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REACH

4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with 3-aminomethyl-3,5,5-trimethylcyclohexylamine

EC number: 500-101-4 | CAS number: 38294-64-3

Life Cycle description
Uses advised against

Toxicological information

Toxicity to reproduction

Administrative data

Endpoint:: extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:: study scientifically not necessary / other information available
Justification for data waiving:: other:

Cross-referenceopen all close all

Cross-reference 1

Reason / purpose for cross-reference:: data waiving: supporting information

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 21 September 1998

Deviations:

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

30 May 2008

Deviations:

GLP compliance:

yes (incl. QA statement)

Limit test:

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han™:RccHan™:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK
- Age at study initiation: 6-8 weeks
- Weight at study initiation: 218 to 270 g (males), 158 to 193 g (females)
- Housing: in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding
- Diet (e.g. ad libitum): pelleted diet (Rodent 2014C Teklad Global Certified Diet, Envigo RMS (UK) Limited., Oxon, UK), ad libitum
- Water (e.g. ad libitum): mains drinking water, ad libitum
- Acclimation period: 9 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 13 May 2016 (first day of treatment) to 09 September 2016 (final day of necropsy)

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were therefore prepared weekly for the first week and then fortnightly thereafter and stored at approximately 4 ºC in the dark and under nitrogen.

VEHICLE
- Concentration in vehicle: 1.67, 16.67, 33.33 mg/mL
- Amount of vehicle (if gavage): 6 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results showed the formulations to be stable for at least twenty-two days. Measured concentrations were within ±10% of nominal concentrations, confie´rming accurate formulation.

Duration of treatment / exposure:

90 d + 28 d recovery

Frequency of treatment:

daily, 7 d/week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on previous dose range finding study (Envigo Research Limited Study Number YY28YK; Fourteen Day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study in the Rat)
- Rationale for animal assignment (if not random): randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups: 28 d

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately before dosing, up to thirty minutes post dosing and one hour after dosing. During the treatment-free period, animals were observed daily.
- Detailed individual clinical observations were performed for each non-recovery animal using a purpose built arena. The following parameters were observed:
Gait, Tremors, Twitches, Convulsions, Bizarre/Abnormal/Stereotypic behavior, Salivation, Pilo-erection, Exophthalmia, Lachrymation, Hyper/Hypothermia, Skin color, Respiration, Palpebral closure, Urination, Defecation, Transfer arousal, Tail elevation

BODY WEIGHT: Yes
- Time schedule for examinations: on Day 1 (prior to dosing) and at weekly intervals thereafter. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION:
- for each cage group at weekly intervals

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily, for each cage group, by visual inspection of the water bottles for any overt changes

OPHTHALMOSCOPIC EXAMINATION: Yes
- The eyes of all control and treated animals were examined pre-treatment and all non-recovery control and non-recovery high dose animals were examined before termination of treatment (during Week 12).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 90 (non-recovery animals), Day 118 (recovery animals)
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: 10/dose group
- Parameters: Hemoglobin (Hb), Erythrocyte count (RBC), Hematocrit (Hct), Erythrocyte indices (mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC)), Total leukocyte count (WBC), Differential leukocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas)), Platelet count (PLT), Reticulocyte count (Retic), Prothrombin time (CT), Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 90 (non-recovery animals), Day 118 (recovery animals)
- Animals fasted: No
- How many animals: 10/dose group
- Parameters: 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), Bile acids , Gamma glutamyl transpeptidase, Triglycerides (Trigs)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and at weekly intervals thereafter, all non-recovery animals were observed for signs of functional/behavioral toxicity. During Week 12 functional performance tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
organ weights: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus, Uterus (with cervix)

HISTOPATHOLOGY: Yes
samples preserved:
Adrenals, Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Cecum, Colon, Duodenum, Epididymides, Esophagus, Eyes, Gross lesions, Heart, Ileum (including Peyer’s patches), Jejunum, Kidneys, Liver, Lungs (with bronchi), Lymph nodes (mandibular and mesenteric), Mammary gland, Muscle (skeletal), Ovaries, Pancreas, Pituitary, Prostate, Rectum, Salivary glands (submaxillary), Sciatic nerve, Seminal vesicles (including coagulating gland), Skin, Spinal cord (cervical, mid thoracic and lumbar), Spleen, Stomach, Testes, Thymus, Thyroid/Parathyroid, Tongue, Trachea, Urinary bladder, Uterus (with cervix), Vagina

All tissues from non-recovery control and 200 mg/kg bw/day dose group animals and any animals that died during the study were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed.
Since there were indications of possible treatment-related mesenteric lymph node and adrenal changes, examination was subsequently extended to include similarly prepared sections of the mesenteric lymph nodes and adrenals from animals in the low, intermediate and recovery dose groups.

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.
Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Incidences of increased salivation were evident in all animals of both sexes treated with 200 mg/kg bw/day from Day 1 (females) and Day 2 (males) until the termination of treatment. Episodes of noisy respiration were also evident in all animals of both sexes treated with 200 mg/kg bw/day from Day 1 (females) and Day 2 (males) onwards. Isolated incidences of a stained snout, labored respiration, decreased respiratory rate, hunched posture and fur loss were also evident in some females treated with 200 mg/kg bw/day and a stained snout or sneezing was also evident in two males treated with 200 mg/kg bw/day. The male treated with 200 mg/kg bw/day that was found dead on Day 48 had only previously shown increased salivation and noisy respiration. During the treatment-free, twenty-eight day period, one male and one female that were previously given 200 mg/kg bw/day continued to show episodes of noisy respiration.
At 100 mg/kg bw/day, increased salivation and noisy respiration were evident throughout the treatment period albeit to a lesser extent than at 200 mg/kg bw/day. One male from this treatment group also had a decreased respiratory rate and hunched posture on Day 22 only.
No such effects were detected in males treated with 10 mg/kg bw/day, however, one female from this treatment group had increased salivation on Day 81 only.
One control female showed increased salivation on Day 22 whilst a further one control female showed increased salivation on Day 77. One female treated with 100 mg/kg bw/day had fur loss between Days 44 and 91.
These incidences were considered to be incidental.

Mortality:

mortality observed, treatment-related

Description (incidence):

One male treated with 200 mg/kg bw/day was found dead on Day 48. There were no further unscheduled deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males treated with 200 and 100 mg/kg bw/day showed a statistically significant reduction (p<0.01) in body weight gain during the first week of treatment. Periods of recovery were evident thereafter, however, generally body weight gain in these males was slightly below controls. A further, statistically significant reduction (p<0.01) in body weight gain was evident during Week 12 for males treated with 200 mg/kg bw/day and actual body weight losses were evident in males treated with 200 and 100 mg/kg bw/day during the final week of treatment. Consequently overall body weight gain was lower than controls in these males. Females treated with 200 mg/kg bw/day showed a slight reduction in body weight gain during the first week of treatment, however, statistical significance was not achieved and recovery was evident thereafter. Significant improvement in body weight gain was evident in males previously treated with 200 mg/kg bw/day during the twenty-eight day treatment-free period. Statistically significant increases (p<0.05-0.01) when compared to controls were evident in these males throughout the treatment-free period.
No such effects were detected in females treated with 100 mg/kg bw/day or in animals of both sexes treated with 10 mg/kg bw/day.
A statistically significant increase (p<0.05) in body weight gain during Week 9 and a statistically significant reduction (p<0.05) in body weight gain during Week 10 was evident in females treated with 200 mg/kg bw/day. These intergroup differences were considered to be incidental and not to be of toxicological importance.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Animals of both sex treated with 200 mg/kg bw/day and males treated with 100 mg/kg bw/day showed a reduction in overall food consumption. Incidences of reduced food conversion efficiency was also evident in males treated with 200 and 100 mg/kg bw/day during the treatment period and generally followed the fluctuations seen in body weight gain. During the twenty-eight day treatment-free period, recovery in both food consumption and food conversion efficiency were evident in animals of both sexes that were previously given 200 mg/kg bw/day.
No such effects were detected in females treated with 100 mg/kg bw/day or in animals of both sexes treated with 10 mg/kg bw/day.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

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

no effects observed

Description (incidence and severity):

