Benzenamine, N-phenyl-, styrenated

Endpoint summary

Description of key information

Repeated dose toxicity: Subacute (28-day) study, oral: gavage, Sprague-Dawley rat m/f, 5/sex/dose, 0, 100, 300, 1000 mg/kg bw/d (OECD 407, GLP): NOAEL = 100 mg/kg bw/d (m/f), based on haematology, clinical biochemistry, gross pathology, histopathology

Repeated dose toxicity: Subacute (6-7 week) study, oral: gavage, Sprague-Dawley rat m/f, 10/sex/dose, 0, 50, 250, 600 mg/kg bw/d (OECD 422, GLP): NOAEL = 600 mg/kg bw/d (systemic toxicity, only adaptive, non-adverse responses)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993-04-08 - 1993-08-31 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

EEC Methods for the determination of toxicity, Directive 84/449/EEC (OJ No. L251, 19.9.84), Part B, Method B7. Subacute toxicity (oral).

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

OECD Guideline for Testing of Chemicals No. 407, "Repeated Dose Oral Toxicity - Rodent: 28-day or 14-day study". Adopted: 12 May 1981

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 4°C in the dark

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD BR VAF PLUS™

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent, England
- Age at study initiation: 28 ± 1 day old
- Weight at study initiation: weight range of ± 13% of the mean weight on arrival
- Fasting period before study: No
- Housing: All rats were initially caged, as far as possible, in groups of five according to sex in metal cages with wire mesh floors. The cages (each containing five rats) constituting each group were distributed in batteries in such a manner that possible environmental influences arising from their spatial distribution were equilibrated, as far as possible, for all treatments
- Diet (e.g. ad libitum): A standard expanded laboratory rodent diet (Special Diet Services Rat and Mouse Maintenance Diet) provided ad libitum. The batches of diet used for the study had been analysed for nutrients, possible contaminants and micro-organisms.
- Water (e.g. ad libitum): drinking water provided ad libitum
- Acclimation period: An one week acclimatisation period was allowed between delivery of the animals and start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Animal room temperature was controlled in the range 19.5 to 24°C
- Humidity (%): relative humidity was controlled in the range 42 to 59% RH
- Air changes (per hr): Air exchange was maintained at a rate of approximately 19 air changes per hour.
- Photoperiod (hrs dark / hrs light): Lighting was controlled to give 12 hours artificial light (0700 - 1900 hours) in each 24-hour period.

IN-LIFE DATES: From: 31 March 1993

The health status of all animals was monitored, by daily observation, throughout the acclimatisation period, to ensure that the rats selected for final assignment to the study were satisfactory.
Two days before the start of treatment, each rat was weighed and forty rats were randomly allocated to four groups, each consisting of five males and five females. This allocation was carried out using a computer program, so that the weight distribution within each group was similar and the initial group mean bodyweights were approximately equalised.

Route of administration:

oral: gavage

Details on route of administration:

The dosages were selected by the Sponsor and on the basis of a preliminary oral toxicity investigation performed at this laboratory.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
A 20% w/v suspension of Styrenated diphenyiamine was freshly prepared each day by mixing the test substance in corn oil by stirring. The formulation was then mixed in a high shear homogeniser. Further formulations, (6.0 and 2.0% w/v) were prepared in a similar manner.
The chemical stability and homogeneity of test substance formulations in corn oil were assessed prior to the start of treatment by HRC Department of Analytical Chemistry and were found to be satisfactory.
Concentration analyses of formulations prepared for administration on Day 1 were also performed. Additional samples of all concentrations were taken on Day 6 due to an analytical problem with the low and intermediate formulations (2.0 and 6.0% w/v respectively) on Day 1.
The absorption of the test substance was not determined in this study.
On completion of the study, the remaining test substance was returned to the supplier for re-analysis.
Data concerning the analytical purity and homogeneity of the test substance and its stability under the specified conditions of storage are the responsibility of the Sponsor.

VEHICLE
- Concentration in vehicle: 20%, 6%, 2% w/v
- Amount of vehicle (if gavage): 5 ml/kg/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The chemical stability and homogeneity of test substance formulations in corn oil were assessed prior to the start of treatment by HRC Department of Analytical Chemistry and were found to be satisfactory.
Concentration analyses of formulations prepared for administration on Day 1 were also performed. Additional samples of all concentrations were taken on Day 6 due to an analytical problem with the low and intermediate formulations (2.0 and 6.0% w/v respectively) on Day 1

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 / sex / dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dosages were selected by the Sponsor and on the basis of a preliminary oral toxicity investigation performed at this laboratory.
- Rationale for animal assignment (if not random): Two days before the start of treatment, each rat was weighed and forty rats were randomly allocated to four groups, each consisting of five males and five females. This allocation was carried out using a computer program, so that the weight distribution within each group was similar and the initial group mean bodyweights were approximately equalised.

Positive control:

not required

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed daily for signs of ill health, behavioural changes or toxicosis. Any observed changes were recorded.
All animals were checked early in each working day and again in the late afternoon to look for dead or moribund animals in order that a post mortem examination could be undertaken during the working part of that day. On Saturdays, Sundays and public holidays, a similar procedure was followed except that the final check was carried out at approximately mid-day. Any animal showing signs of severe debility or toxicosis was sacrificed for reasons of animal welfare.

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed prior to dosing on Day 1 (Week 0) and subsequently at weekly intervals throughout the study.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food consumed in each cage was measured at weekly intervals throughout the study.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily monitoring by visual appraisal was maintained throughout the dosing period. In addition, water consumption was measured, by weight, over daily periods during Week 3, for all groups of rats.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: the orbital sinus of all rats prior to termination (Week 4).
- Anaesthetic used for blood collection: Yes (identity): light ether anaesthesia
- Animals fasted: Yes: Food was withdrawn overnight prior to collection of samples
- How many animals: all rats
Due to analytical problems with the OSM 3 Haemoximeter (used for methaemoglobin analysis) animal nos. 1(female) to 15(female) were re-bled, for this parameter only, within one hour of the initial samples being taken. These animals had access to food during this time. The validity of the methaemoglobin estimations was not considered to have been affected.
The collected blood samples were divided as follows:
EDTA anticoagulant tubes for haematological investigations
Citrate anticoagulant tubes for coagulation test
Heparin anticoagulant microtainer tubes* for biochemical tests
* Microtainer, brand plasma separator tube, Becton Dickinson, Rutherford, New Jersey, U.S.A.
All the tubes were then mechanically agitated for at least five minutes and the microtainer tubes were subsequently centrifuged for a minimum period of two minutes (3000 'g')
The estimations performed have been listed below, together with an abbreviated title, the methods and the units of measurement applicable.

Haematology
The following parameters were analysed with an Ortho ELT-1500 Analyser, using standard Ortho methodology:
Packed cell volume (PCV): %
Haemoglobin (Hb): g/dl
Red blood cell count (RBC): x 10exp6/mm³
Absolute indices:
Mean corpuscular haemoglobin concentration (MCHC) Calculated: Hb (g/dl) x 100 / PCV (%): %
Mean corpuscular volume (MCV) Calculated: PCV (%) x 10 / RBC (x 10exp6/mm³): fl
Mean corpuscular haemoglobin (MCH) Calculated: Hb x 10 / RBC: pg/l
Platelet count (Pits) x 10³/mm³
Total white blood cell count (WBC): x 10³/mm³
Heinz bodies (Hz B): + to +++
The following estimations were measured using the appropriate methodology:
Methaemoglobin (Met Hb) - OSM 3 Haemoximeter: % Hb
Thrombotest (TT) - Method of Owren, P.A. (Lancet, 1959, ii, 754): s
Differential white blood cell count (Dift) - namely: x 10³/mm³
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
The percentage distribution of each cell type was determined by Standard microscopy of a blood smear stained with modified Wright's stain counting 100 cells. Percentage values were then converted to absolute values by computer inevitably involving a 'rounding off' in a proportion of the results. Hence, the measured total WBC may differ slightly from the calculated total for the differential count.
Additionally, blood film slides were examined for morphological abnormalities. If abnormal cells (see below) were observed when examining the stained slides, their presence was recorded and included in the haematology appendix.
P Polychromasia
H Hypochromasia
A Anisocytosis
R Rouleaux formation
S Separate film report (generated for additional abnormalities)
nad No abnormality detected
1 Slight
2 Moderate
3 Marked
4 Gross
In addition to protocol requirements, Reticulocytes (Retic, %) were measured. This deviation from protocol was not considered to have affected the integrity, or the result of the study.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see above
- Animals fasted: Yes
- How many animals: all rats
The following parameters were analysed with a Hitachi 737 Clinical Chemistry Analyser:
Glucose - using BCL Test Kit (hexokinase mediated): mg/dl
Total protein: g/dl
Albumin (Alb): g/dl
Globulin (Glob), Calculated: Total protein (g/dl) minus Alb (g/dl): g/dl
Albumin/Globulin ratio (A/G), Calculated from Total protein and Albumin concentrations
Urea nitrogen (Urea Nitr): mg/dl
Creatinine: mg/dl
Alkaline phosphatase (AP) - reaction temperature 30°C: mU/ml
Glutamic-pyruvic transaminase (GPT), also known as 'alanine Aminotransferase (ALT)' - using BCL Test Kit, reaction temperature 30°C: mU/ml
Glutamic-oxaloacetic transaminase (GOT), also known as 'aspartate aminotransferase (AST)‘ - using BCL Test Kit, reaction temperature 30°C: mU/ml
Total bilirubin (Bilirubin): mg/dl
Sodium (Na): mEq/l
Potassium (K): mEq/l
Calcium (Ca): mEq/l
Chloride (Cl): mEq/l
Inorganic phosphorus (P): mEq/l
Cholesterol (Chol): mg/dl

