Registration Dossier

Administrative data

Description of key information

The UVCB substance causes adverse effects on liver in rats as indicated by slight to moderate fatty changes at doses between 75 and 300 mg/kg bw, single cell necrosis and changes in clinical chemistry observed upon subacute exposure. These changes are accompanied by a liver enlargement of up to 54% in male rats. The same hazard profile was observed for a related UVCB substance. For this substance, no additional hazards were identified in the subchronic oral toxicity study.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD testing guideline compliant study with well-characterized test material
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 10-12 weeks
- Weight at study initiation: 353 g (males), 210 g (females)
- Fasting period before study: none
- Housing: groups of 5 animals/sex/cage (premating males and females and postmating males), otherwise single cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%,
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 March 2014 To: 1 May 2014
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) are be prepared daily within 6 hours prior to dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Test item is insoluble in water, but soluble in corn oil
- Amount of vehicle (if gavage): 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
28 days (males), ca 53 days (females)
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
25, 75 and 225 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10
5 animals/sex/group were selected for functional observations, locomotor activity, clinical pathology, organ weights and histopathology
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a 14-day range-finding study in rats
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: mortality, clinical observations

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the first exposure and at the end of the study as part of the functional observation battery (FOB)

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of male and female animals were recorded shortly before the start of
administration of the test item at randomization and at the start of the study (day 0). Males
were weighed weekly until sacrifice. Females were weighed once per week during the premating period. Mated females were
weighed on days 0, 7, 14 and 21 during presumed gestation and on day 0 and 4 of lactation.
Non-mated females were weighed once per week after the mating period. In addition, the
animals were weighed on their scheduled necropsy date in order to calculate the correct organ to body weight ratios.


FOOD CONSUMPTION:
Feed consumption was measured per cage over weekly intervals during the study, with exemption of the mating period, during which no feed consumption was registered.


HAEMATOLOGY
Prior to sacrifice, 5 animals/sex/group were fasted overnight (water was freely available) and blood was taken from the aorta during necropsy, whilst under CO2/O2 anaesthesia. K3-EDTA was used as anticoagulant. In each sample the following determinations were carried out:
haemoglobin
packed cell volume
red blood cell count
reticulocytes
total white blood cell count
differential white blood cell counts (neutrophils, lymphocytes, eosinophils,
basophils, monocytes)
prothrombin time
thrombocyte count
mean corpuscular volume (MCV; calculated)
mean corpuscular haemoglobin (MCH; calculated)
mean corpuscular haemoglobin concentration (MCHC; calculated)
activated partial prothoplastin time (APTT)
red blood cell districution width (RDW)

CLINICAL CHEMISTRY
Prior to sacrifice, 5 animals/sex/group were fasted overnight (water was freely available) and blood was taken from the aorta during necropsy, whilst CO2/O2 anaesthesia. Blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation. The following measurements were made in the plasma:
alkaline phosphatase activity (ALP),
aspartate aminotransferase activity (ASAT),
alanine aminotransferase activity (ALAT),
gamma glutamyl transferase activity (GGT),
total protein,
albumin,
ratio albumin to globulin (calculated),
urea,
creatinine,
glucose (fasting),
bilirubin (total),
cholesterol (total),
triglycerides,
phospholipids,
calcium (Ca),
sodium (Na),
potassium (K),
chloride (Cl),
inorganic phosphate (PO4),
bile acids

Thyroid hormone analyses:
Thyroid stimulating hormone (TSH) μIU/mL
total triiodothyronine (T3) ng/dL
total thyroxine (T4) μg/dL


NEURO-BEHAVIOURAL TESTING (FOB) AND SPONTANEOUS MOTOR ACTIVITY
During neuro-behavioural testing, the observer was unaware of the treatment of the animals. FOB and spontaneous motor activity were assessed in all study animals during the predose phase and in 5 animals/sex/group at the end of the study.
Sacrifice and pathology:
GROSS NECROPSY AND HISTOLOGY OF PARENTAL ANIMALS
All surviving male and female parent animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anaesthesia at necropsy and then examined grossly for pathological changes. Male animals were sacrificed after the mating period. Female animals were sacrificed at or shortly after day 4 of lactation. A necropsy was performed on animals that died intercurrently (if not precluded by autolysis) or that had to be killed because they were moribund. Prior to preservation of organs/tissues, the following organ weights were recorded: adrenals, brain, heart, kidneys, liver, ovaries, prostate, seminal vesicles, spleen, thymus, thyroid, uterus. Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes which were preserved in Bouin's fixative:

- ovaries (after counting the corpora lutea)
- uterus (after counting of the implantation sites)
- testes
- epididymides
- seminal vesicles
- prostate
- all gross lesions
In addition of 5 animals/sex/group following organs were preserved:
adrenals
bone marrow (femur)
brain (including sections of cerebrum, cerebellum, medulla/pons)
caecum
cervix
clitorial gland
colon
coagulation gland
duodenum
eyes
femur including joint
heart
ileum
jejunum (including Peyer's patches)
kidneys
liver
lungs
mesenterial and axillary lymph nodes
ovaries
peripheral nerve (sciatic or tibial)
pituitary gland
preputial gland
prostate
rectum
seminal vesicles (including coagulation gland)
skeletal muscle (thigh)
spinal cord (cervical, mid-thoracic and lumbar)
spleen
stomach*
thymus
thyroid (including parathyroid)
trachea
urinary bladder
uterus
vagina
* Non glandular (“forestomach”) and glandular (fundus, pylorus) parts were examined
microscopically.

Microscopic examination was performed on the collected organs of all animals of the control (group 1) and high dose group (group 4). If treatment-related changes were observed in the high-dose group, the evaluation of these tissues/organs was extended to the intermediate-dose groups (2 and 3).
In addition, reproductive organs of males that failed to sire (did not mate or mated females were not pregnant) and females that were non-mated or non-pregnant, of the low- and mid-dose groups, were microscopically examined. Furthermore, organs showing gross lesions of animals of all groups were microscopically examined.
Statistics:
If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 2; many-to one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
various liver parameters, see tables 1 and 2
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
liver, see tables 3 and 4
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
liver
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
liver
Histopathological findings: neoplastic:
not examined
Details on results:
Clinical biochemistry details
Compared to controls, animals at 225 mg/kg bw/day had relevant increases in alkaline phosphatase (ALP, both sexes), bilirubin (both sexes), glucose (males), cholesterol (females) and decreased albumin (both sexes), total protein (females) inorganic phosphate (both sexes) and total thyroxine (T4,
both sexes).
At 75 mg/kg bw/day, animals had increased ALP (both sexes, not significantly different for males), glucose (males, not statistically significant), total bilirubin (both sexes) and decreased albumin (both sexes) and total protein (females).
Calcium was lower for all treated animals of both sexes. T4 measurements were significantly lower for females of Groups 2 and 3 as well. The toxicological relevance of these effects is questionable, however, as no clear dose response relationship was noted for either parameter and no relevant effects were seen in the thyroid for females at the microscopic examination.
The statistically significant decrease in cholesterol seen for males at 75 mg/kg bw/day was not considered to be toxicologically relevant as it occurred in the absence of a dose-dependent distribution.
Thyroid stimulating hormone (TSH) was much higher than controls for all groups (both sexes, not always statistically significant). These data showed high variability with one or more individuals in each treated group showing extremely high values (Group 2, male no. 11, Group 3 male nos. 22, 25, and female no. 62 and Group 4 male no. 33). When recalculated excluding the outliers, group means remained higher than controls (males Group 2: 0.518, Group 3: 0.548, Group 4: 0.475; females Group 3: 0.386), though no dose-dependent distribution was apparent. In the absence of a clear relationship with total T3 or T4, and in the absence of adverse findings seen in the thyroids during the microscopic examination, no toxicological relevance was attributed to higher TSH values.