Visual inspection of water bottles did not reveal any inter-group differences.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmoscopic examination of animals of both sexes from the non-recovery control and 200 mg/kg bw/day dose groups during Week 12 of the treatment period did not indicate any treatment-related differences.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Animals of both sexes treated with 200 and 100 mg/kg bw/day showed a statistically significant increase (p<0.05-0.01) in neutrophils. A statistically significant increase (p<0.01) in neutrophils was also present in females previously treated with 200 mg/kg bw/day at the end of the twenty-eight day treatment-free period. Males treated with 200 mg/kg bw/day also showed a statistically significant increase (p<0.01) in total leukocyte count. The majority of individual values for both parameters at 200 and 100 mg/kg bw/day were outside of historical control ranges.
No toxicologically significant effects were detected in animals of both sexes treated with 10 mg/kg bw/day.
Females treated with 200 mg/kg bw/day showed a statistically significant increase (p<0.05) in platelet count. The majority of individual values were within historical control range and in the absence of any associated histopathological correlates the intergroup difference was considered not to be of toxicological significance.
Males treated with 10 mg/kg bw/day showed statistically significant reductions (p<0.05) in mean corpuscular hemoglobin and mean corpuscular volume. The majority of individual values were within historical control ranges and in the absence of a similar effect in 200 or 100 mg/kg bw/day males or any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.
Following the treatment-free period, females that were previously given 200 mg/kg bw/day showed statistically significant increases (p<0.01) in hemoglobin, erythrocyte count and hematocrit and statistically significant reductions (p<0.05) in mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration. In the absence of a similar effect in 200 mg/kg bw/day females at the end of the treatment period or any associated histopathological correlates the intergroup difference was considered not to be of toxicological significance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects detected in the blood chemical parameters examined.
Males treated with 200 and 100 mg/kg bw/day showed a statistically significant increase (p<0.05) in urea and statistically significant reductions (p<0.05-0.01) in total protein, albumin, alkaline phosphatase, triglycerides and bile acids. Males treated with 200 mg/kg bw/day also showed a statistically significant increase (p<0.01) in albumin/globulin ratio and a statistically significant reduction (p<0.05) in cholesterol. Recovery males that were previously given 200 mg/kg bw/day showed a statistically significant reduction (p<0.05) in total protein and a statistically significant increase (p<0.05) in alkaline phosphatase. Females treated with 200 and 100 mg/kg bw/day showed statistically significant increases (p<0.05-0.01) in urea and albumin/globulin ratio and statistically significant reductions (p<0.05-0.01) in chloride concentration, triglycerides, cholesterol and bile acids. The effect on albumin/globulin ratio also extended to females treated with 10 mg/kg bw/day (p<0.05). Females treated with 200 mg/kg bw/day also showed statistically significant reductions (p<0.05) in sodium concentration and alkaline phosphatase. Recovery females that were previously given 200 mg/kg bw/day showed statistically significant increases (p<0.05) in chloride concentration and bile acids. The majority of individual values for all parameters for both sexes were within historical control ranges and although some of these intergroup differences may indicate minor perturbations in hepatic metabolism, in the absence of any associated microscopic hepatic changes evident these differences were considered not to represent an adverse effect of treatment.
Animals of both sexes treated with 200 and 100 mg/kg bw/day showed statistically significant increases (p<0.05-0.01) in alanine aminotransferase and aspartate aminotransferase. These intergroup differences may again indicate minor perturbations in hepatic metabolism and the majority of individual values did exceed historical control ranges, however, in the absence of any associated microscopic hepatic changes evident, the intergroup differences were considered not to represent an adverse effect of treatment.
Following the treatment-free period, females that were previously given 200 mg/kg bw/day showed statistically significant reductions (p<0.05-0.01) in potassium concentration and inorganic phosphorus. The majority of individual values were within historical control ranges and in the absence of similar effects in 200 mg/kg bw/day females at the end of the treatment period or any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no toxicologically significant changes in functional performance.
Males from all treatment groups showed a statistically significant increase (p<0.01) in overall activity. A true dose related response was not evident and in the absence of any clinical signs of neurotoxicity, the intergroup difference was considered not to be of toxicological importance.

There were no treatment-related changes in sensory reactivity.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No toxicologically significant effects were detected in the organ weights measured.
At the end of the treatment period, males from all treatment groups showed statistically significant reductions (p<0.05-0.01) in kidney and thymus weights both absolute and relative to terminal body weight and absolute brain weight. Males treated with 10 mg/kg bw/day also showed a statistically significant reduction in relative brain weight, however, males treated with 200 and 100 mg/kg bw/day showed a statistically significant increase (p<0.05-0.01) in relative brain weight. Males treated with 200 mg/kg bw/day also showed a statistically significant increase (p<0.05) in spleen weight both absolute and relative to terminal body weight. The majority of individual values for both absolute and relative weights were within historical control ranges and no associated micropscopic changes were evident in these organs, therefore, the intergroup differences were considered not to be of toxicological significance.
Following the treatment-free period, females that were previously given 200 mg/kg bw/day showed a statistically significant increase (p<0.05) in brain weight both absolute and relative to terminal body weight. All of the individual values were within historical control ranges and in the absence of similar effects in 200 mg/kg bw/day females at the end of the treatment period or any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No toxicologically significant macroscopic abnormalities were detected in surviving animals.
The male treated with 200 mg/kg bw/day that was found dead on Day 48 had a dark liver and dark lungs. Histopathological examination revealed moderate prostatic inflammation, unilateral lymphoid aggregates and urothelial hyperplasia in the kidneys and adrenal cortical hypertrophy. The adrenal change was likely to indicate stress and was probably secondary to the prostatic inflammation. The cause of death in this animal was undetermined, and none of the microscopic or macroscopic changes were considered to be treatment-related.
A number of animals across most dose groups including controls showed reddened lungs. Such findings are common in this type of study and were considered unrelated to treatment with the test item. One male treated with 200 mg/kg bw/day had an enlarged spleen. One female treated with 10 mg/kg bw/day had increased renal pelvic space. In the absence of any associated treatment-related microscopic changes, these findings were considered to be incidental.
One female that was previously given 200 mg/kg bw/day had a fluid filled (dark) right uterine horn at the end of the treatment-free period. In the absence of a similar effect at the end of the treatment period, the intergroup difference was considered to be incidental.
One non-recovery control male had enlarged fluid filled kidneys and the left kidney was also pale and malformed. A further non-recovery control male had a hard and pale left seminal vesicle. One recovery control female had increased renal pelvic space in both kidneys. In the absence of treatment, these were considered to be incidental findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The following treatment-related microscopic abnormalities were detected:
Mesenteric Lymph Nodes: Histiocytosis with granulomas was evident in animals of both sexes from all treatment groups, varying from minimal to moderate in severity and following a dose-dependent response. At 100 and 200 mg/kg bw/day the more severe grades of histiocytosis were often accompanied by minimal abscesses. Following the twenty-eight day recovery period, histiocytosis with granulomas and abscesses were evident in both sexes previously treated with 200 mg/kg bw/day. When compared with the non-recovery animals, the incidence of abscesses was lower, whereas histiocytosis persisted at much the same incidence and severity.
The following microscopic abnormalities were evident, however, these were considered to reflect individual variation rather than an effect of treatment.
Adrenals: A slightly increased incidence of cortical vacuolation was evident in males treated with 200 mg/kg bw/day when compared with controls. The change was minimal in all cases, and was not apparent in females. As cortical vacuolation is a background finding with a variable incidence, this intergroup difference is considered to be chance and to represent normal variation rather than an effect of treatment.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

clinical signs

haematology

histopathology: non-neoplastic

mortality

Dose descriptor:

LOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

clinical signs

haematology

histopathology: non-neoplastic

mortality

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

immune system

Organ:

mesenteric lymph node

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

n Data not appropriate for statistical analysis

. not examined

Group Mean Body Weight Values

Group

(sex)

Day Numbers Relative to Start Date

1(M)

Mean

248

11.3

278.6

17.2

302.8

325.4

26.8

345.6

29.8

357.7

31.9

374.1

32.2

384.4

33.3

394.7

34.4

2(M)

Mean

243.3

11.1

272.4

15.4

297.1

316.3

23.9

338

26.9

351.4

29.2

366.3

33.1

376.8

32.8

387.8

32.7

3(M)

Mean

239.7

260.9

15.3

283.5

18.7

300.6

24.3

321.1

27.2

335.6

28.3

349.7

28.9

361.9

368.5

31.5

4(M)

Mean

241.5

11.6

254.5

14.3

277.0

22.1

295.1

26.6

318.7

32.1

327.6

35.8

341.8

37.6

353.5

41.4

362.8

44.0

Group

(sex)

Day Numbers Relative to Start Date

105

112

119

1(M)

Mean

407.9

36.6

416.9

39.3

425.6

38.9

434.1

39.7

437.1

40.2

434.5

44.4

445.1

47.4

447.3

453.7

2(M)

Mean

396.9

33.3

404.3

35.2

415

37.8

424.7

39.1

424.8

39.2

3(M)

Mean

379.8

29.8

387.7

28.5

397.6

30.4

404.1

398.9

31.1

4(M)

Mean

372.8

43.4

378.5

45.5

387

47.7

390.5

48.7

383.6

398.3

55.4

412.6

58.8

417.6

59.3

431.9

61.3

Group

(sex)

Day Numbers Relative to Start Date

1(F)

Mean

173.8

8.5

186.9

10.6

199.1

12.4

212

12.9

225.7

17.5

231.8

18.8

238.4

19.9

242.4

18.8

248.9

2(F)

Mean

169.8

7.7

181.5

10.1

193

10.9

204.1

14.5

215.1

14.4

221.3

228.2

227.5

15.9

235.9

14.8

3(F)

Mean

179

7.4

191.7

10.6

200.4

13.2

217.1

15.4

231.4

15.1

236.6

17.1

242.5

15.9

247.3

18.9

255.5

4(F)

Mean

174.5

7.4

184.2

10.4

195.6

13.6

206

219.1

14.8

226.4

16.8

232

17.3

236.4

17.5

240.3

20.8

Group

(sex)

Day Numbers Relative to Start Date

105

112

119

1(F)

Mean

251.6

18.8

257.2

20.3

261.4

18.9

262

19.3

266.2

20.5

261.1

20.4

261.9

20.6

261.9

264.1

2(F)

Mean

238.8

13.1

243

14.3

243.7

15.8

246.9

15.9

248

16.7

3(F)

Mean

259.3

261.2

17.8

263.4

18.2

267.3

18.5

269.5

20.4

4(F)

Mean

247.3

18.1

248

18.3

250.9

21.8

253.6

19.9

254.5

19.1

262.9

24.4

265.9

265

25.7

265

28.2

Group Mean Body Weight Gains

Group

(sex)

Day Numbers Relative to Start Date

1 – 8

8-15

15-22

22-29

29-36

36-43

43-50

50-57

57-64

1(M)

