HD group) and seminal vesicles- dark abnormal colour (male no. 35 of HD group), mesenteric lymph node- dark discolouration (female no. 78 of HD group), mammary gland- grey abnormal colour (female no. 42 of control group), skin- black abnormal colour (male no. 40 of HD group), corpora lutea- abnormal colour – dark (female no. 42 of control group) and implantation sites- enlarged- Both sides (female no. 42 of control group).
Macroscopic findings correlating with histopathological observations were observed in following animals from the HD group:
 Animal no. 35 (Male – HD group)- the enlarged and dark coloured (abnormal color, dark) thymus observed at necropsy histologically correlated with severe thymic hemorrhages.
 Animal no. 38 (Male – HD group) – the small spleen (small complete) observed at necropsy histologically correlated with splenic atrophy.
 Animal no. 40 (Male – HD group) – a black mass surrounding the kidney was observed and histologically correlated with severe peri-renal hemorrhage.
 Animal no. 78 (Female – HD group)- the enlarged pericardium histologically correlated with the mixed cell infiltration observed in the heart.
The above mentioned findings were deemed incidental. All other observed gross lesions reflected the animal physiology or, in absence of corresponding histopathological findings, were considered to be of no toxicological relevance.
*Organ Weight
In males sacrificed at the end of treatment period, there were statistically significantly higher thyroid/parathyroid and liver weight (absolute and relative to body weight) in MD and HD group when compared with the controls. There was also statistically significantly lower relative (to body weight) liver weights observed in LD group when compared with the controls. In the light of absence of histopathological findings and T4 hormone levels in males, this effect on thyroid/parathyroid weight is not considered to be adverse.
In females sacrificed at the end of treatment period, statistically significantly higher absolute and relative liver weights (relative to body weight) were observed in LD, MD and HD group when compared with the controls.
This effect on male and female liver weight was considered as test item related as histopathologically hepatocytes vacuolization (fatty changes) was observed in all dose groups and in both males and females. The hepatocytes vacuolization sometimes associated with hepatocytes hypertrophy which was correlated with increased liver weights in male and females.
*Histopathology
Under the conditions of this experiment, repeated dose exposure to 2,6-di-tert-butyl-4-nonylphenol induced microscopic changes at all doses in the liver of treated animals.
There were 7 animals (6 from the HD group and one control animal) found dead or sacrificed in moribund condition during the treatment period.
The lymphocyte depletion observed in spleen, mesenteric lymph node, axillary lymph nodes and thymus of the decedents and moribund animals could be induced by the test item application or, could be stress related.
In the heart there was a mixed inflammatory cell infiltrates in few decedents from the high dose group. The observed heart changes were deemed most likely incidental not treatment related. Further, the inflammatory cell infiltrates and hemorrhages observed in the pericardium of one female decedent were also considered incidental not treatment related.
Furthermore, the hemorrhages observed in different organs (i.e. thymus, epididymis) were considered to be of traumatic nature and not test item related.
In liver, hepatocellular vacuolation was observed in both surviving and decedent animals. The hepatocellular vacuolation in all groups showed dose dependency and was considered adverse.
Further, in liver, glycogen accumulation was observed in surviving females from the mid and high dose groups. This finding could also be related to the animal feeding schedule. However, the glycogen accumulation in hepatocytes, although test item related, was considered as non-adverse.
In kidneys, hyaline droplets were observed in the cytoplasm of tubular epithelial cells. The observed hyaline droplets represent an accumulation of secondary lysosomes containing alpha-2u globulin in the cytoplasm of tubular epithelial cells. This process is considered to be a male rat specific phenomenon not occurring in humans and is of no toxicological relevance for humans.
In lung, accumulation of vacuolated alveolar macrophages (histiocytosis) were observed in decedent and surviving animals of all groups including controls. This histopathologic change could be related to aspiration of small amounts of vehicle (corn oil) and, in decedents, to an ongoing inflammatory process whereas a relationship with the administration of the 2,6-Di-tert-butyl-4-nonylphenol cannot be fully excluded. However, based on the incidence, severity and distribution the observed lung change was considered to be most likely incidental in nature.
In axillary lymph nodes, accumulation of vacuolated alveolar macrophages (histiocytosis) were observed in decedent and surviving animals. There was also a minor degree of lymphocytes depletion observed. This histopathologic change was considered most likely stress related, whereas a relationship with the administration of 2,6-Di-tert-butyl-4-nonylphenol cannot be fully excluded.
The vacuolization of adrenal gland cells from the Zona fasciculata was considered to be most likely secondary to stress from various causes and was deemed incidental and non-adverse.
The histopathologic changes observed in the esophagus of one animal were considered related to the route of administration (gavage) of the test item and were deemed incidental.
Microscopic evaluation of reproductive organs in surviving animals and decedent revealed no histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, oviducts, uterus and cervix, and vagina. In decedents (animal no. 78 and 80), intrauterine hemorrhage was observed and considered most likely to be incidental in nature.
In testes, a detailed qualitative examination of hematoxylin-PAS (Periodic Acid Schiff) stained slides after taking into account the tubular stages of the spermatogenic cycle (Sperm staging) revealed no treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure.
In non-pregnant females and mating partners, no treatment related histomorphological effects were observed in the reproductive organs of the non-pregnant control female (no. 42) and its pairing partner (no. 2), the non-pregnant mid dose group females 63 and 65 and their pairing partners 23 and 25 and the non-pregnat high-dose group females 72 and 78 and their pairing partners (no. 32 and 38).
Summing up, in high dose animals the repeated dose administration of 2,6-di-tert-butyl-4-nonylphenol induced adverse changes in the liver.
In addition, in high and mid dose groups several overlapping histopathologic changes were observed, whereas in the low dose group only hepatocellular vacuolation was present. In addition, the comparison between low dose and control animals revealed that hepatocellular vacuolation was present at slightly higher incidence (2/5 in low dose animals vs. 1/5 animals in controls) but similar severity in low dose animals. Therefore the hepatocellular vacuolation in animals from the low dose group was regarded as low adverse effect and a LOAEL (low adverse effect level) was established at 100 mg/kg bw/day for repeated dose administration of 2,6-di-tert-butyl-4-nonylphenol in male and female Wistar rats.







