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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2012-2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP and OECD guideline compliant study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): Reaction products of benzeneamine, N-phenyl with nonene (branched)
- Substance type: viscous, slightly yellow
- Physical state: liquid
- Analytical purity: UVCB-substance, purity not applicable
- Purity test date: 2013
- Lot/batch No.: 240312/K7 Lot. Nr. 020
- Expiration date of the lot/batch: 19 Feb 2014
- Stability under test conditions: stable
- Storage condition of test material: Ambient (room temperature); avoiding temperatures <10 °C

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Age at study initiation: 42 ± 1 day(s)
- Weight at study initiation: average 160g (males) and 130 g (females)
- Fasting period before study: no
- Housing: 5 animals per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2012-12-12 To: 2013-03-14

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: For each concentration, respective amount of test item was weighted into a beaker. Then, the respective volume of the carrier was added. Finally, each formulation was stirred with a magnetic stirrer until the test item was completely dissolved. All test item formulations were prepared at least once weekly.

VEHICLE
- Justification for use and choice of vehicle (if other than water): substance is miscible in vehicle
- Concentration in vehicle: 2.5 - 25 g/100 mL, depending on dose
- Amount of vehicle (if gavage): 4 ml/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior the start of the study, maximal stability of the test substance in the carrier was assessed.
At the beginning and towards the end of the study, one sample of the low, mid and high dose preparations was taken for determination of concentration in the carrier, respectively. Since all test substance preparations were true solutions, no homogeneity analysis was done.
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose-range-finding study (28-days)

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for moribund and dead animals was made twice daily on working day and once daily on Saturday, Sunday and public holidays. If animals were in a moribund state, they were sacrificed and dissected.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All rats were checked daily for any abnormal clinical signs prior administration, as well as, within 2 hours and 5 hours post administration. Detailed clinical observation (DCO) was performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high). The following parameters were examined:

1. abnormal behavior during “handling”
2. fur
3. skin
4. posture
5. salivation
6. respiration
7. activity/arousal level
8. tremors
9. convulsions
10. abnormal movements
11. impairment of gait
12. lacrimation
13. palpebral closure
14. exophthalmos
15. feces (appearance/ consistency)
16. urine
17. pupil size


BODY WEIGHT: Yes
- Time schedule for examinations:
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between body weight on the respective day of weighing and body weight on day 0 was calculated as body weight change.


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: At the end of the administration period, on study day 91.
- Dose groups that were examined: control and highest dose group animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the administration period, on study day 92.
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- How many animals: 5 animals per test group and sex
- Parameters checked below were examined.
Leukocyte count
(WBC)
Erythrocyte count
(RBC)
Hemoglobin
(HGB)
Hematocrit
(HCT)
Mean corpuscular volume
(MCV)
Mean corpuscular hemoglobin
(MCH)
Mean corpuscular hemoglobin concentration
(MCHC)
Platelet count
(PLT)
Differential blood count
Reticulocytes
Heinz bodies (indicator for methb-formation)
Prothrombin time (Hepato Quick’s test) (HQT)





CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the administration period, on study day 92.
- Animals fasted: Yes
- How many animals: 5 per sex and dose
- Parameters checked below were examined.

Alanine aminotransferase
(ALT)

Aspartate aminotransferase
(AST)

Alkaline phosphatase
(ALP)

g-Glutamyltransferase
(GGT)

Sodium
(NA)
Potassium
(K)
Chloride
(CL)
Inorganic phosphate
(INP)
Calcium
(CA)
Urea
(UREA)
Creatinine
(CREA)
Glucose
(GLUC)
Total bilirubin
(TBIL)
Total protein
(TPROT)
Albumin
(ALB)
Globulins
(GLOB)
Triglycerides
(TRIG)
Cholesterol
(CHOL)
Magnesium
(MG)
Bile acids
(TBA)




URINALYSIS: Yes
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes / No / No data
- Parameters checked below were examined.
pH
Protein
Glucose
Ketones
Urobilinogen
Bilirubin
Blood
Specific gravity
Sediment
Color, turbidity
Volume


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: days 85 - 90
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity / (FOB)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)

The following weights were determined in all animals sacrificed on schedule:

1. Anesthetized animals
2. Adrenal glands
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Ovaries
9. Spleen
10. Testes
11. Thymus
12. Thyroid glands
13. Uterus with cervix



HISTOPATHOLOGY: Yes (see table)
Other examinations:
Heinz body formation as a marker for methaemoglobin formation was determined.
Statistics:
Blood parameters For parameters with bidirectional changes:
Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians
For parameters with unidirectional changes:
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians * for p < 0.05
** for p < 0.01 SIEGEL, S. (1956):
Non-parametric statistics for the behavioural sciences.
McGraw-Hill New York Holm (1979): A Simple Sequentially Rejective Multiple Test Procedure. Scand. J. Statist. 6, 65-70