Mean

30.6

6.6

24.3

7.1

22.6

4.8

20.2

5.1

12.1

3.3

16.5

5.4

10.3

3.4

10.4

3.4

13.2

4.5

2(M)

Mean

29.1

5.2

24.7

19.2

4.2

21.7

13.4

3.6

14.9

4.4

10.5

4.4

3.4

9.1

4.2

3(M)

Mean

21.2**

7.8

22.6

8.2

17.1

8.7

20.5

4.7

14.5

4.6

14.1

4.3

12.2

4.7

6.6

6.7

11.3

5.7

4(M)

Mean

13.0**

9.4

22.6

12.3

18.1

23.6

6.6

8.6

14.2

5.4

12.4

6.6

9.3

4.7

10.1

4.6

Group

(sex)

Day Numbers Relative to Start Date

64-71

71-78

78-85

85-91

91-98

98-105

105-112

112-119

1(M)

Mean

4.4

8.7

3.6

8.6

4.8

5.3

5.5

4.1

10.6

4.3

2.2

2.7

6.4

4.6

2(M)

Mean

7.4

4.7

10.7

3.9

9.7

4.9

0.1

3.2

3(M)

Mean

7.9

9.9

3.5

6.5

5.2

8.3

4(M)

Mean

5.6

7.6

8.5

4.5

3.5**

6.9

9.6

12.6*

7.4

14.2

4.7

6.3

14.3**

6.2

Group

(sex)

Day Numbers Relative to Start Date

1 – 8

8-15

15-22

22-29

29-36

36-43

43-50

50-57

57-64

1(F)

Mean

13.1

4.9

12.3

12.9

5.6

13.7

7.8

6.1

5.9

6.6

4.1

4.9

6.5

4.2

2.7

5.5

2(F)

Mean

11.7

4.8

11.5

4.6

11.1

5.6

5.8

6.2

6.9

4.2

0.7

6.3

8.4

6.4

2.9

5.7

3(F)

Mean

12.7

6.5

8.7

9.3

16.7

11.4

14.3

5.6

5.2

4.6

5.9

5.3

4.8

6.8

8.2

6.2

3.8

5.4

4(F)

Mean

9.8

5.2

11.4

8.7

10.4

7.4

13.1

6.1

7.4

5.9

5.6

6.3

4.4

6.9

3.9

8.1

7.1*

Group

(sex)

Day Numbers Relative to Start Date

64-71

71-78

78-85

85-91

91-98

98-105

105-112

112-119

1(F)

Mean

5.6

8.3

4.2

5.9

0.6

3.7

4.2

6.4

1.3

7.4

0.8

3.5

3.8

2.2

4.4

2(F)

Mean

4.2

0.7

5.7

3.2

4.9

1.1

4.5

3(F)

Mean

1.9

5.4

2.2

6.7

3.9

4.7

2.2

3.8

4(F)

Mean

0.7*

6.8

2.9

7.1

2.7

6.1

0.9

5.7

0.6

7.2

5.7

0.9

5.2

Group Mean Hematological Values

Non-recovery animals:

RBC

Hct

MCH

MCV

MCHC

WBC

Neut

Group

(sex)

g/dl

10^12/l

g/dl

10^9/l

1(M)

Mean

15.9

0.4

9.037

0.326

48.01

1.52

17.63

0.64

53.16

1.68

33.15

0.42

7.67

1.68

1.17

0.553

2(M)

Mean

15.38

0.34

9.136

0.371

46.63

1.01

16.85*

0.69

51.06*

1.57

0.45

7.35

1.53

1.31

0.571

3(M)

Mean

16.07

0.5

9.328

0.391

48.44

1.68

17.23

0.67

51.97

1.59

33.15

0.37

7.36

1.28

1.985**

0.64

4(M)

Mean

16.05

0.62

9.149

0.344

48.66

1.75

17.54

0.54

53.21

1.36

0.32

10.02**

2.15

3.751**

1.793

Lymph

Mono

Eos

Bas

PLT

APTT

Group

(sex)

10^9/l

Seconds

10^9/l

Seconds

1(M)

Mean

6.435

1.485

0.011n

0.023

0.052

0.049

0.000n

9.16

0.43

589.2

53.3

13.9

0.86

2(M)

Mean

5.955

1.399

0.000n

0.088

0.053

0.000n

8.74

0.46

565.9

90.6

13.11

0.66

3(M)

Mean

5.253

1.136

0.000n

0.122

0.074

0.000n

9.02

0.38

558.1

58.6

13.98

2.02

4(M)

Mean

6.212

1.813

0.000n

0.058

0.07

0.000n

9.14

0.43

653.9

108.7

13.03

0.95

RBC

Hct

MCH

MCV

MCHC

WBC

Neut

Group

(sex)

g/dl

10^12/l

g/dl

10^9/l

1(F)

Mean

14.93

0.39

8.059

0.395

42.95

1.34

18.56

0.7

53.38

1.71

34.75

0.3

4.83

0.75

0.894

0.547

2(F)

Mean

15.16

0.33

8.129

0.265

43.45

1.06

18.67

0.59

53.49

1.63

34.92

0.31

4.68

0.92

0.514

0.337

3(F)

Mean

0.67

8.059

0.377

43.05

1.96

18.63

0.57

53.44

1.34

34.87

0.35

5.52

1.7

1.569*

1.002

4(F)

Mean

14.75

1.23

8.042

0.765

42.71

3.96

18.35

0.52

53.14

1.1

34.54

0.44

6.32

2.6

1.644*

0.648

Lymph

Mono

Eos

Bas

PLT

APTT

Group

(sex)

10^9/l

Seconds

10^9/l

Seconds

1(F)

Mean

3.887

0.614

0.000n

0.048

0.031

0.000n

8.81

0.35

571.6

15.18

1.98

2(F)

Mean

4.107

0.751

0.000n

0.059

0.000n

8.93

0.59

550.1

88.1

15.39

1.78

3(F)

Mean

3.908

1.071

0.000n

0.043

0.056

0.000n

8.79

0.64

626.1

76.4

14.44

1.36

4(F)

Mean

4.607

2.235

0.000n

0.069

0.066

0.000n

9.03

0.75

655.4*

104.8

14.61

1.83

Recovery animals:

		Hb	RBC	Hct	MCH	MCV	MCHC	WBC	Neut
Group (sex)		g/dl	10^12/l	%	pg	fl	g/dl	10^9/l	10^9/l
1(M)	Mean SD N	16.24 0.48 10	9.428 0.377 10	48.86 1.39 10	17.25 0.56 10	51.86 1.25 10	33.23 0.32 10	7.34 1.26 10	1.359 0.708 10
4(M)	Mean SD N	15.88 1.57 9	9.019 0.726 9	47.31 4.73 9	17.61 1.14 9	52.39 3.04 9	33.57 0.58 9	7.09 1.99 9	1.593 0.818 9

		Lymph	Mono	Eos	Bas	CT	PLT	APTT
Group (sex)		10^9/l	10^9/l	10^9/l	10^9/l	Seconds	10^9/l	Seconds
1(M)	Mean SD N	5.907 1.019 10	0.000n 0 10	0.077 0.064 10	0.000n 0 10	10.03 0.83 10	610.4 104.8 10	15.18 1.4 10
4(M)	Mean SD N	5.432 1.433 9	0.008n 0.023 9	0.056 0.057 9	0.000n 0 9	9.84 0.55 9	536.4 89.8 9	14.94 1.82 9

		Hb	RBC	Hct	MCH	MCV	MCHC	WBC	Neut
Group (sex)		g/dl	10^12/l	%	pg	fl	g/dl	10^9/l	10^9/l
1(F)	Mean SD N	15.42 0.59 10	8.145 0.522 10	44.86 2.32 10	18.97 0.6 10	55.14 1.32 10	34.39 0.59 10	3.87 0.69 10	0.604 0.18 10
4(F)	Mean SD N	16.37** 0.51 10	8.866** 0.251 10	48.41** 1.32 10	18.35* 0.53 10	54.31 1.26 10	33.81* 0.28 10	4.32 1.02 10	1.046** 0.402 10

		Lymph	Mono	Eos	Bas	CT	PLT	APTT
Group (sex)		10^9/l	10^9/l	10^9/l	10^9/l	Seconds	10^9/l	Seconds
1(F)	Mean SD N	3.225 0.557 10	0.000n 0 10	0.133 0.306 10	0.000n 0 10	8.98 0.38 10	588.1 54.8 10	15.28 1.79 10
4(F)	Mean SD N	3.235 0.975 10	0.000n 0 10	0.043 0.042 10	0.000n 0 10	8.81 0.56 10	545.2 115.5 10	15.49 1.45 10

Group Mean Blood Chemical Values

Non-recovery animals:

Urea

Glucose

total Prot.