Macroscopic findings
At 225 mg/kg bw/day treatment related macroscopic findings were seen in the liver including accentuated lobular pattern of the liver (6 males, 2 females), pale discoloration (7 males, 1 female), and an enlarged liver (1 male). Taken together with relevant clinical biochemistry and microscopic findings, these were considered to be toxicologically relevant.
Pale discoloration of the liver was also seen for 2 males at 75 mg/kg bw/day, this was also considered to be treatment related but since microscopic evidence indicated this was an adaptive response, it was not considered to be adverse at this level.
One male at 25 mg/kg bw/day (no. 13) was noted with a liver that was enlarged and with black discoloration. At the microscopic examination, this animal was found with centrilobular vacuolation,
congestion and hepatocellular degeneration/necrosis. These were not considered toxicologically relevant as they occurred in the absence of other relevant findings and were seen for a single animal.
The macroscopic findings noted for the female that was euthanized after struggling with an extended parturition (no. 50) included pale discoloration of the whole body and 11 dead and autolytic pups were
found in the uterus.

Organ weights
Relative kidney weights were significantly higher for males at 225 mg/kg bw/day only. Absolute and relative liver weights were significantly higher for males of all treatment groups (approximately 20-54%
increase in relative weight for males). Liver weights were also higher for females at 75 and 225 mg/kg bw/day, but the difference from controls was only significantly different for females at 75 mg/kg
bw/day.

Histopathology
Test item-related microscopic findings were noted in the liver of both sexes starting at 75 mg/kg bw/day, the thyroid gland of the males in all treated groups, and in the kidney of females at 225 mg/kg bw/day.
Hepatocellular hypertrophy of the liver was noted in 6/6 males (6 minimal) and 1/5 females (1 minimal) treated at 75 mg/kg bw/day and in 8/8 males (2 minimal, 6 slight) and 6/6 females (2 minimal, 4 slight) at 225 mg/kg bw/day.
Hepatocellular vacuolation of zone 1 (periportal) and/or zone 2 (midzonal) of the liver was noted in 2/5 females (2 minimal) treated at 75 mg/kg bw/day and in 8/8 males (3 minimal, 5 slight) and 6/6 females
(3 minimal, 1 slight, 2 moderate) at 225 mg/kg bw/day. This finding consisted of microvesicular vacuolation of the midzonal hepatocytes (zone 2) in the males, whereas in the females the vacuolation ranged from microvesicular vacuolation of the periportal hepatocytes (zone 1) to combined micro-and macrovesicular vacuolation of periportal and midzonal hepatocytes (recorded as slight and moderate degrees) in the 225 mg/kg bw/day group.
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Test-item related effects on liver at 75 and 225 mg/kg bw
Critical effects observed:
not specified

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Group 1 (control group) did not contain the test substance.

Table 1: Clinical chemistry values for males at the end of treatment
control 25 mg/kg bw 75 mg/kg bw 225 mg/kg bw

ALAT
MEAN 55.3 61.1 57.3 63.6
U/L ST.DEV 22.9 29.9 14.2 13.3
N 5 5 5 5
ASAT MEAN 78.8 84.6 86.1 84.0
U/L ST.DEV 14.4 14.0 4.1 8.4
N 5 5 5 5
ALP MEAN 144 176 220 409 **
U/L ST.DEV 52 43 32 64
N 5 5 5 5
Total protein MEAN 60.7 58.3 58.3 58.3
g/L ST.DEV 1.5 2.0 3.0 2.2
N 5 5 5 5
Albumin MEAN 31.2 30.1 29.5 ** 28.4 **
g/L ST.DEV 0.5 0.9 0.8 0.8
N 5 5 5 5
Total bilirubin MEAN 2.1 2.5 3.4 * 5.6 **
umol/L ST.DEV 0.2 0.3 1.0 1.0
N 5 5 5 5
Urea MEAN 5.4 5.1 5.1 5.8
mmol/L ST.DEV 1.8 0.6 0.7 0.9
N 5 5 5 5
Creatinine MEAN 39.4 39.4 41.0 39.2
umol/L ST.DEV 3.2 1.2 2.5 1.3
N 5 5 5 5
Glucose MEAN 8.59 10.47 11.08 11.27 *
mmol/L ST.DEV 0.96 1.32 2.37 1.46
N 5 5 5 5
Cholesterol MEAN 1.94 1.81 1.44 * 1.55
mmol/L ST.DEV 0.44 0.23 0.18 0.18
N 5 5 5 5
Sodium MEAN 142.2 141.6 141.9 141.3
mmol/L ST.DEV 1.4 0.4 1.4 0.5
N 5 5 5 5
Potassium MEAN 4.31 4.10 4.24 4.32
mmol/L ST.DEV 0.30 0.18 0.14 0.24
N 5 5 5 5
Chloride MEAN 104 105 105 105
mmol/L ST.DEV 1 1 2 1
N 5 5 5 5
Calcium MEAN 2.55 2.44 * 2.38 ** 2.45 *
mmol/L ST.DEV 0.05 0.05 0.07 0.05
N 5 5 5 5
Inorg.Phos MEAN 2.08 1.88 1.90 1.79 *
mmol/L ST.DEV 0.25 0.05 0.14 0.07
N 5 5 5 5