URINALYSIS: Yes
- Animals fasted: Not specified
The following estimations were measured using the appropriate methodology:
Volume (Vol): ml
pH - using pH meter
Specific Gravity (SG) - using Atago UR-1 Refractometer, sample compared with water (nominal value of 1000)
Protein - using Roche Cobas Centrifugal Analyser, utilising modified method of Macart, M. and Gerbaut, L. (Clin. Chim. Acta, 1984, 141 77): mg/dl
The following tests were also performed using qualitative indicators (+) of analyte concentration:
Total reducing substances (TRS)
Glucose
Ketones
Bile pigments
Haem pigments*
Urobilinogen
ts*
* Positive or negative finding only
+ Clinitest (total reducing substances) and Multistix (remaining parameters) are diagnostic reagents obtained from Ames Company, Stoke Poges, Berkshire, England
Results have been reported according to the following convention:
0 Negative
tr 'Trace' of analyte
+ 'Small amountT of analyte
+ + 'Moderate amount' of analyte
+++ 'Large amount' of analyte
Microscopic examination of urine samples was carried out by centrifuging samples at approximately 500 'g' for 10 minutes and spreading the resulting deposit on a microscope slide. The deposit was then examined for the presence of the following:
Epithelial cells (E)
Polymorphonuclear leucocytes (P)
Mononuclear leucocytes (M)
Erythrocytes (R)
Organisms (O)
Renal tubule casts (C)
Sperm (sp)
Other abnormal constituents (A)
The frequency of the above parameters in the centrifugal deposit has been recorded as follows:
0 None found in any field examined
1 Few in some fields examined
2 Few in all fields examined
3 Many in all fields examined

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Termination
After 28 days of treatment (Day 29) all surviving animals were killed by carbon dioxide asphyxiation and a complete autopsy undertaken. The order of sacrifice was determined using a pre-set cage sequence. Specified organs were weighed and relevant tissue samples were fixed for microscopic examination.
Organ weight: The following organs from each animal were dissected free of fat and weighed:
adrenals, liver, brain, ovaries, kidneys, spleen, testes (with epididymides)
Macroscopic pathology: The macroscopic appearance of the tissues of all rats was recorded and samples of the following tissues were preserved in 10% buffered formalin:
adrenals* aorta, brain (medullary, cerebellar and cortical sections), caecum, colon, duodenum, eyes (Davidson's fluid as fixative), femur (with joint), head, heart*, ileum, jejunum, kidneys*, larynx, liver*, lungs, lymph nodes (cervical and mesenteric), mammary glands, oesophagus, ovaries, pancreas, pharynx, pituitary, prostate, rectum, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin, spleen*, sternum (for bone and marrow sections), stomach, testes (including epididymis)*, thymus (where present), thyroid (with parathyroid), tongue, trachea, urinary bladder, uterus, vagina, any macroscopically abnormal tissue*
* tissues required for histopathological examination for rats from Groups 1 and 4

HISTOPATHOLOGY: Yes
Microscopic pathology: Fixed tissue samples required for microscopic examination were prepared by embedding in paraffin wax (mp 56°C); sections were cut at 4 µm and stained with haematoxylin and eosin.
Microscopic examination of prepared slides (from tissues indicated under Macroscopic pathology) was carried out for all animals that were sacrificed during the study, in an attempt to ascertain the cause of death, and all rats of Group 1 (Control group) and Group 4 (high dosage group) killed on Day 29.
Examinations were extended, following documented approval from the Sponsor, to include the heart, liver and kidneys from rats of both sexes of the intermediate and low dosage groups.

Statistics:

STATISTICAL ANALYSES
All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.
The following sequence of statistical tests was used for bodyweight gains, organ weight and clinical pathology data:
If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75%) the proportion of values different from the mode was analysed by Fisher's exact test (1) followed by Mantel's test for a trend in proportions (2). Otherwise:
Bartlett's test (3) was applied to test for heterogeneity of variance between treatments. If significant heterogeneity was found at the 1 % level, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.
If no significant heterogeneity was detected (or if a satisfactory transformation was found), an one-way analysis of variance was carried out followed by Williams' test (5) for a dose-related response.
If significant heterogeneity of variance was present and could not be removed by a logarithmic transformation, the Kruskal-Wallis analysis of ranks (4) was used. This analysis was followed by the non-parametric equivalent of Williams' test (Shirley's test, (6)).
Significant differences between control animals and those treated with the test substance have been expressed at the 5% (* P <0.05 for Williams’ test or +P <0.05 for Student's Y test) or \% (** P <0.01 for Williams' test or + +P <0.01 for Student's 't test) level.
References
1. FISHER, R.A. (1932) Statistical methods for research workers, 4th ed, Oliver and Boyd.
2. MANTEL, N. (1963)7. Amer. Statist. Ass.9 58, 690.
3. BARTLETT, M.S. (1937) Proc. Roy. Soc. A, 160 268.
4. KRUSKAL, W.H. and WALLIS, W.A. (1952/3) J. Amer. Statist. Ass.y 47, 583 and 48 907.
5. WILLIAMS, D.A. (1971/2) Biometrics, 27 103 and 28, 519.
6. SHIRLEY, E. (1977) Biometrics, 33 386.
7. ANGERVALL, L. and CARLSTROM, E. (1963) J. Theoreu Biol 4 254.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No clinical signs were noted for groups of treated rats during the first three weeks of the study. During Week 4, clinical signs noted among rats treated at 1000 mg/kg/day included abnormal gait (walking on toes) for one male and all five females, abdominal distension for all females and red/brown staining of the urogenital region for two females.
Fur loss was recorded for all female rats treated at 1000 mg/kg/day during Weeks 2, 3 and 4. Fur loss is generally associated with fighting and is often seen in rat studies which are group boused. However, it should be noted that in this study there was a high incidence of this finding among females from the high dosage group.
No clinical signs of toxicity were seen for rats from the intermediate and low dosage groups.
Increased salivation following dosing was commonly seen throughout the study in the majority of rats receiving 1000 mg/kg/day and sporadically during the first half of the treatment period and more commonly during Weeks 3 and 4 in animals receiving 300 mg/kg/day. This sign was generally associated with wet fur in rats of both sexes receiving 1000 mg/kg/day and for females and one male treated at 300 mg/kg/day. In rats treated at 100 mg/kg/day, increased salivation following dosing was observed intermittently during the study. These findings are commonly observed in rat, orally dosed, studies and were considered to be a result of the unpalatability of the test substance. Therefore, these findings are considered to be of no toxicological importance.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