Albumin

A/G

Na+

K+

Ca++

Group

(sex)

mg/dl

g/dl

Ratio

mmol/l

1(M)

Mean

37.5

4.6

174.2

19.3

6.965

0.271

3.85

0.14

1.241

0.063

148.1

2.5

4.541

0.15

104.8

1.8

2.57

0.33

1.4

0.21

2(M)

Mean

7.8

187.5

22.4

6.891

0.308

3.79

0.1

1.243

0.074

147.4

2.3

4.592

0.735

105.3

1.8

2.557

0.192

1.5

0.26

3(M)

Mean

45.8*

9.3

194.8

38.6

6.711*

0.411

3.69*

0.16

1.235

0.099

147.1

1.7

4.585

0.767

104.8

1.4

2.66

0.108

1.42

0.32

4(M)

Mean

44.6*

7.4

193.7

6.535**

0.245

3.75*

0.08

1.366**

0.085

146.4

1.9

4.918

0.732

103.5

1.6

2.714

0.095

1.48

0.29

yGT

ASAT

ALAT

Creat

Tri

Chol

Bili

Bile acids

Group

(sex)

IU/l

mg/dl

µmol/l

1(M)

Mean

1.4

0.7

97.1

20.1

17.8

115

20.9

0.71

0.05

230.3

81.9

84.2

10.8

0.114

0.023

9.76

7.23

2(M)

Mean

0.9

0.57

89.2

20.3

67.6

11.4

114.7

24.5

0.762

0.17

278.5

81.2

97.4

20.4

0.121

0.01

9.25

6.13

3(M)

Mean

1.6

1.17

128.2*

43.3

108.5**

31.5

88.8*

15.7

0.776

0.237

127.8**

44.6

74.5

13.6

0.116

0.02

3.73*

4(M)

Mean

0.9

0.57

169.7**

176.7**

39.8

93.9*

22.2

0.831

0.227

126.3**

47.2

68.6*

12.5

0.122

0.018

4.22*

1.11

Urea

Glucose

total Prot.

Albumin

A/G

Na+

K+

Ca++

Group

(sex)

mg/dl

g/dl

Ratio

mmol/l

1(F)

Mean

41.4

12.8

160.8

14.7

7.141

0.359

4.26

0.24

1.483

0.069

148.8

2.1

4.776

1.435

105.1

1.3

2.735

0.161

1.44

0.24

2(F)

Mean

4.2

165.9

18.4

7.245

0.338

4.45

0.25

1.585*

0.08

148.1

2.7

4.116

0.442

105.3

2.1

2.781

0.092

1.39

0.26

3(F)

Mean

49.2*

8.8

160.1

24.7

7.415

0.382

4.51

0.19

1.562*

0.116

147

2.6

4.34

0.858

103.5*

1.8

2.863

0.086

1.39

0.56

4(F)

Mean

55.8**

158.5

18.9

6.763

0.786

4.14

0.39

1.601*

0.135

146.3*

2.1

4.914

0.722

103.0*

1.9

2.788

0.117

1.24

0.22

yGT

ASAT

ALAT

Creat

Tri

Chol

Bili

Bile acids

Group

(sex)

IU/l

mg/dl

µmol/l

1(F)

Mean

0.22

0.44

92.9

45.5

65.4

16.9

48.2

10.9

0.815

0.197

164.9

69.2

0.074

0.034

16.51

8.93

2(F)

Mean

0.1

0.32

79.1

12.3

58.2

9.4

57.9

9.9

0.821

0.063

137.6

32.3

65.6

5.5

0.09

0.019

20.33

13.11

3(F)

Mean

0.3

0.48

117.0*

19.4

124.9**

30.5

43.3

0.865

0.17

103.6**

47.2

58.9**

7.9

0.095

0.013

7.98**

4.02

4(F)

Mean

0.4

0.52

109.3*

26.6

112.2**

46.7

37.4*

9.1

0.824

0.138

80.9**

33.1

50.1**

7.8

0.088

0.023

6.75**

2.27

Recovery animals:

		Urea	Glucose	total Prot.	Albumin	A/G	Na+	K+	Cl	Ca++	P
Group (sex)		mg/dl	mg/dl	g/dl	g/dl	Ratio	mmol/l	mmol/l	mmol/l	mmol/l	mmol/l
1(M)	Mean SD N	46.1 5.3 10	159.2 15.7 10	7.16 0.219 10	3.95 0.14 10	1.232 0.076 10	145.7 1.3 10	6.662 4.874 10	104.8 1.9 10	2.742 0.232 10	1.82 0.29 10
4(M)	Mean SD N	52.2 8.9 9	159.7 12.7 9	6.923* 0.26 9	3.89 0.17 9	1.282 0.11 9	146.7 2.3 9	5.191 1.111 9	105.3 1.4 9	2.782 0.093 9	1.86 0.27 9

		yGT	ASAT	ALAT	AP	Creat	Tri	Chol	Bili	Bile acids
Group (sex)		IU/l	IU/l	IU/l	IU/l	mg/dl	mg/dl	mg/dl	mg/dl	µmol/l
1(M)	Mean SD N	0.00n 0 10	149 187.6 10	102.2 120.4 10	103 23.5 10	0.656 0.051 10	232.9 89.9 10	95.1 21.6 10	0.092 0.049 10	8.53 5.21 10
4(M)	Mean SD N	0.00n 0 9	88.7 15.7 9	71.1 10.4 9	121.4* 11.5 9	0.737 0.187 9	178 68.8 9	82 11.7 9	0.106 0.027 9	11.57 5.84 9

		Urea	Glucose	total Prot.	Albumin	A/G	Na+	K+	Cl	Ca++	P
Group (sex)		mg/dl	mg/dl	g/dl	g/dl	Ratio	mmol/l	mmol/l	mmol/l	mmol/l	mmol/l
1(F)	Mean SD N	55.1 9.9 10	168.2 46.7 10	7.979 0.797 10	4.59 0.42 10	1.364 0.15 10	147.9 1.5 10	7.097 5.293 10	103 2.3 10	2.599 0.184 10	1.71 0.47 10
4(F)	Mean SD N	52.7 4.5 10	152.4 28.3 10	7.871 0.382 10	4.62 0.25 10	1.43 0.063 10	148.6 3 10	4.029** 0.349 10	105.5* 2.7 10	2.735 0.118 10	1.10* 0.52 10

		yGT	ASAT	ALAT	AP	Creat	Tri	Chol	Bili	Bile acids
Group (sex)		IU/l	IU/l	IU/l	IU/l	mg/dl	mg/dl	mg/dl	mg/dl	µmol/l
1(F)	Mean SD N	0.00n 0 10	112.9 84.3 10	60.9 14.9 10	39.5 23.5 10	0.719 0.09 10	167.7 87.6 10	85.2 16 10	0.079 0.051 10	9.95 8.97 10
4(F)	Mean SD N	0.10n 0.32 10	85 22.7 10	61.9 13.4 10	47.2 9.7 10	0.72 0.095 10	121.1 27.3 10	73.9 15.2 10	0.105 0.02 10	19.80* 9.65 10

Histopathology

Non-recovery animals

	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg bw/d)	0	10	100	200	0	10	100	200
Mesenteric lymph node Histiocytosis/Granulomas
Minimal	0	4	1	0	1	5	0	0
Slight	0	1	5	3	1	1	4	2
Moderate	0	0	3	4	0	0	6	4
Marked	0	0	1	3	0	0	0	4
Total	0	5	10	10	2	6	10	10
Abscess
Minimal	0	0	2	5	0	0	1	4
Total	0	0	2	5	0	0	1	4
N	10	10	10	10	10	10	10	10
Adrenals Vacuolation, cortical, diffuse
Minimal	1	0	2	4
total	1	0	2	4
N	10	10	10	10

Recovery animals:

	1M	4M	1F	4F
Dose (mg/kg bw/d)
Mesenteric lymph node Histiocytosis/Granulomas
Slight	0	1	0	3
Moderate	0	4	0	4
Marked	0	4	0	3
Total	0	9	0	10
Abscess
Minimal	0	1	0	2
Total	0	1	0	2
N	10	10	10	10

Summary Incidence of Necropsy Findings – fertility related parameters

Males

	0 (control)	10 mg/kg bw/d	100 mg/kg bw/d	200 mg/kg bw/d
Group:	1	2	3	4
Seminal Vesicles (Including Coagulating Gland)
Submitted	20	10	10	19
No Visible Lesions	19	10	10	19
Hard; left	1	0	0	0
Pale; left	1	0	0	0

Females

	0 (control)	10 mg/kg bw/d	100 mg/kg bw/d	200 mg/kg bw/d
Group:	1	2	3	4
Uterus And Cervix
Submitted	20	10	10	20
No Visible Lesions	20	10	9	19
Damaged On Removal	0	0	1	0
Fluid Filled; dark; right horn	0	0	0	1

Group Mean Organ Weights with Corresponding Relative (% of Bodyweight) Organ Weights – fertility related parameters

Non-recovery animals

		0 (control)	10 mg/kg bw/d	100 mg/kg bw/d	200 mg/kg bw/d	0 (control)	10 mg/kg bw/d	100 mg/kg bw/d	200 mg/kg bw/d
Group:		1	2	3	4	1	2	3	4
Sex		M	M	M	M	F	F	F	F
Epididymides	Mean (g) S.D. N	1.73247 0.19320 10	3.12171 4.72007 10	1.53095 0.17581 10	1.65394 0.25675 10	-	-	-	-
	Mean (%) S.D. N	0.390 0.036 10	0.711 1.018 10	0.384 0.031 10	0.436 0.061 10	-	-	-	-
Testes	Mean (g) S.D. N	3.90426 0.37256 10	3.79877 0.31706 10	3.82483 0.46413 10	3.75560 0.43943 10	-	-	-	-
	Mean (%) S.D. N	0.883 0.110 10	0.898 0.078 10	0.959 0.091 10	0.988 0.081 10	-	-	-	-
Ovaries	Mean (g) S.D. N	-	-	-	-	0.10444 0.02286 10	0.10428 0.01783 10	0.12207 0.02816 10	0.10916 0.02254 10
	Mean (%) S.D. N	-	-	-	-	0.039 0.009 10	0.042 0.007 10	0.046 0.012 10	0.044 0.009 10
Uterus And Cervix	Mean (g) S.D. N	-	-	-	-	0.83856 0.21425 10	0.78873 0.31097 10	0.79504 0.25003 10	0.69453 0.23548 10
	Mean (%) S.D. N	-	-	-	-	0.311 0.076 10	0.318 0.122 10	0.298 0.103 10	0.279 0.086 10