Dose descriptor:
LOAEL
Remarks:
general toxicity
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
93.1% purity
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Conclusions:
The LOAEL (Low Observed Adverse Effect Level) of 2,6-Di-tert-butyl-4-nonylphenol in this study for general toxicity is considered to be 100 mg/kg bw/day and NOAEL (No Observed Adverse Effect Level) for reproductive toxicity screening is considered to be 300 mg/kg bw/day.
Executive summary:

The aim of this study was to assess the possible effects of 2,6-Di-tert-butyl-4-nonylphenol on male and female fertility and embryo-foetal development after repeated dose administration in Wistar rats.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group. Female animals were treated up to 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed. Animals of an additional control group were handled identically as the dose groups but received corn oil, the vehicle used in this study.

Inadvertently, an unintended test item was administered on a single study day (Day 5) to 3 female MD animals (no. 68-70 at 50 mg/kg in 4 mL/kg corn oil) and in all male and female HD animals (no. 31-40 and no. 71-80, at 100 mg/kg in mL/kg corn oil).

The deviation was concluded not to have affected the study quality and results.

The 4 groups comprised 10 male and 10 female Wistar rats. Before initiation of dosing, all females were screened for two weeks for regular estrous cyclicity and animals (10 females / group) with regular estrous cycle (4-5 day cycle) were used in the study.

The following doses were evaluated:

Control: 0 mg/kg bw/day

Low Dose: 100 mg/kg bw/day

Medium Dose: 300 mg/kg bw/day

High Dose: 1000 mg/kg bw/day

The test item formulations were prepared once in 10 days. The test item was dissolved in corn oil and administered daily during the study. Dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 4 mL/kg body weight.