A protrusion of the right eye was observed on Day 22 to 26 in one male (no. 20,male) receiving 1000 mg/kg/day. Following the blood sampling procedure performed on Day 27, bleeding from the right eye was noted for this animal. This persisted and showed no signs of stopping and the animal was killed on humane grounds later on Day 27.
There were no other mortalities during the treatment period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Group mean bodyweights and bodyweight gains for rats of both sexes were comparable to those of controls during the first three weeks of the study. Bodyweight gains for Week 4 were statistically significantly lower than controls for males receiving 1000 mg/kg/day and females treated at either 1000 or 300 mg/kg/day.
For females treated at 100 mg/kg/day and males receiving 300 or 100 mg/kg/day, bodyweights and bodyweight gains were comparable to that of controls throughout the study.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Slightly higher than control food consumption was recorded in Weeks 3 and 4 for female rats receiving 1000 mg/kg/day, although overall food consumption was similar to that of the controls. Food consumption values for remaining treated rats were comparable to those of controls throughout the study.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Gravimetric analysis of water consumption was initiated on Day 15 following a suspected treatment-related effect noted during visual assessment during Week 2.
Measurements performed during Week 3 showed higher than control water consumption for females receiving 1000 mg/kg/day (63% higher than controls). Differences from control recorded for remaining groups of treated rats were considered to represent variation, especially as there was only one cage per sex per group, and not an effect of treatment.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Thrombotest times were markedly longer for males and females receiving 1000 mg/kg/day; slightly but statistically significantly higher values were also noted for males receiving 300 mg/kg/day.
Red blood cell counts (RBC) were statistically significantly higher for females receiving 300 mg/kg/day. However, there was no dose relationship and this small difference was considered to be due to variation. Statistically significantly lower mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) values were recorded for males and females treated at 1000 mg/kg/day and females receiving 300 mg/kg/day. In the absence of any treatment-related effect on the red blood cells the variation in these indices is also considered to be due to chance.
Neutrophil levels were statistically significantly higher for males treated at 1000 mg/kg/day whilst group mean methaemoglobin levels were found to be statistically significantly lower for these animals in comparison with concurrent controls. In view of the variability of individual values these differences were not believed to be related to treatment with Styrenated diphenylamine.
There were no other statistically significant differences between control and treated animals.
The occurrence of slight polychromasia in young laboratory rats is not uncommon and at the incidence seen in this study (two control males) is not believed to be related to treatment.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

For rats at the high dosage group, the following parameters were affected by treatment; higher alkaline phosphatase, glutamic-pyruvic transaminase (females only), glutamic-oxaloacetic transaminase (females only), bilirubin, and lower cholesterol and albumin. Additionally, a general disturbance of the electrolytes was apparent which mainly included higher sodium and chloride (females only) ion concentrations and lower calcium ion concentrations. These findings are detailed below.
Alkaline phosphatase (AP) levels were statistically significantly higher for rats of both sexes treated at 1000 or 300 mg/kg/day. In addition, higher than control glutamic-pyruvic transaminase (GPT) and glutamic-oxaloacetic transaminase (GOT) levels were recorded for female rats treated at 1000 mg/kg/day.
Group mean bilirubin levels were statistically significantly higher for males and females treated with 1000 mg/kg/day in comparison with controls.
Cholesterol levels were statistically significantly lower for males and females receiving either 1000 or 300 mg/kg/day and for males treated at 100 mg/kg/day.
For rats of both sexes receiving 1000 and 300 mg/kg/day, group mean albumin levels were statistically significantly lower in comparison with controls whilst globulin levels for animals treated at 1000 mg/kg/day were statistically significantly higher; total protein levels remained unaffected. The resultant lowering of the A/G ratio was statistically significant for males and females receiving 1000 or 300 mg/kg/day. The A/G ratio for males treated at 100 mg/kg/day was also statistically significantly lower than control, but this small difference was not considered to be a result of treatment.
A general disturbance of the electrolyte values was apparent which included statistically significantly higher sodium levels for rats of both sexes treated at 1000 mg/kg/day and chloride levels for females treated at 1000 mg/kg/day, and statistically significantly lower calcium ion levels for males and females receiving 1000 or 300 mg/kg/day. Phosphorus levels for males treated at 1000 mg/kg/day were statistically significantly higher in comparison with controls and potassium levels were statistically significantly lower thail control for females at this dosage.
A statistically significantly higher group mean glucose level was recorded for females treated at 1000 mg/kg/day. However, there was large variation in individual values and the difference was not believed to be attributable to treatment with Styrenated diphenylamine.
There were no other statistically significant differences between control and treated animals.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Markedly higher than control urinary volumes with correspondingly lower specific gravity was apparent in the females receiving 1000 mg/kg/day. Males at this dosage showed a similar but much lesser effect. The specific gravity was also lower than control for males treated at 300 mg/kg/day.
A statistically significantly higher group mean pH value was recorded for females of the high dosage group (1000 mg/kg/day).
For males treated at 1000 mg/kg/day, group mean protein was statistically significantly lower in comparison with concurrent controls. As there was considerable individual variation, this difference was considered to be due to chance.
There were no other statistically significant differences between treated and control rats.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistically significantly higher than control liver weight was recorded for rats of both sexes treated at 1000 mg/kg/day. Although absolute liver weights for remaining treated groups were only marginally higher than control, for females receiving 300 mg/kg/day liver weight relative to terminal bodyweight was statistically significantly higher than for control.
Adrenal weight was statistically significantly higher for males and females treated at 1000 mg/kg/day relative to controls. The higher than control mean value for females treated at 1000 mg/kg/day was partially due to a particularly high value for one female (no. 36, female). For both males and females there was overlap of individual values between the groups. Also, as there was no histopathological change (see overleaf), this apparent finding was not considered to be related to treatment with Styrenated diphenylamine.
There were no other statistically significant differences between treated and control rats.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The macroscopic examination performed at termination revealed the following changes:
Liver - Enlarged in 3/4 male rats and 5/5 female rats treated at 1000 mg/kg/day and 3/5 male rats treated at 100 mg/kg/day, compared with none in the control group. Swollen in 1/4 male rats and 3/5 female rats treated at 1000 mg/kg/day, compared with none in the control group. Pale in 3/4 male rats and 3/5 female rats treated at 1000 mg/kg/day and 1/5 male rats treated at 300 mg/kg/day, compared with none in the control group. Pale subcapsular areas in 1/4 male rats and 3/5 female rats treated at 1000 mg/kg/day, compared with none in the control group.
Kidneys - Enlarged in 2/5 female rats treated with 1000 mg/kg/day, compared with none in the control group.
Skin - Alopecia in 4/5 female rats treated at 1000 mg/kg/day, compared with none in the control group.
Caecum - Distended in 2/5 female rats treated with 1000 mg/kg/day, compared with none in the control group. However, in the absence of histopathological change or any associated clinical signs, this finding was not considered to be toxicologically important.
Mesenteric lymph nodes - Congested in 3/4 male rats treated with 1000 mg/kg/day, compared with none in the control group. The histopathological finding associated with this finding (see overleaf) was not considered to be treatment-related.
There were no other changes of note.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were reported in the liver, heart and kidneys:
Liver - Minimal bile duct hyperplasia was reported in 4/5 female rats receiving 1000 mg/kg/day. It was not reported in any other treated or control female rats and it was not reported in any male rats.
Fine vacuolation of periportal and sometimes midzonal hepatocytes was reported in treated male and female rats, but not in any control rats.
In male rats, it was reported in 5/5 rats receiving 1000 mg/kg/day and 2/5 rats receiving 300 mg/kg/day. In female rats, it was reported in 5/5 rats receiving 1000 mg/kg/day, 5/5 rats receiving 300 mg/kg/day and 1/5 rats receiving 100 mg/kg/day.
In this study there was no other evidence of any degenerative liver changes. Periportal vacuolation is occasionally reported in untreated control female rats and so its low incidence of 1/5 female rats receiving 100 mg/kg/day is of uncertain significance.
Heart - Moderate myocarditis and moderate myocardial degeneration was reported in one male rat receiving 1000 mg/kg/day. This finding was not reported in any other treated or control rats. It was considered treatment-related in spite of its low incidence, due to the severity of the lesion in this one rat.
Kidney - Moderate focal interstitial inflammation in the cortex and medulla and marked cystic dilatation and basophilia of cortical and medullary tubules was reported in one female rat receiving 1000 mg/kg/day. It was considered treatment-related, in spite of its low incidence, due to the severity of the lesion in this one rat.
The increased weight in adrenals reported in male and female rats receiving 1000 mg/kg/day did not show any histological treatment-related changes,
Erythrophagocytosis was reported in the mesenteric lymph nodes of 4/5 male rats receiving 1000 mg/kg/day. This is a spontaneous lesion, frequently seen in this age of laboratory-maintained rat and as such is considered unlikely to be associated with treatment.
All other findings reported were considered to be within the background histopathological findings found in this age and strain of laboratory-maintained rat.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

gross pathology

haematology

histopathology: non-neoplastic

Dose descriptor:

NOEL

Remarks on result:

not determinable

Remarks:

lowest dose already induce non-adverse effects

Dose descriptor:

LOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

gross pathology

haematology

histopathology: non-neoplastic

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

urinalysis

water consumption and compound intake

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

not specified

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

The study was conducted under GLP according to OECD guideline 407 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation and performance. Hence, the results can be considered as reliable to assess the repeated dose oral toxicity (short-term) in rats.
Treatment-related changes were reported in the liver, heart and kidneys.
Fine vacuolation of periportal and sometimes midzonal hepatocytes was reported in the livers of treated male and female rats. Minimal bile duct hyperplasia was reported in the livers of treated female rats.
Moderate myocarditis and myocardial degeneration were reported in the heart of one treated male rat.
Moderate focal interstitial fibrosis in the cortex and medulla and marked cystic dilatation and basophilia of cortical and medullary tubules were reported in the kidneys of one treated female rat.
Based on the results of this study, it was concluded that 100 mg/kg/day represents a no-observed adverse effect level (NOAEL) in the rat for Styrenated diphenylamine. A no-observed effect level (NOEL) for Styrenated diphenylamine could not be established.

Styrenated diphenylamine produced evidence of liver toxicity at 1000 mg/kg/day, including higher alkaline phosphatase, glutamic-pyruvic transaminase, glutamic-oxaloacetic transaminase and bilirubin levels, lower plasma albumin and cholesterol, increased liver weights, macroscopic and microscopic changes. The altered blood coagulation, as represented by markedly longer thrombotest times and the animal with persistent bleeding that was sacrificed, was considered likely to be as a consequence of the liver effect.
An effect on the kidney at this dosage of 1000 mg/kg/day was also considered likely, as disturbed plasma electrolytes, high urinary volumes (with specific gravity and pH of urine affected), and macroscopic and microscopic findings (females only) were noted. The increased water consumption seen for female rats could be related.
The toxicological significance of the microscopic finding in the heart of one male rat treated at 1000 mg/kg/day was uncertain.
At 300 mg/kg/day, a reduced continuation of effects was seen. Slightly longer thrombotest times were recorded for males and some biochemistry and urinalysis parameters were also affected, and there were macroscopic and microscopic effects on the liver. The bodyweight gains for female rats at this dosage were also lower than control during Week 4.
Findings seen at the 100 mg/kg/day dosage lever included lower than control cholesterol levels for males, macroscopically enlarged livers for 3/5 males and the microscopic finding of fine vacuolation in hepatocytes for one female rat. However, macroscopically enlarged liver was not seen at the intermediate dosage of 300 mg/kg/day and the microscopic finding is also seen occasionally in control animals.

According to Regulation 1272/2008, Table 3.9.1, Categories for specific target organ toxicity-repeated exposure, Category 2 is required for substances that, on the basis of evidence from studies in experimental animals can be presumed to have the potential to be harmful to human health following repeated exposure. Substances are classified in category 2 for target organ toxicity (repeat exposure) on the basis of observations from appropriate studies in experimental animals in which significant toxic effects, of relevance to human health, were produced at generally moderate exposure concentrations. Guidance dose/concentration values are provided below (see 3.9.2.9) in order to help in classification.
Guidance Value Ranges (dose/concentration) to assist in Category 2 classification are i.a. 10 < C ≤ 100 mg/kg body weight/day for an Oral (rat) study over 90 days. For subacute studies, the limit value is 300 mg/kg bw. The Regulation states further, that the guidance values and ranges mentioned in paragraphs 3.9.2.9.6 and 3.9.2.9.7 are intended only for guidance purposes, i.e. to be used as part of the weight of evidence approach, and to assist with decisions about classification. They are not intended as strict demarcation values. Substances are classified as specific target organ toxicants following repeated exposure by the use of expert judgement; assessment shall take into consideration not only significant changes in a single organ or biological system but also generalised changes of a less severe nature involving several organs.
As stated above, there are treatment-related changes reported in the liver, heart and kidneys.
The toxicological significance of the microscopic finding in the heart of one male rat treated at 1000 mg/kg/day was uncertain and should hence not be seen as evidence for classification as STOT RE, as is was only a single incidence and further only noted at 1000 mg/kg/day, which is way above the guidance value of 300 mg/kg. The same applies for the kidneys, as relevant effects were only noted at 1000 mg/kg.
Initial effects for liver involvement were noted already at 100 mg/kg. However, they were only minor, macroscopically enlarged liver was not seen at the intermediate dosage of 300 mg/kg/day and the microscopic finding is also seen occasionally in control animals. Hence, a clear dose-response could not be established for the lower dose levels. Further, Centrilobular hepatocyte enlargement is commonly observed in rodent liver following the administration of any xenobiotic and is considered to be adaptive and not to represent an adverse health effect. These findings are suggestive of an adaptive response to mixed function oxidase induction in the liver. Last but not least, the vacuolation in the liver of animals in the high dose group may be due to a perturbation of metabolic activity.
In consequence, clear evidence for classification as STOT RE Cat. 2 is not given.

Executive summary:

This study was performed to assess the systemic toxicity of Styrenated diphenylamine to the rat. The method followed was that outlined in Annex V, Part B, Method B7 in the EEC Directive 84/449/EEC and OECD Guideline for Testing of Chemicals No. 407, "Repeated Dose Oral Toxicity -Rodent: 28-day or 14-day study".

Styrenated diphenylamine, a viscous amber liquid intended for use as a rubber protectant, was administered by oral gavage, once daily, to groups of five male and five female rats for twenty-eight consecutive days, at fixed dosage levels of 100, 300 and 1000 mg/kg/day. The test material was prepared as dilutions in corn oil at concentrations of 2.0, 6,0 and 20% w/v. A further group of rats (five males and five females) was held as a concurrent control receiving the vehicle (corn oil) alone.

Bodyweights, food and water consumption and clinical observations were recorded during the study. Blood samples for clinical investigations were taken on Day 27. All surviving animals were killed and examined macroscopically on Day 29, Histopathological examination of specified tissues was then initiated.

The following comments are made in summary:

Mortality. One male rat (no. 20♂) from the high dosage group was sacrificed on humane grounds on Day 27 as a result of persistent bleeding from the right eye following the blood sampling.

Clinical signs. Treatment related clinical signs were not seen until Week 4, were confined to animals receiving 1000 mg/kg/day and included normal gait (walking on toes), abdominal distention and red/brown staining of the urogenital region. A high incidence of fur loss was also noted amongst female rats of this group.

Bodyweights. During Week 4 only, lower than control bodyweight gains were recorded for male and female rats treated at 1000 mg/kg/day and female rats treated at 300 mg/kg/day.

Food consumption. Slightly higher than control food consumption was recorded in Weeks 3 and 4 for female rats receiving 1000 mg/kg/day.

Water consumption. During Week 3 higher than control water consumption was noted for females treated at 1000 mg/kg/day.

Haematology. Markedly longer thrombotest times were recorded for male and female rats treated at 1000 mg/kg/day and slightly higher than control values for males treated at 300 mg/kg/day.

Biochemistry. For rats at the high dosage group, the following parameters were affected by treatment; higher alkaline phosphatase, glutamic-pyruvic transaminase (females only), glutamic-oxaloacetic transaminase (females only), bilirubin, and lower cholesterol and albumin. Additionally, a general disturbance of the electrolytes was apparent which mainly included higher sodium ion and chloride ion (females only) concentrations and lower calcium ion concentrations.

Among rats treated at 300 mg/kg/day, higher alkaline phosphatase, lower cholesterol, lower albumin and lower calcium were recorded. Lower than control cholesterol was recorded for male rats treated at 100 mg/kg/day.

Urinalysis. Higher urinary volume and pH (females only) and lower specific gravity was recorded for rats treated at 1000 mg/kg/day. Lower than control specific gravity was also recorded for males treated at 300 mg/kg/day.

Organ weights. Higher bodyweight - relative liver weights were recorded for males and females treated at 1000 mg/kg/day and females at 300 mg/kg/day.