Recovery animals

		0 (control)	200 mg/kg bw/d	0 (control)	200 mg/kg bw/d
Group:		1	4	1	4
Sex		M	M	F	F
Epididymides	Mean (g) S.D. N	1.68946 0.21805 10	1.73983 0.21805 9	-	-
	Mean (%) S.D. N	0.375 0.052 10	0.409 0.069 9	-	-
Testes	Mean (g) S.D. N	3.85441 0.48796 10	3.76723 0.24391 9	-	-
	Mean (%) S.D. N	0.852 0.095 10	0.889 0.141 9	-	-
Ovaries	Mean (g) S.D. N	-	-	0.09953 0.01985 10	0.11018 0.02005 10
	Mean (%) S.D. N	-	-	0.038 0.007 10	0.042 0.009 10
Uterus And Cervix	Mean (g) S.D. N	-	-	0.94423 0.40225 10	0.98089 0.49808 10
	Mean (%) S.D. N	-	-	0.360 0.151 10	0.374 0.196 10

Conclusions:

The oral (gavage) administration of BADGE-IPD for up to ninety consecutive days, to Wistar rats of both sexes at dose levels of 10, 100 or 200 mg/kg bw/day resulted in adverse treatment-related effects in animals of both sexes treated with 200 and 100 mg/kg bw/day. These findings included the death of one male treated with 200 mg/kg bw/day, clinical signs of toxicity, reduced body weight development and food consumption, changes in the hematology parameters measured and microscopic changes in the mesenteric lymph nodes. These changes were considered to represent an adverse effect of treatment at these levels. The effects evident in both sexes at 10 mg/kg bw/day were confined to minimal histiocytosis with granulomas in the mesenteric lymph nodes. Based on the level of severity of these changes evident in both sexes treated with 10 mg/kg bw/day, the 'No Observed Adverse Effect Level (NOAEL) was considered to be 10 mg/kg bw/day.

Executive summary:

The present study was designed to investigate the systemic toxicity of the test item BADGE-IPD in accordance with OECD Guideline 408 "Subchronic Oral Toxicity - Rodent: 90 Day Study” (Adopted 21 September 1998).

The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for up to ninety consecutive days, at dose levels of 10, 100 and 200 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Propylene Glycol). Two recovery groups, each of ten males and ten females, were treated with the high dose (200 mg/kg bw/day) or the vehicle alone for up to ninety consecutive days and then maintained without treatment for a further twenty-eight days.

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all non-recovery group animals at the end of the treatment period and for all surviving recovery group animals at the end of the treatment-free period. Ophthalmoscopic examination was also performed on all animals prior to the start of treatment and on all non-recovery control and high dose animals during Week 12 of the study.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

Mortality

One male treated with 200 mg/kg bw/day was found dead on Day 48. There were no further unscheduled deaths.

Clinical Observations

Increased salivation and noisy respiration was evident in animals of either sex treated with 200 mg/kg bw/day and to a lesser extent in animals of either sex treated with 100 mg/kg bw/day throughout the treatment period. Isolated incidences of a stained snout, labored respiration, decreased respiratory rate, hunched posture and fur loss were also evident in females treated with 200 mg/kg bw/day, a stained snout and sneezing was also evident in some males treated with 200 mg/kg bw/day and a decreased respiratory rate and hunched posture was also evident in some males treated with 100 mg/kg bw/day. During the treatment-free period, one male and one female that were previously given 200 mg/kg bw/day showed episodes of noisy respiration. No such effects were detected in males treated with 10 mg/kg bw/day however one female from this treatment group had increased salivation on Day 81 only.

Behavioral Assessment

Weekly behavioral assessments revealed instances of noisy respiration in some animals of both sexes treated with 200 mg/kg bw/day. No such effects were detected in animals of both sexes treated with 100 or 10 mg/kg bw/day.

Functional Performance Tests

There were no toxicologically significant changes in functional performance.

Sensory Reactivity Assessments

There were no treatment-related changes in sensory reactivity.

Body Weight

Males treated with 200 and 100 mg/kg bw/day showed a reduction in body weight gain during the first week of treatment. Periods of recovery were evident thereafter, however, generally body weight gain in these males was slightly below controls. Consequently overall body weight gain was lower than controls in these males. Significant improvement in body weight gain was evident in males previously treated with 200 mg/kg bw/day during the twenty-eight day treatment-free period. Females treated with 200 mg/kg bw/day showed a slight reduction in body weight gain during the first week of treatment, however, recovery was evident thereafter. No such effects were detected in females treated with 100 mg/kg bw/day or in animals of both sexes treated with 10 mg/kg bw/day.

Food Consumption

Animals of both sex treated with 200 mg/kg bw/day and males treated with 100 mg/kg bw/day showed a reduction in overall food consumption. Incidences of reduced food conversion efficiency were also evident in males treated with 200 and 100 mg/kg bw/day during the treatment period. Improvement in food consumption was evident during the twenty-eight day treatment-free period. No such effects were detected in females treated with 100 mg/kg bw/day or in animals of both sexes treated with 10 mg/kg bw/day.

Water Consumption

Visual inspection of water bottles did not reveal any inter-group differences.

Ophthalmoscopy

Hematology

Animals of both sexes treated with 200 and 100 mg/kg bw/day showed an increase in neutrophils. Males treated with 200 mg/kg bw/day also showed an increase in total leukocyte count. The effect on neutrophils was also present in females previously treated with 200 mg/kg bw/day at the end of the twenty-eight day treatment -free period. No such effects were detected in both sexes treated with 10 mg/kg bw/day.

Blood Chemistry

There were no toxicologically significant effects detected in the blood chemical parameters examined.

Necropsy

No toxicologically significant macroscopic abnormalities were detected in surviving animals.

The male treated with 200 mg/kg bw/day that was found dead on Day 48 had a dark liver and lungs.

Organ Weights

No toxicologically significant effects were detected in the organ weights measured.

Histopathology

The following treatment-related microscopic abnormalities were detected:

Mesenteric Lymph Nodes: Histiocytosis with granulomas was evident in animals of both sexes from all treatment groups, varying from minimal to moderate in severity and following a dose-dependent response. At 100 and 200 mg/kg bw/day the more severe grades of histiocytosis were often accompanied by minimal abscesses. Following the twenty-eight day recovery period, histiocytosis with granulomas and abscesses were evident in both sexes previously treated with 200 mg/kg bw/day. When compared with the main study, the incidence of abscesses was lower, whereas histiocytosis persisted at much the same incidence and severity.

Histiocytosis in the mesenteric lymph nodes is known to occur in response to the oral administration of some test items and is likely to represent accumulation of material, indicating delayed clearance of an exogenous or endogenous material.

The following microscopic abnormalities were evident, however, these were considered to reflect individual variation rather than an effect of treatment.

Adrenals: A slightly increased incidence of cortical vacuolation was evident in males treated with 200 mg/kg bw/day when compared with controls. The change was minimal in all cases, and was not apparent in females. As cortical vacuolation is a background finding with a variable incidence, this intergroup difference is considered to be by chance and to represent normal variation rather than an effect of treatment.

Conclusion

Cross-reference 2

Reason / purpose for cross-reference:: data waiving: supporting information

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 September - 4 October 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP/Guideline study

Qualifier:

according to guideline

Guideline:

other: USEPA, OPPTS 870.3050 (2000)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes

Limit test:

Species:

rat

Strain:

Fischer 344/DuCrj

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and Sex
Rats, male and female (nulliparous and nonpregnant)

Strain and Justification
F344/DuCrl rats were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data and the reliability of the commercial supplier.

Supplier and Location
Charles River (Kingston, New York)

Age at Study Start
Approximately 6 weeks

Physical and Acclimation
During the acclimation period each animal was evaluated by a laboratory veterinarian, or a trained animal/toxicology technician under the direct supervision of a laboratory veterinarian, to determine the general health status and acceptability for study purposes. The animals were housed two-three per cage per cage in stainless steel solid bottom cages with corncob bedding, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), prior to randomization. Animals were acclimated to the laboratory (fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International - AAALAC International) for at least one week prior to the start of the study.

Housing
After assignment, animals were housed one per cage in stainless steel cages. Cages had solid floors with corncob bedding. Cages contained a feed crock and a pressure activated lixit valve-type watering system. The following environmental conditions were maintained in the animal room.
Temperature: 22°C with a tolerance of ± 1°C (and a maximum permissible excursion of ± 3°C)
Humidity: 40-70%
Air Changes: 10-15 times/hour (average)
Photoperiod: 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

Enrichment
Enrichment for rats included the use of aspen shaving bedding and open areas on the cage sides for visualization of other rats.

Randomization and Identification
Before administration of the test material began, animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study. Animals placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) that were correlated to unique alphanumeric identification numbers.

Feed and Water
Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form. Feed and municipal water were provided ad libitum. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provided adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility. There were no contaminants in either the feed or water that would have adversely impacted the results or interpretation of this study.