Urinalysis parameters (except pH, urine volume, specific gravity, color and turbidity Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians * for p < 0.05
** for p < 0.01 SIEGEL, S. (1956):
Non-parametric statistics for the behavioural sciences.
McGraw-Hill New York


Urine pH, volume, specific gravity, color and turbidity Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians. Urine color and turbidity are not evaluated statistically. * for p < 0.05
** for p < 0.01 SIEGEL, S. (1956):
Non-parametric statistics for the behavioural sciences.
McGraw-Hill New York

Weight parameters Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians * for p ≤ 0.05
** for p ≤-0.01 HETTMANNSPERGER, T.P. (1984): Statistical Inference based on Ranks, John W

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
salivation after dosing
Mortality:
no mortality observed
Description (incidence):
salivation after dosing
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
males of the high dose group
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
possibly treatment related: prolonged PTT in males of high-dose group
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
increased ALP, TG at 300 mg/kg bw; increased albumin at 1000 mg/kg bw, decreased bile acids
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Higher levels of Ca-oxalate at 1000 mg/kg bw
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
liver showed prominent acinar pattern for females of high dose group
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
liver and thyroid
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No adverse findings were observed during the administration period.

Salivation in different grades was observed in males of all compound groups. In summary 4/10 males of the low dose, 10/10 males of the mid dose and high dose group showed salivation. First occurrence of salivation was noted on administration day 14 in the high dose and on day 33 in the low/mid dose group. Thereafter, all affected animals showed transient incidence/duration/grade of salivation until the end of administration period (day 92). The observed salivation onset was within approximately 20 minutes post application and was not present two hours later. Additionally, one male (No. 33) of the high dose group exhibited respiration sounds on application day 78 and on day 80 to 88.

In male animals, the observed salivation showed a dose-dependency and was therefore seen treatment-related but not adverse. With regard to the respiration sounds in animal No. 33 of the high dose group, observation was judged rather secondary to salivation and therefore not treatment-related nor adverse in nature.

In females of all compound groups, salivation in different grades was also noted. In summary 4/10 females of the low dose, 9/10 animals of the mid dose and 7/10 females of the high dose group were affected. First occurrence of salivation was noted on administration day 31 in the high dose and on day 33 in the low/mid dose group. Thereafter, affected animals showed transient incidence/duration/grade of salivation until the end of administration period (day 93). Onset of symptom was within approximately 20 minutes post application and was not present two hours later. Additionally, one female (No. 55) of the low dose group showed respiration sounds on application day 47 to 54 and on day 56 to 60.

Despite the lacking dose-dependency, noted salivation was judged treatment-related but not adverse in females. Regarding the event of respiration sounds in one single animal of the high dose group, observation was seen rather secondary to salivation and therefore not treatment-related nor adverse in nature.


BODY WEIGHT AND WEIGHT GAIN
Male animals of the high dose group exhibited a significantly and moderately lower body weight versus control, starting on administration day 14. The observed significant difference in body weight ranged between -6.6% (day 14) and -14.6% (day 91) versus control.
With regard to body weight gain, males of the highest dose group showed a significant and moderate impairment, starting on administration day 7. Difference of body weight gain ranged between -19.5% (day 14) and -24.2% (day 84/91) versus control.


HAEMATOLOGY
No Heinz bodies were found by microscopic investigation of methylene violet stained slides of controls and dosed individuals.

In males of high dose group (1000 mg/kg bw/d) prothrombin time (HQT = Hepatoquick’s time) was prolonged, whereas in females of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) the prothrombin time was shortened. The HQT means in females were within the historical control range and that one of the males was above the range. Therefore, the reduced prothrombin time in females was regarded as incidental and not treatment-related, whereas regarding the prolonged prothrombin time in males an adverse effect cannot be excluded.

CLINICAL CHEMISTRY
At the end of the study, in rats of both sexes of test groups 2 and 3 (300 and 1000 mg/kg bw/d), alkaline phosphatase (ALP) activity was increased. ALP activity was already higher in females of test group 1 (100 mg/kg bw/d), but the mean was only marginally above the historical control range and therefore this elevated value was regarded as treatment-related, but not adverse. In females of test group 3 (1000 mg/kg bw/d) γ-glutamyltransferase (GGT) activity was increased.

In rats of both sexes of test group 3 (1000 mg/kg bw/d) albumin levels were decreased. Females of test groups 1 and 2 (100 and 300 mg/kg bw/d) had also lower albumin concentrations. Comparing with historical control ranges even the female controls have low albumin levels. In females of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) total protein levels were also decreased, although not dose-dependently. However, the total protein means of females in test group 1 and 2 were within the historical control range. Because albumin represents the main fraction of the total proteins and the latter were within the historical control ranges in females of test groups 1 and 2, even the albumin alteration in these test groups were regarded as incidental and not treatment-related. Globulin levels were increased in females of test groups 2 and 3 (300 and 1000 mg/kg bw/d), but the means were within the historical control range.