Macroscopic pathology. Enlarged and/or swollen livers were noted among rats treated at 1000 mg/kg/day and for 3/5 males treated at 100 mg/kg/day. Pale subcapsular areas were also noted among rats at 1000 mg/kg/day. The liver appeared pale among rats dosed at 1000 mg/kg/day and for 1/5 males at 300 mg/kg/day.

Enlarged kidneys were noted for two females treated at 1000 mg/kg/day.

Alopecia was noted among female rats treated at 1000 mg/kg/day.

Microscopic pathology. Treatment-related changes were reported in the liver, heart and kidneys.

Fine vacuolation of periportal and sometimes midzonal hepatocytes was reported in the liver of treated male and female rats. Minimal bile duct hyperplasia was reported in the liver of treated female rats.

Moderate myocarditis and myocardial degeneration were reported in the heart of one treated male rat.

Moderate focal interstitial fibrosis in the cortex and medulla and marked cystic dilation and basophilia of cortical and medullary tubules were reported in the kidneys of one female receiving 1000 mg/kg/day.

Other findings. All other differences from control were not considered to be related to treatment with Styrenated diphenylamine.

Conclusion. Based on the results of this study it was concluded that 100 mg/kg/day represents a no-observed adverse effect level (NOAEL) in the rat for Styrenated diphenylamine. A no-observed effect level (NOEL) for Styrenated diphenylamine could not be established.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2005-02-21 - 2005-12-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

OECD Guidelines for Testing of Chemicals, No. 422: “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Sprague-Dawley Crl:CD (SD) IGS BR strain rats

Details on species / strain selection:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately six weeks
- Weight at study initiation: At the start of treatment, the males weighed 184 to 239g, the females weighed 145 to 188g.
- Fasting period before study: no
- Housing: Initially, all animals were housed in groups of five in polypropylene cages with stainless steel grid floors and tops, suspended over polypropylene trays lined with absorbent paper. During the mating phase, animals were transferred to similar cages on an one male : one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in polypropylene cages with solid floors and stainless steel lids, furnished with softwood flakes (Datesand Ltd. Cheshire, UK). Environmental enrichment was provided in the form of wooden chew blocks (B & K Universal Ltd, Hull, UK) and cardboard fun tunnels (Datesand Ltd, Cheshire, UK) except for mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.
- Diet (e.g. ad libitum): A pelleted diet (Rodent PMI 5002 (Certified) diet, BCM IPS Limited, London, UK) was used throughout the treatment period ad libitum.
- Water (e.g. ad libitum): Mains drinking water was supplied ad libitum from polycarbonate bottles attached to the cage.
- Acclimation period: On receipt, the animals were examined for signs of ill-health or injury. The animals were acclimatised for up to ten days, during which time their health status was assessed.

DETAILS OF FOOD AND WATER QUALITY:
The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
The animals were housed in air-conditioned rooms within the Laboratories Limited Barrier Maintained Rodent Facility
- Temperature (°C) / Humidity (%): Environmental conditions were continuously monitored by a computerised system and print-outs of hourly mean temperatures and humidities are included in the study records. The temperature and relative humidity controls were set to achieve target values of 21±2°C and 55 ±15% respectively. Occasional deviations from these targets were considered not to affect the purpose or integrity of the study.
- Air changes (per hr): The rate of air exchange was at least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): The low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test material, and the results of the study are believed to be of value in predicting the likely toxicity of the test material to man and to screen for potential adverse effects on reproduction.
The test material was administered daily by gavage using a steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 ml/kg/day of corn oil.
The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at regular intervals.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of the study, the test material was prepared at the appropriate concentrations as a solution in Corn Oil. The stability and homogeneity of the test material formulations were
determined. Results show the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately +4ºC in the dark.

VEHICLE
- Concentration in vehicle: 0, 12.5, 62.5, or 150 mg/ml
- Amount of vehicle (if gavage): 4 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test material formulations were determined by the Analytical Laboratory. Results are given in in the attachment and show the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately +4°C in the dark.
Samples were taken of each test material formulation and were analysed for concentration of CAS No 68442-68-2 at the Analytical Laboratory. The method used for analysis of formulations and the results obtained are given in the attachment. The results indicate that the prepared formulations were within acceptable limits for the purpose of this study.

Duration of treatment / exposure:

Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum.

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

600 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 / sex / dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen based on the results of a preliminary range-finder.
- Rationale for animal assignment (if not random): The animals were allocated to dose groups using a randomisation procedure based on stratified bodyweights and the group mean bodyweights were then determined to ensure similarity between the dose groups. The animals were uniquely identified within the study, by an ear punching system routinely used in these laboratories.

Positive control:

not required

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before and after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before and after dosing, and one hour after dosing at weekends (except for females during parturition where applicable).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity (except for females which did not achieve pregnancy). Detailed individual clinical observations were performed for each animal using a purpose-built arena. The following parameters were observed: Gait, Tremors, Twitches, Convulsions, Bizarre/Abnormal/Stereotypic behaviour, Salivation, Pilo-erection, Exophthalmia, Lachrymation, Hyper/Hypothermia, Skin colour, Respiration, Palpebral closure, Urination, Defecation, Transfer arousal, Tail elevation,

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 (prior to the start of treatment) and then weekly for males until termination. Females were weighed weekly until mating was evident. Bodyweights were then recorded on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION:
During the maturation period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

FOOD EFFICIENCY:
Food efficiency (the ratio of bodyweight change/dietary intake) was calculated retrospectively for males and for females during maturation and the first two weeks of gestation. Due to offspring growth and milk production, food efficiency could not be accurately calculated during the final week of gestation and during lactation.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt change.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group on Day 14 (day prior to pairing). Blood samples were obtained from the lateral tail vein.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: No. Animals were not fasted prior to sampling.
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Methaemoglobin (Meth)
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices:
- mean corpuscular haemoglobin (MCH)
mean corpuscular volume (MCV)
mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count:
- neutrophils (Neut)
- lymphocytes (Lymph)
monocytes (Mono)
eosinophils (Eos)
basophils (Bas)
Platelet count (PLT)
Prothrombin time (CT) was assessed by 'Thrombomax HS with calcium' and Activated partial thromboplastin time (APTT) was assess by 'Actin FS' using samples collected into sodium citrate solution (0.11 mol/l).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group on Day 14 (day prior to pairing). Blood samples were obtained from the lateral tail vein.
- Animals fasted: No. Animals were not fasted prior to sampling.
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea
Glucose
Total protein (Tot.Prot.)
Albumin
Albumin/Globulin (A/G) ratio (by calculation)
Sodium (Na+)
Potassium (K+)
Chloride (Cl-)
Calcium (Ca++)
Inorganic phosphorus (P)
Aspartate aminotransferase (ASAT)
Alanine aminotransferase (ALAT)
Alkaline phosphatase (AP)
Creatinine (Creat)
Total cholesterol (Chol)
Total bilirubin (Bili)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity (except for females which did not achieve pregnancy). Functional performance tests were also performed on five selected males and females per dose level, prior to termination (Day 4 post partum for females), together with an assessment of sensory reactivity to various stimuli. Due to an interim death, only four females were assessed from the 250 mg/kg/day dose group. This was not considered to affect the purpose or integrity of the study.
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity

Functional Performance Tests
Motor Activity. Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period).
Forelimb/Hindlimb Grips Strength. An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed: Grasp response, Vocalisation, Toe pinch, Tail pinch, Finger approach, Touch escape, Pupil reflex, Startle reflex, Blink reflex

IMMUNOLOGY: No

OTHER: Reproduction Screening

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum.
In addition, the corpora lutea of all ovaries from pregnant females were counted at necropsy. The uterine implantation sites were counted. In the case of non-pregnant females, the procedure was enhanced by staining the uteri with a 1% ammonium polysulphide solution.
All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
The following organs, removed from adult animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals
Brain
Epididymides
Heart
Kidneys
Liver
Ovaries
Spleen
Testes
Thymus