TSH
MEAN 0.143 1.116 0.706 0.794
uIU/mL ST.DEV 0.070 1.347 0.253 0.740
N 5 5 5 5
Total T3 MEAN 85.1 74.3 77.9 73.6
ng/dL ST.DEV 9.8 4.4 5.9 12.8
N 5 5 5 5
Total T4 MEAN 4.28 3.97 3.79 3.04 *
ug/dL ST.DEV 1.10 0.74 0.27 0.24
N 5 5 5 5
 Bile acids  MEAN  24.2 15.8  11.7*  10.7**
 umol/ml  ST.DEV  10.3  4.3  4.2  2.2
   N  5

 */** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 2: Clinical chemistry values for females at the end of treatment
control 25 mg/kg bw 75 mg/kg bw 225 mg/kg bw
END OF TREATMENT
ALAT
MEAN 48.5 56.9 56.1 63.1
U/L ST.DEV 12.0 17.9 16.3 19.5
N 5 5 5 5
ASAT MEAN 77.3 77.9 75.3 87.1
U/L ST.DEV 11.3 7.1 7.9 14.4
N 5 5 5 5
ALP MEAN 99 99 206 * 292 **
U/L ST.DEV 55 24 62 55
N 5 5 5 5
Total protein MEAN 61.5 60.4 55.8 ** 57.0 *
g/L ST.DEV 0.3 2.7 2.3 3.1
N 5 5 5 5
Albumin MEAN 31.6 29.8 26.5 ** 27.3 **
g/L ST.DEV 0.6 1.8 1.5 2.2
N 5 5 5 5
Total bilirubin MEAN 1.9 1.9 2.8 ** 4.4 **
umol/L ST.DEV 0.5 0.3 0.4 0.4
N 5 5 5 5
Urea MEAN 7.1 7.5 7.4 7.4
mmol/L ST.DEV 1.3 0.9 1.3 0.9
N 5 5 5 5
Creatinine MEAN 43.4 44.0 45.4 41.9
umol/L ST.DEV 2.0 1.5 2.0 3.2
N 5 5 5 5
Glucose MEAN 6.91 6.72 7.95 7.41
mmol/L ST.DEV 1.19 0.93 0.33 0.88
N 5 5 5 5
Cholesterol MEAN 1.65 1.94 2.62 2.77 *
mmol/L ST.DEV 0.21 0.31 0.94 0.76
N 5 5 5 5
Sodium MEAN 139.9 140.2 140.0 139.2
mmol/L ST.DEV 1.6 2.2 1.1 2.5
N 5 5 5 5
Potassium MEAN 3.53 3.55 3.79 3.79
mmol/L ST.DEV 0.21 0.18 0.18 0.21
N 5 5 5 5
Chloride MEAN 98 100 99 100
mmol/L ST.DEV 1 1 1 2
N 5 5 5 5
Calcium MEAN 2.60 2.51 * 2.46 ** 2.44 **
mmol/L ST.DEV 0.06 0.03 0.05 0.02
N 5 5 5 5
Inorg.Phos MEAN 2.54 2.21 2.16 1.86 **
mmol/L ST.DEV 0.29 0.16 0.29 0.16
N 5 5 5 5