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

All dosing solutions were prepared by mixing the test material in propylene glycol (PG) at concentrations of 0, 5, 16.67, or 50 mg/ml and administered at a dose volume of 6 ml/kg body weight to achieve the targeted dose levels. Dose solutions were not corrected for purity. Dose volumes were adjusted at least weekly based on individual body weights. The control rats were dosed with PG at 6 ml/kg body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose Confirmation and Homogeneity
Dose confirmation analyses of all dose solutions from the first mix were determined pre-exposure. The homogeneity of the low-dose and the high-dose test solutions was determined concurrent with dose confirmation. The method used for analyzing the test material in propylene glycol was liquid chromatography with ultraviolet detection (LC/UV). The actual concentrations of test material in propylene glycol ranged from 95.8 to 102% of targeted values and were considered acceptable. The results of the analyses indicated the preparations were homogeneously mixed based on relative standard deviations of ≤ 4.2%

Stability
BADGE IPD (#33) was stable for at least 16 days in PG at concentrations ranging from 0.025 to 250 mg/ml. The established concentration range spanned those used in this study, and dose solutions were used within the stability duration. No additional stability analyses were conducted.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 30, 100 and 300 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

5 males and 5 females/dose level

Control animals:

yes, concurrent vehicle

Details on study design:

Groups of five male and five female F344/DuCrl rats were administered BADGE IPD (#33) by gavage at targeted dose levels of 0, 30, 100, or 300 mg/kg body weight/day (mg/kg/day; mkd) in propylene glycol for 28 days to evaluate the potential for systemic toxicity. Test material administration for all animals began on September 6, 2012 (test day 1). Rats were necropsied on October 4, 2012 (test day 29).

Positive control:

No data.

Observations and examinations performed and frequency:

Daily Observations
A cage-side examination was conducted at least once a day, approximately at the same time each day (usually in the morning). This examination was typically performed with the animals in their cages and was designed to detect significant clinical abnormalities that were clearly visible upon a limited examination, and to monitor the general health of the animals. The animals were not hand-held for these observations unless deemed necessary. Significant abnormalities that were observed for included, but were not limited to: decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor and twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal/urine quantity. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily.

Clinical Observations
Clinical observations were conducted on all animals at least once daily. Animals were observed approximately one hour after dosing. These examinations included a careful, hand-held examination of the animal.

Detailed Clinical Observations
Detailed clinical observations (DCO) were conducted on all animals pre-exposure and once per week throughout the study. The DCO was conducted on all animals, at approximately the same time each day according to an established format. The examination included cage-side, hand-held and open-field observations that were recorded categorically or using explicitly defined scales (ranked).

Ophthalmology
The eyes of all animals were examined by a veterinarian pre-exposure and prior to the scheduled necropsy using indirect ophthalmoscopy. One drop of 0.5% tropicamide ophthalmic solution was instilled topically in each eye to produce mydriasis prior to the indirect ophthalmic examinations. Eyes were also be examined by a prosector during the necropsy using a moistened glass slide pressed to the cornea.

Body Weights/Body Weight Gains
All rats were weighed pre-exposure, twice during the first week and at least weekly during the dosing period. Body weight gains were calculated relative to test day 1.

Feed Consumption
The amount of feed consumed was determined twice during the first week and at least weekly for all animals by weighing feed containers at the start and end of a measurement cycle. Feed consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of feed container - final weight of feed container)/[(# of days in measurement cycle) (# of animals per cage)]

Sacrifice and pathology:

Clinical Pathology
Animals were fasted overnight prior to blood collection. Blood samples were obtained from the orbital sinus following anesthesia with CO2/O2 at the scheduled necropsy.

Hematology
Sample Preparation
Blood samples for a complete blood count were mixed with ethylenediaminetetraacetic acid (EDTA). Blood smears were prepared, stained with Wright-Giemsa stain, cover-slipped, and evaluated.
Hematologic parameters were assayed using the Advia 120 Hematology Analyzer (Siemens Healthcare Diagnostics, Tarrytown, New York).
Assays
Hematocrit (HCT)
Hemoglobin (HGB) concentration
Red blood cell (RBC) count
Total white blood cell (WBC) count
Differential WBC count
Platelet (PLT) count
Reticulocyte (RET) count
RBC indices:
Mean Corpuscular Hemoglobin (MCH)
Mean Corpuscular Volume (MCV)
Mean Corpuscular Hemoglobin Concentration (MCHC)

Coagulation
Sample Preparation
Blood samples were collected in sodium citrate tubes, centrifuged, plasma collected, and assayed using the ACL9000 Analyzer (Instrumentation
Laboratory, Bedford, Massachusetts).
Assay
Prothrombin time (PT)

Clinical Chemistry
Sample Preparation
Blood samples were collected and serum was separated from cells as soon as possible. Serum parameters were measured using a cobas c311 Clinical Chemistry Analyzer (Roche Diagnostics, Indianapolis, Indiana).
Enzyme Activities of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Gamma-glutamyl transpeptidase (GGT)
Concentrations of:
Albumin (ALB)
Albumin/Globulin Ratio (A/G) - calculated
Cholesterol (CHOL)
Creatinine (CREA)
Electrolytes (NA, K, PHOS, CL and CA)
Globulin (GLOB) - calculated
Glucose (GLUC)
Total bile acids (TBA)
Total bilirubin (TBIL)
Total protein (TP)
Triglycerides (TRIG)
Urea nitrogen (UN)

Urinalysis
Urine samples were obtained from all animals the week prior to the scheduled necropsy. Animals were housed in metabolism cages and the urine collected overnight (approximately 16 hours). Feed and water were available during this procedure.
Assays
Color, appearance, specific gravity (refractometer) and urine volume
Semiquantitative analysis of the following was conducted using Siemens Multistix Reagent Strips on the Clinitek Advantus Analyzer (Siemens Healthcare Diagnostics, Tarrytown, New York):
pH
Bilirubin
Glucose
Protein
Ketones
Blood
Urobilinogen
Microscopic Exam:
Urine samples were collected from each animal by manual compression of the urinary bladder. The urine samples were pooled from each group, and the microsediments were characterized microscopically.

Anatomic Pathology
Necropsy
Fasted rats submitted alive for necropsy were weighed, anesthetized by the inhalation of CO2/O2, and blood samples were obtained from the orbital sinus. Their tracheas were exposed and clamped, and the animals were euthanized by decapitation. A complete necropsy was conducted on all animals by a veterinary pathologist assisted by a team of trained individuals. The necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, reexamined and selected tissues were incised. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10% formalin using a hand-held syringe and blunt needle.

The brain, liver, kidneys, heart, adrenals, testes, epididymides, prostate + seminal vesicles with coagulating glands (and fluids), prostate, thymus, and spleen were trimmed and weighed immediately. The thyroid glands with parathyroids were weighed post fixation. The ratios of organ weight to terminal body weight were calculated.

Representative samples of tissues were collected and preserved in neutral, phosphate-buffered 10% formalin. Transponders were removed and placed in jars with the tissues.

Histopathology
The number of sections from all preserved tissues were processed by standard histological procedures from control and high-dose group animals. Paraffin embedded tissues were sectioned approximately 6 μm thick, stained with hematoxylin and eosin and examined by a veterinary pathologist using a light microscope. The following tissues from the remaining groups were processed, sectioned, and stained: liver, kidneys, relevant gross lesions and target organs (liver, cecum, cecal lymph node, mesenteric lymph node, mesenteric tissue, rectum, colon, prostate (males), and seminal vesicles/coagulating glands (males)). Relevant gross lesions were microscopically examined from all animals. All target tissues were microscopically examined from the low- and intermediate-dose group animals to define a NOEL.

Selected histopathologic findings were graded to reflect the severity of specific lesions to evaluate: 1) the contribution of a specific lesion to the health status of an animal, 2) exacerbation of common naturally occurring lesions as a result of the test material, and 3) dose-response relationships for treatment-related effects. Very slight and slight grades were used for conditions that were altered from the normal textbook appearance of an organ/tissue, but were of minimal severity and usually with less than 25% involvement of the parenchyma. This type of change was neither expected to significantly affect the function of the specific organ/tissue nor have a significant effect on the overall health of the animal. A moderate grade was used for conditions that were of sufficient severity and/or extent (up to 50% of the parenchyma) that the function of the organ/tissue may have been adversely affected, but not to the point of organ failure. The health status of the animal may or may not have been affected, depending on the organ/tissue involved, but generally lesions graded as moderate were not life threatening. A severe grade would have been used for conditions that were extensive enough to cause significant organ/tissue dysfunction or failure. This degree of change in a critical organ/tissue would have been life threatening.

Other examinations:

No additional information available.

Statistics:

Means and standard deviations were calculated for all continuous data. All parameters examined statistically (feed consumption is addressed below) were first tested for equality of variance using Bartlett's test (Winer, 1971). If the results from Bartlett's test were significant at alpha = 0.01, then the data for the parameter may have been subjected to a transformation to obtain equality of the variances. The transformations examined were the common log, the inverse, and the square root, in that order. The data were reviewed and an appropriate form of the data was selected. The selected form of the data was then subjected to the appropriate parametric analysis as described below.

In-life body weights were evaluated using a repeated measures (RM) analysis of variance (ANOVA), the multivariate approach, for time (the repeated factor), sex, and dose (Winer, 1971). In the repeated measures ANOVA with a pre-exposure data point, the time-dose interaction assessed the true effect of treatment (Bonate, 2000).

The first examination in the RM-ANOVA was of the time-sex-dose interaction. If significant at alpha = 0.02, the analysis was repeated separately for each sex without examining the results of other factors. The time-dose interaction was examined next. If the time-dose interaction was statistically significant at alpha = 0.05, linear contrasts tested the time-dose interaction for the comparisons of each dose group to the control group. A Bonferroni correction (Miller, 1966) was applied to the alpha level to compensate for the multiple comparisons with the control group. This correction controlled the experiment-wise error rate. The corrected comparison-wise alpha level of 0.02 was reported so direct comparison could be made to the Pillai’s Trace p-values generated.