HISTOPATHOLOGY: Yes
Samples of the following tissues were preserved from five males and five females from each dose group, in buffered 10% formalin. The tissues marked with x were also removed from the remaining animals:
Adrenals
Aorta (thoracic)
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Brain (including cerebrum, cerebellum and pons)
Caecum
Coagulating gland (x)
Colon
Duodenum
Epididymides * (x)
Eyes
Gross lesions
Heart
Ileum
Jejunum
Kidneys
Liver
Lungs (with bronchi) #
Lymph nodes (cervical and mesenteric)
Mammary gland
Muscle (skeletal)
Ovaries (x)
Pancreas
Pituitary (x)
Prostate (x)
Oesophagus
Rectum
Salivary glands (submaxillary)
Sciatic nerve
Seminal vesicles (x)
Skin (hind limb)
Spinal cord (cervical)
Spleen
Stomach
Thyroid/parathyroid
Trachea
Testes * (x)
Thymus
Urinary bladder
Uterus/Cervix (x)
Vagina

* = preserved in Bouin’s fluid and then in 70% IMS after forty-eight hours
# = lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

The tissues from five selected control and 600 mg/kg/day dose group animals and those animals dying during the study, were prepared as paraffin blocks, sectioned at nominal thickness of 5 µm and stained with haematoxylin and eosin for subsequent microscopic examination. The tissues marked with x from the remaining control and 600 mg/kg/day were also processed.
Since there were indications of treatment-related changes in the liver and thyroid glands examination was subsequently extended to include similarly prepared sections of liver and thyroid gland tissues from five selected males and females from the 250 and 50 mg/kg/day dose groups.
Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE Pathology computerisation system for tabulation and report production.

Other examinations:

see chapter "toxicity to reproduction"

Statistics:

Treatment of Data
Data were processed to give group mean values and standard deviations where appropriate.
For bodyweights and food consumptions during gestation, group mean values were calculated using data from females which produced live young.
For bodyweights and food consumptions during lactation, group mean values were calculated using data from females with live young at Day 5 of lactation.

Statistical Analysis
Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain, litter weights, offspring bodyweights and quantitative functional performance data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney 'U' test.
The non-parametric methods were also used to analyse implantation loss, offspring sex ratio and landmark developmental markers.
The haematology variable basophils was not analysed since consistently greater than 30% of the data were recorded as the same value.
Probability values (p) are presented as follows:
p < 0.001 ***
p < 0.01 **
p < 0.05 *
p >0.05 (not significant)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinically observable signs of toxicity were detected.
Transient episodes of increased salivation were detected soon after dosing for animals of either sex treated with 600 mg/kg/day from Day 5, with the observation also evident up to one hour after dosing for males. Isolated incidents of increased salivation were also apparent at 250 mg/kg/day, during the later stages of the study. This observation is often reported following the oral administration of a slightly unpalatable or irritant test material formulation, and in isolation, is considered to be of no toxicological importance.
Incidents of fur loss, scab formation and staining of the external body surface, detected throughout the treatment groups, are common low incidence findings in laboratory maintained animals and are considered unrelated to treatment.
One female treated with 50 mg/kg/day displayed damage to the tail tip and one control male displayed an injury to the eye. These were physical injuries and unrelated to treatment. Additionally, one control female displayed an incident of diuresis.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no deaths related to treatment.
One female treated with 250 mg/kg/day was killed in extremis on gestation Day 25, following difficulties encountered during parturition. In isolation and in the absence of any histopathological correlates, this death was considered unrelated to treatment.
There were no further deaths.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No adverse effect on bodyweight change was detected for treated males in comparison to controls throughout the treatment period, or for females throughout the maturation, gestation or lactation phases of the study.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No adverse effect on dietary intake was detected for males throughout the treatment period, or for females during maturation and gestation. Food efficiencies were unaffected.
A slight reduction in dietary intake was evident for females treated with 600 mg/kg/day during lactation, compared with controls, however differences failed to attain statistical significance. Lower litter size was apparent at this dose level and it is considered that the observed differences in food intake probably reflected the lower litter demand on the parental females rather than any adverse effect of treatment.

Food efficiency:

not specified

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

no effects observed

Description (incidence and severity):

Daily visual inspection of water bottles revealed no intergroup differences.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related haematological changes were detected prior to mating.
Females treated with 600 mg/kg/day showed a statistically significant reduction in mean cell volume (MCV) when compared with control females. The significance was minimal (p<0.05) and in the absence of supporting data to suggest this was related to treatment, this finding was considered to have arisen incidentally.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males treated with 600 mg/kg/day showed slightly higher alkaline phosphatase levels than control males (p<0.05). Additionally, reduced cholesterol levels were detected for males treated with 600 and 250 mg/kg/day in comparison to controls (p<0.05 and p<0.01, respectively).
The statistically significant reductions in urea levels detected for males treated with 600 mg/kg/day were of minimal significance (p<0.05) and in the absence of any histopathological correlates, was considered to have arisen incidentally and unrelated to test material toxicity.
No treatment-related blood chemical changes were detected in females treated with 600 and 250 mg/kg/day, or for animals of either sex treated with 50 mg/kg/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Behavioural Assessment: Weekly open field arena observations did not reveal any treatment-related effects during the study. A higher number of males treated with 600 mg/kg/day showed urination compared with controls during the first week of the study, however, each animal only showed a single occasion of urination. In the absence of similar effects during later weekly assessments, or any other supporting evidence during the study, this observation was considered to be coincidental and unrelated to treatment. All remaining inter and intra groups variations in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.
Functional Performance Tests: No treatment-related effects were detected in the functional performance parameters investigated. Statistical analysis of the data did not reveal any significant inter group differences.
Sensory Reactivity Assessments: No treatment-related effects were detected in sensory reactivity. All inter and intra group differences in sensory reactivity scores were considered to be result of normal variation for arts of the strain and age used and were of no toxicological importance.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Liver weights, both absolute and relative to terminal bodyweight were elevated for animals of either sex treated with 600 mg/kg/day, although statistical significance was only achieved for absolute weights for females.
Adrenal weights, both absolute and relative to terminal bodyweights were higher than controls for both sexes receiving 600 mg/kg/day and males receiving 250 mg/kg/day. However, differences were slight with statistical significance was restricted to absolute adrenal weights for males receiving 600 mg/kg/day.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related macroscopic abnormalities were detected at terminal kill.
The observations detected, including small, flaccid or reddened testes, small epididymis (from males which failed to achieve pregnancy in their female partners) and sloughing of the glandular gastric epithelium, were isolated findings, and in the absence of a dose related response, were considered unrelated to test material toxicity.
The interim death female treated with 250 mg/kg/day showed red staining around the ano-genital region and a small green coloured mass in the position of the right kidney which was not apparent. One dead foetus, which showed evidence of brachynathnia and microstomia, was found in the left uterine horn. In the absence of any similar findings among other animals at this dose level or for those treated with 600 mg/kg/day, this death was considered to be coincidental and unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There was one unscheduled death during the course of the study. One female treated with 250 mg/kg/day was killed in extremis on gestation Day 25, due to difficulties encountered during parturition. There were no histopathological changes in the tissues of this animal to directly suggest any reasons why parturition was problematical, although the animal did have only one functional kidney which may have been a contributory factor.
The following treatment-related changes were observed:
LIVER: Centrilobular hepatocyte enlargement was observed in relation to treatment for animals of either sex treated with 600 and 250 mg/kg/day. Two females treated with 50 mg/kg/day were similarly affected.
Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and, in the absence of associated inflammatory or degenerative changes, is generally considered to be adaptive in nature.
THYROID GLAND: Follicular cell hypertrophy was observed as a consequence of treatment for males treated with 600 and probably also at 250 mg/kg/day, but not for 50 mg/kg/day males.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

see chapter "Toxicity to reproduction"

Key result

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

Remarks on result:

other: Noted effects are either considered non-treatment related or non-adverse as they are adaptive responses at the highest dose tested.