Triglyceride levels were increased in females of test groups 2 and 3 (300 and 1000 mg/kg bw/d).

Total bile acid levels were decreased in both sexes of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d). However, the bile acid level decrease in test group 1 was the only changed parameter. Therefore, the total bile acid change in this test group was regarded as treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002) .



URINALYSIS
In rats of both sexes of test group 3 (1000 mg/kg bw/d) higher incidences of calcium oxalate crystals were found in the urine sediment. In males of test group 1 (100 mg/kg bw/d) more transitional epithelial cells were found in the sediment, but this change was not dose-dependent and therefore, it was regarded as incidental and not treatment-related.


ORGAN WEIGHTS
see tables 1 and 2

GROSS PATHOLOGY
In the liver three females of test group 3 (1000 mg/kg bw/d) showed a prominent acinar pattern which was regarded to be treatment related.

HISTOPATHOLOGY: NON-NEOPLASTIC
See tables 3 and 4


OTHER FINDINGS

Effect levels

Dose descriptor:
NOEL
Effect level:
< 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: increased liver weight, histopathology findings in liver (centrilobular hypertrophy grade, single cell necrosis, midzonal fatty change) and thyroid (hypertrophy, altered colloid)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Absolute changes in organ weights

 

Male animals

Female animals

Test group

(mg/kg bw/d)

1

(100)

2

(300)

3

(1000)

1

(100)

2

(300)

3

(1000)

Terminal body weight

97%

94%**

84%**

 

 

 

Heart

97%

91%**

88%**

 

 

 

Liver

114%**

120%**

120%**

112%

118%**

137%**

* : p <= 0.05, **: p <= 0.01

Table 2: Relative changes in organ weights (note reduced body weight gain in males at 1000 mg/kg bw)

 

Male animals

Female animals

Test group

(mg/kg bw/d)

1

(100)

2

(300)

3

(1000)

1

(100)

2

(300)

3

(1000)

Adrenal glands

111%

106%

135%**

 

 

 

Brain

102%

105%

118%**

 

 

 

Kidneys

104%

99%

111%**

100%

115%**

112%**

Liver

118%**

127%**

142%**

112%

124%**

143%**

Spleen

 

 

 

110%*

117%**

120%**

Testes

103%

107%

115%**

 

 

 

Thyroid glands

121%*

122%*

133%*

 

 

 

* : p <= 0.05, **: p <= 0.01

Table 3: Histopathology in liver

Liver

Male animals

Female animals

Test group

(mg/kg bw/d)

0

(0)

1

(100)

2

(300)

3

(1000)

0

(0)

1

(100)

2

(300)

3

(1000)

No. of animals

10

10

10

10

10

10

10

10

Hypertrophy, centrilobular/midzon.

0

6

9

10

0

8

10

10

  • Grade 1

 

6

1

 

 

7

7

 

  • Grade 2

 

 

6

3

 

1

3

5

  • Grade 3

 

 

2

7

 

 

 

5

 

 

 

 

 

 

 

 

 

Single cell necrosis

0

2

4

3

0

0

0

0

  • Grade 1

 

2

4

3

 

 

 

 

 

 

 

 

 

 

 

 

 

Fatty change, midzonal

0

9

10

10

0

0

0

0

  • Grade 1

 

6

3

2

 

 

 

 

  • Grade 2

 

3

3

5

 

 

 

 

  • Grade 3

 

 

3

3

 

 

 

 

  • Grade 4

 

 

1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fatty change, peripheral

0

0

0

0

0

0

6

9

  • Grade 1

 

 

 

 

 

 

2

2

  • Grade 2

 

 

 

 

 

 

4

3

  • Grade 3

 

 

 

 

 

 

 

3

  • Grade 4

 

 

 

 

 

 

 

1

Table 4: Histopathology in thyroid

Thyroid glands

Male animals

Female animals

Test group

(mg/kg bw/d)

0

(0)

1

(100)

2

(300)

3

(1000)

0

(0)

1

(100)

2

(300)

3

(1000)

No. of animals

10

10

10

10

10

10

10

10

Hypertrophy/

hyperplasia

0

3

8

8

0

5

5

10

  • Grade 1

 

3

3

1

 

4

4

5

  • Grade 2

 

 

5

7

 

1

1

5

 

 

 

 

 

 

 

 

 

Altered colloid

1

0

4

3

0

1

1

1

  • Grade 1

 

 

2

 

 

1

 

1

  • Grade 2

 

 

2

3

 

 

1

 