Continued below

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males injesting 300 mg/kg/day had treatment-related decreases

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males ingesting 100 and 300 mg/kg/day and females ingesting 300 mg/kg/day had treatment-related decreases.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Male and female rats in the 100 and 300 mg/kg/day dose groups had treatment-related decreases in hemoglobin concentration and hematocrit, and increases in reticulocytes.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males and females given 300 mg/kg/day had statistically-identified treatment-related increases in alanine aminotransferase and aspartate aminotransferase activities.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Females given 300 mg/kg/day had statistically-identified (sexes combined for statistical analysis) increased urine volume and decreased specific gravity

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ weight decrements interpreted to be reflective of the decreased body weight/fat reserves and feed consumption in rats given 300 mg/kg/day.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Males and females given 100 or 300 mg/kg/day had a treatment-related increase in the size of the cecum, which was attributed to an increase in the amounts of watery luminal contents.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Primary treatment-related histological effects occurred within the liver, lymph nodes (mesenteric and cecal), gastrointestinal tract, and mesenteric fat in animals given 100 or 300 mg/kg/day.

Histopathological findings: neoplastic:

no effects observed

Details on results:

Mortality
All rats survived the full duration of the study.

Detailed Clinical Observations
There were no treatment-related findings for any animal for the ranked portion of the detailed clinical observations.

Clinical and Cage-Side Observations
Three animals given 300 mg/kg/day, two males (#5465 and #5469) and one female (#5488), had treatment-related soft feces and/or fecal soiling occurring between test days (TD) 11-15. All other observations were considered spurious and unrelated to treatment due to the isolated occurrences and lack of a dose-response relationship.

Ophthalmology
Pre-exposure examination on all animals placed on study indicated all but three rats were within normal limits. These three animals, two males (#5460 and #5462) assigned to the 100 mg/kg/day group and one female (#5476) assigned to the 30 mg/kg/day group had cloudy corneas during the pre-exposure examination, but were considered suitable for study purposes.

In addition to pre-exposure observations, cloudy cornea was noted in three male rats (#5454 in the control group, #5457 in the 30 mg/kg/day group, and #5464 in the 100 mg/kg/day group) and one female rat (#5474 in the 30 mg/kg/day group) prior to study termination. These observations were interpreted to be unrelated to treatment due to their low incidence and lack of a dose-response relationship.

Body Weights/Body Weight Gains
Males given 300 mg/kg/day had treatment-related decreases in body weight and body weight gains that were 11.1 and 22.7% less than controls, respectively by TD 28 (Text Tables 1 and 2). The body weights and body weight gains of female rats given 300 mg/kg/day were similar to controls throughout the study. Body weights and body weight gains of male and female rats given 30 or 100 mg/kg/day were also similar to controls.

Feed Consumption
Males given 300 mg/kg/day had treatment-related decreases in feed consumption that ranged 13.2 - 22.9% less than controls from TD 1-15 (Text Table 3). Females given 300 mg/kg/day and males given 100 mg/kg/day also had treatment-related decreases in feed consumption that were 13.1 and 13.3% lower than controls, respectively on TD 1-4 (Text Tables 3 and 4). Feed consumption for females given 100 mg/kg/day and the males and females given 30 mg/kg/day was similar to controls.

Clinical Pathology
Hematology
Treatment-related alterations in hematologic parameters are summarized in Text Tables 5-7. Male and female rats in the 100 and 300 mg/kg/day dose groups had treatment-related decreases in hemoglobin concentration and hematocrit, and increases in reticulocytes. In addition, rats in the 300 mg/kg/day group had increased white blood cell counts (Text Table 5).

Microscopic evaluation of peripheral blood smears demonstrated treatment-related very slight increases in polychromasia in both sexes given 300 mg/kg/day, and in males given 100 mg/kg/day (Text Table 6) corroborating the increased reticulocyte counts noted at these dose levels. Polychromasia and reticulocytosis are associated with increased erythropoietic activity in response to decrements in hemoglobin/hematocrit (Latimer et al., 2003).

The mean total white blood cell count was significantly increased in animals given 300 mg/kg/day. Differential white blood cell counts indicated that treatment-related leukocytosis was due, in part, to an increase in large unstained cells. The percentages of eosinophils were decreased in males and females given 300 mg/kg/day and were interpreted to be treatment related (Text Table 7).

Prothrombin Time
There were no statistically significant or treatment-related differences in the prothrombin times of male or female rats at any dose level as compared to controls.

Clinical Chemistry
Treatment-related and/or statistically identified alterations in clinical chemistry parameters are summarized in Text Table 8. Males and females given 300 mg/kg/day had statistically-identified treatment-related increases in alanine aminotransferase and aspartate aminotransferase activities. Increases in both enzymes represent possible hepatocellular leakage; histology corroborated hepatocellular necrosis in this dose group. Additional statistically-identified serum clinical chemistry parameters which demonstrate treatment-related alterations to hepatic function in males and females given 300 mg/kg/day included: decreases in total protein, albumin, calcium, cholesterol, blood urea nitrogen, and bile acids. Decreases in calcium were interpreted to be secondary to decreased albumin. Treatment-related decreases in total protein, cholesterol, and blood urea nitrogen were also statistically identified in males and females given 100 mg/kg/day.

A few of the statistically identified clinical chemistry parameters were interpreted to be spurious and unrelated to treatment due to a lack of dose response relationship. These included: triglycerides (females given 100 mg/kg/day), and alkaline phosphatase (males and females given 100 mg/kg/day).

Urinalysis
Statistically-identified and/or treatment-related alterations in urinalysis parameters are summarized in Text Table 9. Males and females given 300 mg/kg/day had statistically-identified (sexes combined for statistical analysis) increased urine volume and decreased specific gravity. However, in males, urinalysis values were near or within historical control ranges and lacked a dose-response relationship; therefore, they were not considered treatment related. In females, these changes were interpreted to be treatment related because urine volume and specific gravity values were consistently altered in majority of the individuals within the dose group and the values were outside of the historical control range. These higher urine volume and lower specific gravity were interpreted to be due to increased urine output secondary to possible increased water intake (water consumption not quantified in this study).

Anatomic Pathology
Organ Weights
Treatment-related alterations in final body weights and organ weights are summarized in Text Table 10. Males and females (combined for statistical analysis) given 300 mg/kg/day had statistically-identified treatment-related decreases in final body weights. Although the quantitative decrease in final body weights of females given 300 mg/kg/day was minimal, it was interpreted to be treatment-related because the actual decrease was likely offset to some extent by treatment-related increases in the size of the cecum (cecum filled with watery contents diagnosed at necropsy - see Gross pathology section for more details). Decreased amounts of abdominal fat in the high-dose females noted at necropsy also supports this interpretation. Decreased final body weights in the high-dose rats correlated with histological observation of atrophy of adipose tissue observed in both sexes.

Organ weight decrements interpreted to be reflective of the decreased body weight/fat reserves and feed consumption in rats given 300 mg/kg/day include: liver (relative and absolute weights in males), heart (relative and absolute weights in males and females), brain (absolute weight in males and females), and thymus (relative and absolute weights in males and females) weights. Additionally, relative spleen weight increases in males given 300 mg/kg/day were interpreted to be reflective of the decreased body weight.

Absolute and relative spleen weights were statistically identified as increased in females given 300 mg/kg/day. Increased spleen weights were interepreted to be treatment related because they were higher than the historical control range. Moreover, the individual spleen weights of the high-dose females were consistently higher than those of the individual control females; however, there was no histopathological correlate for the increased spleen weight.

Relative adrenal weights were statistically identified as increased in males and females given 300 mg.kg/day. In females, increased adrenal weights were interpreted to be treatment-related and secondary to non-specific stress as weights were greater than the historical control weights, and correlated with histologically noted hypertrophy of the zona fasciculata. In males, statistically identified increases in adrenal weights were not considered treatment-related as there was no histological correlate and the increase was within the historical control range.

Absolute thymus weights were decreased in both males and females given 30 mg/kg/day, and were interpreted to be unrelated to treatment because they lacked a dose-response relationship, moreover, the mean thymic weight was disproportionally affected by one animal (#5478) within the dose group.

Gross Pathology
Treatment-related gross pathological effects are summarized in Text Table 11. Males and females given 100 or 300 mg/kg/day had a treatment-related increase in the size of the cecum, which was attributed to an increase in the amounts of watery luminal contents. The increase in cecal size also corresponded to the microscopic observation of slight to moderate epithelial hyperplasia (see Histopathology section). Enlarged mesenteric and cecal lymph nodes were observed in animals given 100 and 300 mg/kg/day; however, changes were more prevalent in the high-dose group. Histologically, increased mesenteric and cecal lymph node size was correlated to sinus histiocytosis present in lymph nodes at both dose levels. The absence of formed fecal pellets within the rectum was observed in both sexes at 300 mg/kg/day and one male given 100 mg/kg/day. This change was attributed to the presence of soft stool. Decreased body fat reserves were observed in four females given 300 mg/kg/day and one female given 100 mg/kg/day. This gross finding correlated with histological atrophy of mesenteric adipose tissue, present in high-dose females and males. All other gross pathological findings were interpreted to be spontaneous changes, unassociated with the administration of the test material.

Histopathology
Treatment-related histopathological effects are presented in Text Table 12 and 13 for males and females, respectively. Primary treatment-related histological effects occurred within the liver, lymph nodes (mesenteric and cecal), gastrointestinal tract, and mesenteric fat in animals given 100 or 300 mg/kg/day. Animals given 30 mg/kg/day did not demonstrate treatment-related histopathological changes.