Key result

Critical effects observed:

no

Conclusions:

The study was conducted under GLP according to OECD guideline 422 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation and performance. Hence, the results can be considered as reliable to assess the repeated dose oral toxicity (short-term) in rats, reproductive effects are discussed in the respective chapter.
The oral administration of CAS No 68442-68-2 to rats for a period of up to fifty-four consecutive days at dose levels of 50, 250 or 600 mg/kg/day resulted in treatment-related systemic changes at 250 and 600 mg/kg/day.
The physical health of the parental animals was unimpaired and daily clinical observations, weekly open field arena observations and functional performance tests did not show any effects of treatment. Bodyweight change and dietary intake was not adversely affected for males or females throughout the study, although a lower dietary intake was observed for females treated with the highest dose during lactation. This was considered to result from the lower demands on the parental females from this dose group, due to fewer offspring.
Biochemical analysis revealed elevated alkaline phosphatase (AP) levels and reduced cholesterol levels, compared with controls, for males treated with 250 and 600 mg/kg/day. The higher levels of AP suggest increased liver activity while the lower cholesterol indicates increased thyroid activity. An increase in liver weights was evident at 600 mg/kg/day and histopathology investigations revealed centrilobular hepatocyte enlargement for animals of either sex treated with 250 and 600 mg/kg/day, with findings possibly extending down to 50 mg/kg/day for females. For males treated with 250 or 600 mg/kg/day, histopathological examinations revealed follicular cell hypertrophy, again indicating increased thyroid activity. Centrilobular hepatocyte enlargement is commonly observed in rodent liver following the administration of any xenobiotic and, in the absence of any degenerative or inflammatory changes, is considered to be adaptive and not to represent an adverse health effect. The lower cholesterol levels and changes in thyroid follicular pathology in this study were restricted to males. Thyroid follicular cell hypertrophy is commonly associated with adaptive liver changes due to the induction of thyroxine metabolising enzymes and resulting changes in thyroid follicular cells in rats are often seen at a higher incidence in males than females. The rodent thyroid shows greater sensitivity than the human, possibly due to the comparatively shorter half-life of thyroxine T4. The thyroid pathology occurring on this study was considered to be secondary to the adaptive changes observed for the liver and was considered not to indicate an adverse health effect in man. Additionally, the increase in adrenal weights observed at 600 mg/kg/day is a non-specific common 'stress response' in rodents, usually associated with increased steroidogenesis and an expansion of the adrenal cortex. The absence of pathological changes minimises the toxicological significance of this finding.
The oral administration of CAS No 68442-68-2 to rats by gavage, at dose levels of 600, 250 and 50 mg/kg/day, resulted in treatment-related effects at all dose levels. These effects however, were considered entirely adaptive in nature, therefore the 'No Observed Adverse Effect Level' (NOAEL) was considered to be 600 mg/kg/day.
Further, due to the adaptive nature of the responses in liver in thyroid, evidence for classification as STOT RE Cat. 2 is not given.

Executive summary:

Introduction. The study was designed to investigate the systemic toxicity and potential adverse effects on reproduction (including offspring development) of the test material and complies with the recommendations of the OECD Guidelines for Testing of Chemicals No. 422 "Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test" (adopted 22 March 1996) under GLP.

 

Methods. The test material was administered by gavage to three groups each of ten male and ten female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for up to fifty-four consecutive days, at dose levels of 50, 250 and 600 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (corn oil).

Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating on five selected males and females from each dose group.

Pairing of animals within each dose group was undertaken on an one male: one female basis on Day 15 of the study, to produce litters.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of developmental landmarks.

Extensive functional observations were performed on five selected parental males from each dose group after the completion of the mating phase, and for five selected parental females from each dose group on Day 4 post partum.

Males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5post partum.All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

Results.

Mortality. No treatment-related deaths were detected.

Clinical Observations. No clinically observable signs of toxicity were detected.

Behavioural Assessments. No treatment-related effects were detected.

Functional Performance Tests. No treatment-related effects were detected.

Sensory reactivity Assessments. No treatment-related effects were detected.

Bodyweights. No adverse effect on bodyweight was observed for males throughout the treatment period, or for females during the maturation, gestation or lactation phases of the study.

Food Consumption. No adverse effect on dietary intake was detected for males throughout the treatment period, or for females during the maturation, gestation or lactation phases of the study.

Water Consumption. No overt intergroup differences were detected.

Haematology. No treatment-related changes were detected prior to mating.

Blood Chemistry. Elevated alkaline phosphatase levels were detected for males treated with 600 mg/kg/day. Males treated with 600 and 250 mg/kg/day also showed reduced cholesterol levels.

No such effects were detected for females treated with 600 or 250 mg/kg/day or for animals of either sex treated with 50 mg/kg/day.

 

Reproductive Screening:

Mating. No adverse effects on mating performance, fertility or gestation were detected.

Offspring Litter Size and Viability. Females treated with 600 mg/kg/day showed a higher percentage of pre-implantation losses in comparison to controls, resulting in the birth of less offspring per litter and lower total litter weights at this dose level. Mean offspring weights for treated animals were comparable to controls.

Offspring Development. Offspring from the600mg/kg/day dose group showed less successful completion of surface righting assessments. There were no treatment-related differences in pinna unfolding.

Litter Observations. There were no clinical signs to suggest an effect of treatment.

Pathology:

Necropsy.

Offspring: No treatment related macroscopic abnormalities were detected for the interim death offspring or for the remaining offspring at terminal kill.

Adults: No treatment-related macroscopic abnormalities were detected for the interim death female or for the remaining animals at terminal kill.

Organ Weights. Elevated liver and adrenal weights, both absolute and relative to terminal bodyweights, were detected for animals of either sex treated with 600 mg/kg/day.

Histopathology. Histopathological examination of adult tissue revealed the following treatment-related changes:

Liver: Centrilobular hepatocyte enlargement was observed for animals of either sex treated with 600 and 250 mg/g/day, with the effect extending into the female 50 mg/kg/day dose group.

Thyroid glands: Follicular cell hypertrophy was observed for males treated with 600 and probably also at 250 mg/kg/day. No such effects were detected for females at these dose levels, or for animals of either sex treated with 50 mg/kg/day.

 

Conclusion. The oral administration of CAS No 68442-68-2 to rats by gavage, at dose levels of 600, 250 and 50 mg/kg/day, resulted in treatment-related effects at all dose levels. These effects however, were considered entirely adaptive in nature, therefore the 'No Observed Adverse Effect Level' (NOAEL) was considered to be 600 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

There are two equally reliable subacute Klimisch 1 studies (acc. OECD 407 and 422) available, the tonnage driven data requirements under REACH are fully met, and the study giving the lower NOAEL = 100 mg/kg was chosen out of precautionary reasons as key study, so the potential hazard will be very likely not underestimated. Hence, the database is of high quality.

System:

other: hepatobiliary and urinary

Organ:

kidney

liver

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

Mode of Action Analysis / Human Relevance Framework

In both available studies, predominantly liver effects noted, which are not considered results of specific organ toxicity but rather related to overall systemic responses. It is hypothesized that these effects are adaptive responses, which may turn into adverse effects. Further, kidney involvement was seen in the OECD 407 study only. The following effects were noted:

 

Liver:

In the available OECD 407 study in rats, Styrenated diphenylamine produced evidence of liver toxicity at 1000 mg/kg/day, including higher alkaline phosphatase, glutamic-pyruvic transaminase, glutamic-oxaloacetic transaminase and bilirubin levels, lower plasma albumin and cholesterol, increased liver weights, macroscopic and microscopic changes. The altered blood coagulation, as represented by markedly longer thrombotest times and the animal with persistent bleeding that was sacrificed, was considered likely to be as a consequence of the liver effect.

The liver was enlarged in 3/4 male rats and 5/5 female rats treated at 1000 mg/kg/day and 3/5 male rats treated at 100 mg/kg/day, compared with none in the control group. It was swollen in 1/4 male rats and 3/5 female rats treated at 1000 mg/kg/day, compared with none in the control group., and pale in 3/4 male rats and 3/5 female rats treated at 1000 mg/kg/day and 1/5 male rats treated at 300 mg/kg/day, compared with none in the control group. Pale subcapsular areas were noted in 1/4 male rats and 3/5 female rats treated at 1000 mg/kg/day, compared with none in the control group.

Minimal bile duct hyperplasia was reported in 4/5 female rats receiving 1000 mg/kg/day. It was not reported in any other treated or control female rats and it was not reported in any male rats.

Fine vacuolation of periportal and sometimes midzonal hepatocytes was reported in treated male and female rats, but not in any control rats. In male rats, it was reported in 5/5 rats receiving 1000 mg/kg/day and 2/5 rats receiving 300 mg/kg/day. In female rats, it was reported in 5/5 rats receiving 1000 mg/kg/day, 5/5 rats receiving 300 mg/kg/day and 1/5 rats receiving 100 mg/kg/day.

In this study there was no other evidence of any degenerative liver changes. Periportal vacuolation is occasionally reported in untreated control female rats and so its low incidence of 1/5 female rats receiving 100 mg/kg/day is of uncertain significance.