TSH
MEAN 0.146 0.490 * 0.488 * 0.224
uIU/mL ST.DEV 0.037 0.233 0.282 0.075
N 5 5 5 5
Total T3 MEAN 71.1 62.2 72.6 73.3
ng/dL ST.DEV 8.0 6.0 8.0 9.0
N 5 5 5 5
Total T4 MEAN 3.15 1.96 * 2.05 * 2.02 *
ug/dL ST.DEV 0.44 0.49 0.58 0.71
N 5 5 5 5
 Bile Acids  MEAN  23.6  6.9**  15.5  12.9
 umol/ml  ST.DEV  11.7  1.7  4.3  4.5
   N  5  5  5  5

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 3: Organ and body weight values for males at the end of treatment
    control 25 mg/kg bw 75 mg/kg bw 225 mg/kg bw

BODY W.
MEAN 388 385 384 376
(GRAM) ST.DEV 28 25 20 20
  N 10 10 10 10
BRAIN MEAN 2.11 2.10 2.17 2.13
(GRAM) ST.DEV 0.08 0.07 0.08 0.08
  N 10 10 10 10
HEART MEAN 1.026 1.026 1.044 1.047
(GRAM) ST.DEV 0.087 0.069 0.058 0.062
  N 10 10 10 10
LIVER MEAN 9.10 10.84 * 11.15 * 13.55 **
(GRAM) ST.DEV 0.79 2.56 0.82 1.18
  N 10 10 10 10
THYROIDS MEAN 0.016 0.016 0.018 0.017
(GRAM) ST.DEV 0.004 0.003 0.003 0.004
  N 10 10 10 10
THYMUS MEAN 0.321 0.324 0.313 0.315
(GRAM) ST.DEV 0.082 0.065 0.048 0.046
  N 10 10 10 10
KIDNEYS MEAN 2.36 2.46 2.42 2.52
(GRAM) ST.DEV 0.25 0.22 0.21 0.14
  N 10 10 10 10
ADRENALS MEAN 0.051 0.053 0.056 0.056
(GRAM) ST.DEV 0.006 0.010 0.006 0.007
  N 10 10 10 10
SPLEEN MEAN 0.620 0.616 0.658 0.608
(GRAM) ST.DEV 0.066 0.074 0.110 0.079
  N 10 10 10 10
TESTES MEAN 3.52 3.64 3.75 3.63
(GRAM) ST.DEV 0.29 0.34 0.27 0.29
  N 10 10 10 10
PROSTATE MEAN 0.695 0.761 0.616 0.642
(GRAM) ST.DEV 0.100 0.153 0.072 0.155
  N 10 10 10 10
EPIDIDYMIDES MEAN 1.158 1.182 1.180 1.147
(GRAM) ST.DEV 0.066 0.092 0.079 0.122
  N 10 10 10 10
SEMINAL VES MEAN 1.625 1.651 1.558 1.601
(GRAM) ST.DEV 0.133 0.242 0.160 0.222
  N 10 10 10 10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Executive summary:

Hepatic toxicity was noted for animals of both sexes at 225 mg/kg bw/day. This was supported by changes in clinical pathology endpoints (increase of ALP, bilirubin, cholesterol and decrease of albumin, total protein and inorganic phosphate, among others), macroscopic abnormalities (accentuated lobular pattern, enlargement and discoloration), increased organ weights (relative weights approximately 50% and 19% higher for males and females, respectively) and microscopic findings including hepatocellular vacuolation and hypertrophy. Taken together, the findings at this dose level were considered to be adverse.

At 75 mg/kg bw/day, morphologic liver findings were regarded to be an adaptive, non-adverse response based on the minimal severity of the hypertrophy (correlated to pale discoloration) and low incidence and minimal degree of vacuolation in the females. However, when taken together withsimilar changes seen in clinical biochemistry parameters as high dose animals, and an approximate

relative liver weight increase of 24% and 17% for males and females, respectively, the findings were also considered toxicologically relevant at 75 mg/kg bw/day.