During the period of administration, the animals were observed each day for signs of toxicity. Animals that died were examined macroscopically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically.

Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals.

Haematological and clinical biochemistry evaluations were performed on blood samples collected at terminal sacrifice from five males and five randomly selected females from each group. Urinalysis was performed on samples collected at terminal sacrifice from five randomly selected males from each group.

Functional observations including sensory reactivity to different stimuli, grip strength, motor activity assessments and other behavior observations were performed in the week before the treatment from all animals and in the last week of treatment in five randomly selected males and females of each group.

After 14 days of treatment, male and female animals were mated (1:1) for a maximum of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually.

Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition, on day 4 and day 13 post-partum. The anogenital distance (AGD) of each pup was measured on PND 0. The number of nipples/areolae in male pups was counted on PND 12.

From 2 pups/litter on day 4 after birth; from all dams and 2 pups /litter at termination on day 13 and from all adults males at termination blood samples were collected from the defined site. All blood samples were stored under appropriate conditions. Blood samples from the day 13 pups and from the adult males were assessed for serum levels for thyroid hormones (T4). Further assessment of T4 in blood samples from adult females and day 4 pups were not deemed necessary. Pup blood was pooled by litter for thyroid hormone analysis.

The males were sacrificed after completion of the mating period on treatment day 29 and the females along with their pups were sacrificed on post-natal day 13. Non-pregnant females were sacrificed on day 26.

The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Pups sacrificed on post-natal day 4 or 13 and those found dead, were carefully examined for gross external abnormalities.

A full histopathological evaluation of the tissues was performed of 5 randomly selected male and female animals of the control and high dose groups, all dead and moribund animals, all non pregnant female animals and their mating partners of the LD and MD groups. In addition, from 5 randomly selected males and females from the LD and MD group liver was processed and evaluated. Furthermore, from the 5 randomly selected males from the LD and MD group lung, axillary lymph nodes and kidney were processed and evaluated. For the testes, a detailed qualitative examination was made taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides. All gross lesions macroscopically identified were examined microscopically in all animals.

On the basis of this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with 2,6-Di-tert-butyl-4-nonylphenol in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg body weight day the following conclusions can be made:

- There were 7 mortalities (6 from HD and 1 from Control group) observed in the study. Histopathologically, the cause of death/morbidity of 3 animals was not evident. Predominant causes of mortality/morbidity of other animals were incidental intra-uterine hemorrhage (hemometra), inflammatory process involving the heart (myocardium and or pericardium) and peri-renal hemorrhage.

- No adverse effects of 2,6-Di-tert-butyl-4-nonylphenol were found on male and female clinical observations, functional observations, urinalysis, gross pathological findings at necropsy up to dose levels of 1000 mg/kg bw/day.

- Test item related effects on body weight development, food consumption in male and female HD group, on haematology, coagulation and clinical biochemistry in male and female MD and HD group and organ weights effect in all groups of males and females were observed.

- There were no effects on estrous cyclicity, anogenital distance, nipple retention, precoital interval and duration of gestation, reproductive indices, pup thyroid weight and male and PND 13 pup T4 hormone, pup survival, pup litter weight data and pup external findings from PND 0-12 and at death were observed up to dose levels of 1000 mg/kg bw/day.

- There were no test item treatment related effects observed on the number of corpora lutea, percent preimplantation loss and post implantation loss in treatment groups. However, statistically significantly lower number of implantation sites were observed in HD group.

- There were no test item treatment related or statistically significant effects observed on litter data parameters like number of male pups, number of female pups, sex ratio, number of live pups, still birth, runt on PND 0 as well as number of male pups, number of female pups, number of live pups and sex ratio on PND 4 and PND 13. However, group mean total number of pups born were statistically significantly lower in HD group when compared with the controls.

- Histopathologically, in liver, hepatocellular vacuolation was observed in both surviving and decedent animals. Hepatocyte vacuolation (fatty changes) was observed in all dose groups and in both males and females. The observed hepatic change indicates hepatocyte functional alteration / disturbances related to the administration of 2,6-Di-tert-butyl-4-nonylphenol and the observed hepatic changes were considered to be adverse.