Table 5: Red blood cell + coagulation parameters in males

control 100 mg/kg bw 300 mg/kg bw 1000 mg/kg bw
RBC Mean 8.32 k 8.40 8.37 8.14
[tera/L] S.d. 0.19 0.45 0.30 0.29
day 92 N 10 10 10 10
  Median 8.32 8.48 8.35 8.11
HGB Mean 8.9 v 8.8 8.8 8.6 **
[mmol/L] S.d. 0.1 0.2 0.2 0.2
day 92 N 10 10 10 10
  Median 9.0 8.8 8.8 8.6
HCT Mean 0.425 v 0.423 0.420 0.411 **
[L/L] S.d. 0.008 0.010 0.011 0.008
day 92 N 10 10 10 10
  Median 0.426 0.424 0.420 0.411
MCV Mean 51.1 k 50.4 50.2 50.6
[fL] S.d. 1.4 2.3 1.2 1.9
day 92 N 10 10 10 10
  Median 50.8 50.3 50.0 51.0
MCH Mean 1.07 k 1.06 1.05 1.06
[fmol] S.d. 0.03 0.05 0.03 0.05
day 92 N 10 10 10 10
  Median 1.08 1.04 1.05 1.06
MCHC Mean 20.98 k 20.93 20.85 20.88
[mmol/L] S.d. 0.28 0.21 0.21 0.38
day 92 N 10 10 10 10
  Median 20.95 20.88 20.91 20.81
RET Mean 1.8 k 1.8 1.9 2.3
[%] S.d. 0.3 0.4 0.4 0.6
day 92 N 10 10 10 10
  Median 1.7 1.8 1.8 2.4
PLT Mean 692 k 716 763 750
[giga/L] S.d. 65 103 71 125
day 92 N 10 10 10 10
  Median 679 697 746 741
HQT Mean 37.8 v 37.5 39.1 41.5 **
[sec] S.d. 2.2 1.1 1.8 1.8
day 92 N 10 10 10 10
  Median 36.9 37.5 38.5 41.3

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

k=KRUSKAL-WALLIS; v=KRUSKAL-WALLIS-WILCOX

Applicant's summary and conclusion

Executive summary:

Regarding clinical examination, incipient general systemic toxicity was observed in males of the high dose group (1000 mg/kg bw/d). These animals showed a significantly and moderately lower body weight (-14.6%), as well as a corresponding significant and moderate impairment of body weight gain (-24.2%).

 

Regarding clinical pathology, liver was the target organ. Mainly the liver cell metabolism was affected. This was indicated by lower albumin and bile acid synthesis in rats of both sexes of test group 3 (1000 mg/kg bw/d for albumin and bile acids) and test group 2 (300 mg/kg bw/d for bile acids, only) as well as by prolonged prothrombin time in males of test group 3 (1000 mg/kg bw/d) due to a decreased synthesis of coagulation factors. Higher alkaline phosphatase (ALP) activities in rats of both sexes of test groups 2 and 3 (300 and 1000 mg/kg bw/d), as well as higher g-glutamyltransferase (GGT) activities in females of test group 3 (1000 mg/kg bw/d) indicated a liver cell swelling. Increased glucose levels in females of test group 3 (1000 mg/kg bw/d) and triglyceride levels in females of test group 2 and 3 (300 and 1000 mg/kg bw/d) may be due to a higher energy demand of these individuals which was accomplished by increased lipolysis and gluconeogenesis.

Regarding pathology, liver was the target organ. Males and females of all treatment groups revealed a liver cell hypertrophy and fatty change in the midzonal area or peripheral zone, respectively. Three females revealed a macroscopically observed prominent acinar pattern. That was regarded to have been caused by the fatty change. Males showed in addition a minimal increase in single cell necrosis, when compared to control animals. Also the liver weight increase in males of all treated test groups and females of test group 2 and 3(300 and 1000 mg/kg bw/d) were regarded to be consequence of the liver cell changes observed.Taken all these findings together and also the findings in clinical pathology according to Hall et al. (2012)[1] the liver cell hypertrophy was regardedto be treatment related and adversein test group 1-3 (100, 300 and 1000 mg/kg bw/d) in males and females.

The hypertrophy/hyperplasia and altered colloid of follicular cell in the thyroid gland of males and females of all treated test groups was regarded to be a secondary event to the liver cell changes and most likely induced by liver enzyme induction. Therefore it was regarded to be treatment-related but not adverse.


[1]HallAP, Elcombe CR, Foster JR, Harada T, Kaufmann W, Knippel A, Küttler K, Malarkey DE, Maronpot RR, Nishikawa A, Nolte T, Schulte A, Strauss V, York MJ (2012) Liver Hypertrophy : A Review of Adaptive (Adverse and Non-adverse) Changes--Conclusions from the 3rd International ESTP Expert Workshop.Toxicol Pathol40: 971-994