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

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Experimental data includes a subacute gavage study (OECD 422) and its 28-day range-finding study, a 90-day study with a related alkylated diphenylamine and its 28-day range-finding study as well as mechanistic (“metabolome”) investigations for both substances. The related UVCB substance is “reaction products with branched nonene” instead of “reaction products with 2,2,4 –trimethylpentene”

The extended dose-range finding study in male rats was performed to verify a previously used read-across and to support dose-range finding for future testing for reproductive toxicity (BASF 2013) and accordingly, 125 and 300 mg/kg bw were chosen as dose levels. Corn oil served as vehicle since the substance is not miscible with water, but miscible with oil. Since liver was expected to be the target organ, haematology, clinical chemistry and liver histopathology were investigated. As part of this study, plasma was subjected to a "metabolome" analysis (see IUCLID section 7.9.3) which investigates changes in plasma concentrations of more than 200 endogenous plasma components.

As a result of the adverse effects on liver, dose levels of 25, 75 and 225 mg/kg bw were chosen for the OECD 422 study. This study was performed at another laboratory with slightly older animals (10-12 weeks versus 6 weeks). This may explain why the liver effects were more prominent in the full study although lower doses were tested. Affected parameters are summarized in the table below.

The data was compared to results of a full 90 -day study (OECD 408) and its dose-range finder with another UVCB substance of similar composition (Reaction products with branched nonene instead of reaction products with 2,2,4 -trimethylpentene) tested at equal and higher doses (100, 300 and 1000 mg/kg bw). The composition of both UVCB substances is shown in a table in the toxicokinetic section. An overview on these results is also given in the table below.

All studies identify liver as the primary target organ for both substances. Both substances cause an adaptive enlargement of liver in combination with adverse findings such as a disturbed lipid metabolism and cellular degeneration. Single cell necrosis is seen in a few animals at the high dose groups only. Alkaline phosphatase levels are increased less than 3fold at 300 mg/kg bw and albumin levels are decreased at equal or less than 10% for subacute and 100 mg/kg bw for subchronic exposure, respectively.

As seen in table 1, the parameters affected by the two substances are identical with two exceptions. The target substance caused a slight reduction in total bilirubin levels and an increased severity of lymphoid cell infiltrates in the liver (2/5 animals at 300 mg/kg bw). But both parameters fit to the general picture of adverse effects on liver. Comparison regarding the overall toxicity shows that for some parameters, the substance is more potent than the nonylated version. The accentuated lobular pattern of the liver (6 males, 2 females), pale discoloration (7 males, 1 female) was observed at 225 mg/kg bw after 28 days whereas only 3/20 animals showed a prominent acinar pattern of liver at 1000 mg/kg bw after 90 days. This is consistent with an increase in male relative liver weight by 54% at 225 mg/kg bw after 28 days versus 27% at 300 mg/kg bw after 90 days.

In contrast, the occurrence of single cell necrosis and the fatty changes in liver was more pronounced after 90 days at comparable dose levels. Slight changes in haematology were only observed after 90 days.

All other affected parameters similarly affected at 28 and 90 days. So overall, it appears that nonylated version is less hazardous. This may be due to the simple fact that the average molecular weight is higher and therefore the dose on a molar base lower.

The mechanistic investigation showed that both substances have the most similar metabolome patterns among all substances existing in the database.

The 90-day study with the nonylated version shows that prolonging the treatment from subacute to subchronic toxicity does not introduce previously unknown effects. Therefore, the NOAEL of 25 mg/kg bw is considered adequate for deriving the DNEL and no full 90 day study for the actual substance is needed.

The registrant is awaiting the final decision for a two-generation study. Liver histopathology can be included for males to verify the subchronic NOAEL.