In the OECD 422 study, biochemical analysis revealed elevated alkaline phosphatase (AP) levels and reduced cholesterol levels, compared with controls, for males treated with 250 and 600 mg/kg/day. The higher levels of AP suggest increased liver activity while the lower cholesterol indicates increased thyroid activity. An increase in liver weights was evident at 600 mg/kg/day and histopathology investigations revealed centrilobular hepatocyte enlargement for animals of either sex treated with 250 and 600 mg/kg/day, with findings possibly extending down to 50 mg/kg/day for females. For males treated with 250 or 600 mg/kg/day, histopathological examinations revealed follicular cell hypertrophy, again indicating increased thyroid activity. Centrilobular hepatocyte enlargement is commonly observed in rodent liver following the administration of any xenobiotic and, in the absence of any degenerative or inflammatory changes, is considered to be adaptive and not to represent an adverse health effect. The lower cholesterol levels and changes in thyroid follicular pathology in this study were restricted to males. Thyroid follicular cell hypertrophy is commonly associated with adaptive liver changes due to the induction of thyroxine metabolising enzymes and resulting changes in thyroid follicular cells in rats are often seen at a higher incidence in males than females. The rodent thyroid shows greater sensitivity than the human, possibly due to the comparatively shorter half-life of thyroxine T4. The thyroid pathology occurring on this study was considered to be secondary to the adaptive changes observed for the liver and was considered not to indicate an adverse health effect in man. Additionally, the increase in adrenal weights observed at 600 mg/kg/day is a non-specific common 'stress response' in rodents, usually associated with increased steroidogenesis and an expansion of the adrenal cortex. The absence of pathological changes minimises the toxicological significance of this finding.

In the OECD 407, initial effects for liver involvement were noted already at 100 mg/kg. However, they were only minor, macroscopically enlarged liver was not seen at the intermediate dosage of 300 mg/kg/day and the microscopic finding is also seen occasionally in control animals. Hence, a clear dose-response could not be established for the lower dose levels. Further, as stated above, centrilobular hepatocyte enlargement is commonly observed in rodent liver following the administration of any xenobiotic and is considered to be adaptive and not to represent an adverse health effect. These findings are suggestive of an adaptive response to mixed function oxidase induction in the liver. Last but not least, the vacuolation in the liver of animals in the high dose group may be due to a perturbation of metabolic activity. This conclusion is supported by similar findings in the OECD 422, in which all effects were similarly considered to be adaptive responses.

 

Kidney:

In the OECD 407 study, the macroscopic examination performed at termination revealed that the kidneys were enlarged in 2/5 female rats treated with 1000 mg/kg/day, compared with none in the control group. Microscopic examinations showed moderate focal interstitial inflammation in the cortex and medulla and marked cystic dilatation and basophilia of cortical and medullary tubules was reported in one female rat receiving 1000 mg/kg/day. It was considered treatment-related, in spite of its low incidence, due to the severity of the lesion in this one rat. An effect on the kidney at this dosage of 1000 mg/kg/day was also considered likely, as disturbed plasma electrolytes, high urinary volumes (with specific gravity and pH of urine affected), and macroscopic and microscopic findings (females only) were noted. The increased water consumption seen for female rats could be related.

Hence, several indications were given that the kidneys were affected.

In the OECD 422 study however, there were no such effects noted at all, no indication for kidney involvement is given. In consequence, no clear evidence for classification as STOT RE is given, as the findings in the OECD 407 study may be as well related to a general poor condition of health. This is supported e.g. by a reduced body weight gain, haematological or clinical biochemistry findings.

 

In summary, it can be concluded that liver effects were related to overall systemic effects and are predominantly based on an adaptive response, which may become adverse with increasing severity. Consistent effects in the OECD 407 study in the kidneys could not be confirmed in the OECD 422 study and may be so related to overall systemic toxicity as well. In consequence, no clear evidence for classification as STOT RE is given. Further, there are no indications for species-specifity of the effects, so they are considered relevant for human risk assessment as well.

Justification for classification or non-classification

In the available OECD 422 study, only adaptive, non-adverse effects were noted, the NOAEL was set so as the highest dose tested, i.e. 600 mg/kg bw/d, and no LOAEL could be established due to the absence of adverse effects. So, due to the adaptive nature of the responses in liver in thyroid, evidence for classification as STOT RE Cat. 2 is not given. Further, no LOAEL below the guidance limit value as given in Regulation 1272/2008, i.e. 300 mg/kg bw/d, could be observed.

 

With regard to the OECD 407 study, also no necessity for classification is given. Styrenated diphenylamine produced evidence of liver toxicity at 1000 mg/kg/day, including higher alkaline phosphatase, glutamic-pyruvic transaminase, glutamic-oxaloacetic transaminase and bilirubin levels, lower plasma albumin and cholesterol, increased liver weights, macroscopic and microscopic changes. The altered blood coagulation, as represented by markedly longer thrombotest times and the animal with persistent bleeding that was sacrificed, was considered likely to be as a consequence of the liver effect.

An effect on the kidney at this dosage of 1000 mg/kg/day was also considered likely, as disturbed plasma electrolytes, high urinary volumes (with specific gravity and pH of urine affected), and macroscopic and microscopic findings (females only) were noted. The increased water consumption seen for female rats could be related.

The toxicological significance of the microscopic finding in the heart of one male rat treated at 1000 mg/kg/day was uncertain.

At 300 mg/kg/day, a reduced continuation of effects was seen. Slightly longer thrombotest times were recorded for males and some biochemistry and urinalysis parameters were also affected, and there were macroscopic and microscopic effects on the liver. The bodyweight gains for female rats at this dosage were also lower than control during Week 4.

Findings seen at the 100 mg/kg/day dosage lever included lower than control cholesterol levels for males, macroscopically enlarged livers for 3/5 males and the microscopic finding of fine vacuolation in hepatocytes for one female rat. However, macroscopically enlarged liver was not seen at the intermediate dosage of 300 mg/kg/day and the microscopic finding is also seen occasionally in control animals.

According to Regulation 1272/2008, Table 3.9.1, Categories for specific target organ toxicity-repeated exposure, Category 2 is required for substances that, on the basis of evidence from studies in experimental animals can be presumed to have the potential to be harmful to human health following repeated exposure. Substances are classified in category 2 for target organ toxicity (repeat exposure) on the basis of observations from appropriate studies in experimental animals in which significant toxic effects, of relevance to human health, were produced at generally moderate exposure concentrations. Guidance dose/concentration values are provided below (see 3.9.2.9) in order to help in classification.

Guidance Value Ranges (dose/concentration) to assist in Category 2 classification are i.a. 10 < C≤100 mg/kg body weight/day for an Oral (rat) study over 90 days. For subacute studies, the limit value is 300 mg/kg bw. The Regulation states further, that the guidance values and ranges mentioned in paragraphs 3.9.2.9.6 and 3.9.2.9.7 are intended only for guidance purposes, i.e. to be used as part of the weight of evidence approach, and to assist with decisions about classification. They are not intended as strict demarcation values. Substances are classified as specific target organ toxicants following repeated exposure by the use of expert judgement; assessment shall take into consideration not only significant changes in a single organ or biological system but also generalised changes of a less severe nature involving several organs.

As stated above, there are treatment-related changes reported in the liver, heart and kidneys.

The toxicological significance of the microscopic finding in the heart of one male rat treated at 1000 mg/kg/day was uncertain and should hence not be seen as evidence for classification as STOT RE, as is was only a single incidence and further only noted at 1000 mg/kg/day, which is way above the guidance value of 300 mg/kg. The same applies for the kidneys, as relevant effects were only noted at 1000 mg/kg.

Initial effects for liver involvement were noted already at 100 mg/kg. However, they were only minor, macroscopically enlarged liver was not seen at the intermediate dosage of 300 mg/kg/day and the microscopic finding is also seen occasionally in control animals. Hence, a clear dose-response could not be established for the lower dose levels. Further, Centrilobular hepatocyte enlargement is commonly observed in rodent liver following the administration of any xenobiotic and is considered to be adaptive and not to represent an adverse health effect. These findings are suggestive of an adaptive response to mixed function oxidase induction in the liver. Last but not least, the vacuolation in the liver of animals in the high dose group may be due to a perturbation of metabolic activity.

In consequence, clear evidence for classification as STOT RE Cat. 2 is not given.