The LOAEL (Low Observed Adverse Effect Level) of 2,6-Di-tert-butyl-4-nonylphenol in this study for general toxicity is considered to be 100 mg/kg bw/day and NOAEL (No Observed Adverse Effect Level) for reproductive toxicity screening is considered to be 300 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Reliable
System:
hepatobiliary
Organ:
liver

Mode of Action Analysis / Human Relevance Framework

Additional information

A dose range finding study was performed in rats with the registered substance administered by oral gavage given daily at 100, 300 and 1000 mg/kg bw for a treatment period of 54 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females (Holalagoudar, 2017). Males were dosed after the mating period until the minimum total dosing period of 28 days were completed. 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 3 male and 3 female Wistar rats. One male was found dead on study day 24. due to a gavaging error.

There were no adverse clinical symptoms observed in male and female animals of test-item and control groups during the study. No effects on body weight, body weight gain and food consumption were observed in both males and females of test-item treated groups compared to the control. The precoital interval and the duration of gestation were not affected in test-item groups compared to the control. Except for blood-filled lung in one male (no. 10) that was found dead on study day 24, no macroscopic findings were noted in the surviving animals killed at the study termination. There were no effects on organ weights (set of reproductive organs) in test-item treated groups compared to the corresponding control group except the uterus with cervix weight in LD, MD and HD groups was markedly higher. But without histopathology, the treatment relevance was not clear. The copulation, delivery and viability indices were not affected in test-item groups. The fertility index was markedly lower in HD group (33%) compared to control (100%). 2 of 3 females were not pregnant. The test-item related effect cannot be excluded. There were no effects of test-item treatment on pre- and post-natal parameters including the number of corpora lutea, implantation sites and live pups, and percentage of pre- and post-implantation losses. There were no effects on litter parameters.  Based on the generated data the dose levels 100, 300 and 1000 mg/kg body weight/ day were selected for the main study.

A key combined repeated dose and reproductive toxicity study according to OECD Test Guidance 422 was performed with the registered substance in Wistar rats dosed by oral gavage at 100, 300 and 1000 mg/kg bw (Takawale et al., 2017). Results can be summarised as follows (reproductive effects are provided in Section 7.8):

- There were 7 mortalities (6 from HD and 1 from Control group) observed in the study. Histopathologically, the cause of death/morbidity of 3 animals was not evident. Predominant causes of mortality/morbidity of other animals were incidental intra-uterine hemorrhage (hemometra), inflammatory process involving the heart (myocardium and or pericardium) and peri-renal hemorrhage.

- No adverse effects of 2,6-Di-tert-butyl-4-nonylphenol were found on male and female clinical observations, functional observations, urinalysis, gross pathological findings at necropsy up to dose levels of 1000 mg/kg bw/day.

- Test item related effects on body weight development, food consumption in male and female HD group, on haematology, coagulation and clinical biochemistry in male and female MD and HD group and organ weights effect in all groups of males and females were observed.

- Histopathologically, in liver, hepatocellular vacuolation was observed in both surviving and decedent animals. Hepatocyte vacuolation (fatty changes) was observed in all dose groups and in both males and females. The observed hepatic change indicates hepatocyte functional alteration / disturbances related to the administration of 2,6-Di-tert-butyl-4-nonylphenol and the observed hepatic changes were considered to be adverse.

The LOAEL (Low Observed Adverse Effect Level) of 2,6-Di-tert-butyl-4-nonylphenol in this study for general toxicity is considered to be 100 mg/kg bw/day and NOAEL (No Observed Adverse Effect Level) for reproductive toxicity screening is considered to be 300 mg/kg bw/day.

Justification for classification or non-classification

Based on a NOAEL < 100 mg/kg bw and according to CLP the test item 2,6-Di-tert-butyl-4-nonylphenol has obligatory labelling requirement for STOT and is classified into Category STOT RE Category 2 (target organ: hepatobiliary).