Table: Comparison of the effects observed upon repeated dose treatment with reaction products with 2,4,4 -trimethylpentene (28 -days and OECD 422) and nonene (28 and 90 days)

(Substance CAS 15721 -78 -5, Bis((1,1,2,2 -tetramethylbutyl)phenyl)amine could also be added, but caused no adverse effects at 100, 300 and 1000 mg/kg bw in the OECD 408, based on the dissiminated information)

    Benzenamine, N-phenyl-, reaction products with 2,4,4-trimethylpentene Reaction products of benzeneamine, N-phenyl with nonene
(branched)
METHOD Test institute Will, NL BASF, DE BASF, DE BASF, DE BASF, DE
  gender males, females males males males females
  number of animals per sex 10 (5 examined) 5 3 10 10
  duration of treatment 28 days (males), 54 - 57 days (females) 28 days 28 days 90 days 90 days
  study type OECD 422 extended range-finder range-finder OECD 408 OECD 408
  rat strain Crl:WI(Han) Wistar Wistar Wistar Wistar
  vehicle for gavage corn oil corn oil corn oil corn oil corn oil
  doses 25, 75, 225 mg/kg bw 125 and 300 mg/kg bw 100, 300 and 1000 mg/kg bw 100, 300 and 1000 mg/kg bw 100,300 and 1000 mg/kg bw
  Organ weights Adrenal glands, brain, epididymides, hearts, kidney, liver, prostate, seminal vesicle, spleen, testes, thymus, thyroid gland, uterus with cervix Adrenal glands, brain, epididymides, hearts, kidney, liver, prostate, seminal vesicle, spleen, testes, thymus, thyroid gland none Adrenal glands, brain, epididymides, hearts, kidney, liver, prostate, seminal vesicle, spleen, testes, thymus, thyroid gland Adrenal glands, brain, hearts, kidney, liver, ovaries, spleen, thymus, thyroid gland, uterus with cervix
  Organs examined by histopathology all liver none all all
RESULTS Mortality none none none none none
  Gross necropsy accentuated lobular pattern of the liver (6 males, 2 females), pale discoloration (7 males, 1 female),
and an enlarged liver (1 male) at 225 mg/kg bw;  Pale discoloration of the liver was also seen for 2 males at 75 mg/kg bw/day
no effects observed no effects observed no effects observed  prominent acinar pattern of liver (n=3)
  Body weight or body weight gain not affected at 25, 75 and 225 mg/kg bw not affected at 125 and 300 mg/kg bw not affected at 100, 300 mg/kg bw and 1000 mg/kg bw at 1000 mg/kg bw body weight -14.6% and body weight gain -24.2% on day 91; no effects
   At 300 mg/kg bw terminal body weights -6%
  Clinical observations none none not examined none none
  Decreased red blood cell (RBC) counts, hemoglobin, and hematocrit no effects yes yes (except RBC) yes
  Increased relative reticulocyte counts no effects yes yes yes
  Increased alkaline phosphatase (ALP) activities yes (2.8x males) yes (2.3x) yes (1.6x) yes (3.3x)
  Decreased total bilirubin levels 2.6x increased (males) 35% decreased no no
  Decreased total albumin yes (-9% in males) yes (-7%) yes (-14%) yes (-2%)
Findings at 300 mg/kg bw (or 225 mg/kg bw) Decreased total bile acid (TBA)
levels
yes (-55% males) (-44% females) yes (-84%) yes (-62%) yes (-54%)
  Increased triglyceride levels no no no yes
  Prolonged prothrombin time in males no no no no
  Increased absolute liver weights 54% males, 5% females 29% 20% 18%
  Increased relative liver weights 54% males, 11% females 22% 27% 24%
  Minimal single cell necrosis in the liver none  1/5  4/10 none
   Fatty changes in hepatocytes 8/8 males (3 minimal, 5 slight) and 6/6 females
(3 minimal, 1 slight, 2 moderate)
 3/5 (minimal/slight) 10/10 males (3 minimal, 3 slight, 3 moderate, 1 severe)  6/10 (minimal, slight; peripheral)
  Increased severity of lymphoid cell infiltrates in the liver not observed  2/5 not observed not observed
  Liver cell hyerptrophy  8/8 males (2 minimal, 6 slight) and 6/6 females (2 minimal, 4 slight) 5/5 (minimal up to moderate)  9/10 (minimal to moderate) minimal to slight (10/10)
  Clinical observations   none   none none
  Decreased red blood cell (RBC) counts, hemoglobin, and hematocrit   not observed not observed yes
  Increased relative reticulocyte counts   not observed not observed yes
  Increased alkaline phosphatase (ALP) activities   yes (1.6x) no (1.1x) yes (1.7)
  Decreased albumin   yes (-7%) not observed yes (-10%)
  Decreased total bilirubin levels   34% decreased not observed no
Findings at 125/100 mg/kg bw decreased total bile acid (TBA)
levels
  yes (-71%) yes (-49%) yes (-60%)
  Increased absolute liver weights   25% 14% 12%
  Increased relative liver weights   17% 18% 12%
  Minimal single cell necrosis in the liver    2/5  2/10 no effects
  centrilobular hepatocellular hypertrophy
hepatocytes)
  Minimal to slight (5/5) minimal (6/10) minimal to slight (8/10)
  Increased severity of lymphoid cell infiltrates in the liver    2/5 not observed not observed
   Fatty changes in hepatocytes   not observed 9/10 (6 minimal, 3 slight, midzonal) not observed
Findings at 75 mg/kg bw Increased alkaline phosphatase (ALP) activities yes
Increased total bilirubin levels yes
Decreased total total protein and albumin yes
Increased absolute liver weights 22% (males), 17% (females)
Increased relative liver weights 22% (males), 17% (females)
 Fatty changes in hepatocytes 2/5 females (minimal, peripheral)
Liver cell hyerptrophy 6/6 males (minimal) and 1/5 females (minimal)
Increased severity of lymphoid cell infiltrates in the liver not observed
  Functional observation battery no effects not examined   no effects no effects
  Opthalmology not examined not examined   no effects no effects
  Heinz bodies not examined not examined   no effects no effects
  Urinalysis not examined not examined   no effects no effects
  Absolute organ weights other than liver no effects no effects   (heart at mid and high dose group, not biologically relevant) no effects
Any other findings Relative organ weights other than liver Relative kidney weights were significantly higher for males at 225 mg/kg bw/day no effects   adrenal glands, brain, kidney, thyroid gland, testes (all high dose group only) kidney (mid and high dose group), spleen (all dose groups)
  Histophathology findings other than liver Hypertrophy/
hyperplasia of thyroid gland at all dose levels
not examined   Hypertrophy/
hyperplasia of thyroid gland at all dose levels
Hypertrophy/
hyperplasia of thyroid gland at all dose levels
  Accumulation of brown pigment in the cortical tubular epithelium of the kidneys of females was noted in 3/5 rats (minimal) treated at 225 mg/kg bw/day   Altered colloid at 300 and 1000 mg/kg bw no effects on colloid
Follicular cell hypertrophy of the thyroid gland was noted at an increased incidence in 4/5 males (2
minimal, 2 slight) treated at 25 mg/kg bw/day, in 5/5 males (2 minimal, 3 slight) at 75 mg/kg bw/day
and in 5/5 males (minimal) at 225 mg/kg bw/day compared to 1/5 males (minimal) of the control group.
For the thyroids, the increased incidence of hypertrophy of the follicular epithelium was considered to
be non-adverse based on the absence of a clear dose response and/or relation in severity of
hypertrophy of the follicular epithelium of the thyroid gland, hypertrophy of the liver, decrease in total
T4 and increase in TSH (hypertrophy of the follicular epithelium of the thyroid glands may reflect an
increase in thyroxin production in response to feedback mechanisms as a result of increased turnover
of thyroxin by hypertrophic hepatocytes). Additionally, the recorded severities/incidences of the
hypertrophy of the follicular epithelium of thyroid were within background levels noted in male rats of
this strain and age.



Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Indentificaton of a NOAEL, most detailed examinations

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

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. No serious irreversible effects were observed at dose levels of less than 150 mg/kg bw upon subacute exposure. As a result the substance is not considered to be classified for repeated dose toxicity under Directive 67/548/EEC, as amended for the 28th time in Directive 2001/59/EC.

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. There no significant toxic effects at doses of less than 300 mg/kg bw upon subacute oral exposure in rats. Fatty changes were at most moderate, and not severe. There was no morbidity. As a result the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the second time in Directive (EC 286/2011).