Primary Treatment-related Histological Effects:
Liver: Males and females given 300 mg/kg/day had multifocal individual hepatocyte necrosis (very slight to moderate) with accompanying inflammation. Inflammation was predominantly histiocytic (granulomatous); occasional neutrophils and lymphocytes were admixed with cellular debris. In all affected animals, hepatic necrosis and accompanying inflammatory lesions were most prominent in the right lateral lobe. Background levels of multifocal aggregates of histiocytes were also increased from very slight to slight in males and females given 300 mg/kg/day. Very slight periportal hepatocyte vacuolization was present in females given 100 and 300 mg/kg/day. Vacuolization was consistent with fatty change (microvesicular) and most
prominent in the right lateral lobe.

Lymph nodes: Males and females given 100 or 300 mg/kg/day had increased numbers of histiocytes within the sinuses (sinus histiocytosis) of the mesenteric and cecal lymph nodes. Histiocytes contained abundant foamy cytoplasm and were more prevalent in high-dose animals.

Gastrointestinal tract: Males and females given 300 mg/kg/day had a very slight or slight, diffuse infiltration of mixed leukocytes within the lamina
propria of the cecum, colon, and rectum. Males and females given 300 mg/kg/day had epithelial hyperplasia (very slight to moderate) of the cecum;
similar changes were present in the colon of males given 300 mg/kg/day. Very slight diffuse atrophy of the nonglandular gastric mucosal epithelium was present in males given 300 mg/kg/day, characterized by thinning of the nonglandular mucosal epithelium with decreased density and separation of the keratin layer. While epithelial changes were diffuse, the mucosal epithelium lining the limiting ridge was within normal limits.

Histological Effects Secondary to Body Weight Effects:
Mesenteric fat: Males and females given 300 mg/kg/day had very slight or slight atrophy of the mesenteric adipose tissue, reflective of decreased body weight/body weight gain and feed consumption.

Prostate: Males given 300 mg/kg/day had decreased prostate secretory material (very slight or slight), reflective of decreased body weight/body weight gain and feed consumption.

Histological Effects Secondary to Stress:
Females given 300 mg/kg/day had very slight hypertrophy of the adrenal zona fasciculata, and diffuse very slight acinar cell hypertrophy of the submandibular salivary glands. Hypertrophy of the salivary gland acinar cells of rats and mice has been shown to be mediated through a stress-related adrenergic mechanism (Chan and Mahler, 1992 and Tada et al., 2008).

All other histopathological changes were interpreted to be spontaneous alterations unassociated with the exposure to the test material.

References
Chan, P. C. and Mahler, J. F. (1992). NTP Technical Report on Toxicity Studies of Glyphosate (CAS No. 1071-83-6) Administered in Dosed Feed to F344/N Rats and B6C3F1 Mice. Natl. Toxicol. Progr. Tech. Rep. Ser. 16, 1-39.

Latimer, K. S., Mahaffey, E. A., and Prasse, K. W. (2003) Duncan & Prasses’s Veterinary Laboratory Medicine: Clinical Pathology – 4th Edition. Iowa State Press, Iowa, pp. 3 -34.

Tada, Y., Yano, N., Takahashi, H., Yuzawa, K., Ando, H., Kubo, Y., Nagasawa, A., Uehara, S., Ogata, A., Nakae, D. (2008). Toxic Effects of L-aspartic Acid at High Dose Levels on Kidneys and Salivary Glands in Fischer 344 Rats Detected in a 90-Day Feeding Study. Food and Chemical Toxicology 46, 2789-2795.

Dose descriptor:

NOEL

Remarks:

systemic effects

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

Critical effects observed:

not specified

Text Table 1. Mean Body Weight (g) – Males – TD 4-28

Test Day	4	8	15	22	28
0 mg/kg/day	137.9	155.2	184.0	207.9	224.2
30 mg/kg/day	137.7	154.7	181.3	205.0	220.0
100 mg/kg/day	137.6	153.2	182.3	208.4	225.4
300 mg/kg/day	132.3	146.9	161.2	182.3	199.3

Bold type indicates the effects were interpreted to be treatment related.

Text Table 2. Mean Body Weight Gain (g) – Males – TD 4-28

Test Day	4	8	15	22	28
0 mg/kg/day	12.2	29.5	58.3	82.2	98.6
30 mg/kg/day	12.7	29.7	56.2	80.0	95.0
100 mg/kg/day	12.2	27.8	57.0	83.1	100.0
300 mg/kg/day	9.1	23.7	38.0	59.1	76.2

Bold type indicates the effects were interpreted to be treatment related.

Text Table 3. Mean Feed Consumption (g) – Males

Test Day	1 -4	4 -8	8 -15	15 -22	22 -28
0 mg/kg/day	10.5	11.4	12.6	12.8	12.7
30 mg/kg/day	10.4	11.0	11.8	12.2	12.7
100 mg/kg/day	9.1*	11.2	12.4	12.8	13.3
300 mg/kg/day	8.1*	9.9	10.4*	12.1	12.5

* Statistically significant from control mean by Dunnett’s Test, Alpha = 0.05.

Bold type indicates the effects were interpreted to be treatment related.

Text Table 4. Mean Feed Consumption (g) – Females

Test Day	1 -4	4-8	8 -15	15 -22	22 -28
0 mg/kg/day	8.4	9.3	9.2	9.3	9.1
30 mg/kg/day	8.6	9.4	9.1	9.3	9.2
100 mg/kg/day	8.1	9.0	9.3	9.5	9.6
300 mg/kg/day	7.3*	8.9	9.2	9.4	9.5

* Statistically significant from control mean by Dunnett’s Test, Alpha = 0.05.

Bold type indicates the effects were interpreted to be treatment related.

Text Table 5. Treatment-related Hematologic Differences

Sex	Males
Dose (mg/kg/day)	Historical Control^@	0	30	100	300
White Blood Cell Count (E3/ul)	5.44 - 7.28	6.51	7.17	7.37	8.89*
Hemoglobin Concentration (g/dl)	15.7 - 17.5	16.1	16.5	15.7*	15.2*
Hematocrit (%)	48.4 - 50.4	48.2	50.4	47.7	46.3*
Reticulocytes (E9/L)	146.1 - 190.3	165.1	180.1	200.5*	224.0*
Sex	Females
Dose (mg/kg/day)	Historical Control^@	0	30	100	300
White Blood Cell Count (E3/ul)	7.06 - 8.44	6.62	7.78	8.62	10.65*
Hemoglobin Concentration (g/dl)	16.0 - 17.0	16.3	15.9	15.7*	15.4*
Hematocrit (%)	47.8 - 48.8	48.0	47.4	46.5	45.5*
Reticulocytes (E9/L)	138.6 - 156.5	143.3	154.5	155.0*	184.1*

@Historical control data obtained from four 28-day gavage studies with PG as vehicle conducted in this laboratory in 2012.

* Statistically different from control mean, males and females analyzed together, by Dunnett’s test, alpha = 0.05.

Bold type indicates the effects were interpreted to be treatment related.

Text Table 6. Treatment-related Peripheral Blood Smear Effects

Sex	MalesAnimals per group affected
Dose (mg/kg/day)	0	30	100	300
Increased polychromasia Very Slight	1/5	0/5	3/5	5/5
Sex	Females
Dose (mg/kg/day)	0	30	100	300
Increased polychromasia Very Slight	0/5	0/5	0/5	4/5

Bold type indicates the effects were interpreted to be treatment related.

Text Table 7. White Blood Cell Differential Effects

Sex	Males
Dose (mg/kg/day)	Historical Control^@	0	30	100	300
Eosinophils (%)	0.7 - 0.9	1.0	0.8	0.8	0.5
Large unstained cells (%)	0.8 - 1.1	0.8	0.8	1.0	1.4
Sex	Females
Dose (mg/kg/day)	Historical Control^@	0	30	100	300
Eosinophils (%)	0.5 - 0.9	0.9	0.9	0.7	0.3
Large unstained cells (%)	0.9 - 1.3	0.9	1.2	1.3	2.3

@Historical control data obtained from four 28-day gavage studies conducted in this laboratory in 2012.

Bold type indicates the effects were interpreted to be treatment related.

Text Table 8. Clinical Chemistry Differences

Sex	Males
Dose (mg/kg/day)	Historical Control@	0	30	100	300
Alanine aminotransferase (u/l)	39 - 64	41	38	42	70*
Alkaline phosphatase (u/l)	157 - 175	158	148	170*	156
Aspartate aminotransferase (u/l)	84 - 101	82	81	90	106*
Total bile acids (umol/l)	4.51 - 17.18	3.70	2.71	3.36	2.29*
Total protein (g/dl)	6.6 - 6.8	6.7	6.6	6.5*	5.8*
Albumin (g/dl)	4.5 - 4.6	4.5	4.5	4.4	4.0*
Cholesterol (mg/dl)	48 - 62	59	58	54*	40*
Triglycerides (mg/dl)	42 - 75	67	64	43*	52
Urea Nitrogen (mg/dl)	18 - 23	19	19	16*	14*
Calcium (mg/dl)	12.6 - 13.1	12.6	12.5	12.7	12.0*
Sex	Females
Dose (mg/kg/day)	Historical Control^@	0	30	100	300
Alanine aminotransferase (u/l)	31 - 57	32	30	34	58*
Alkaline phosphatase (u/l)	133 - 140	123	130	137*	133
Aspartate aminotransferase (u/l)	77 - 112	82	81	90	110*
Total bile acids (umol/l)	8.66 - 16.32	8.87	7.47	6.21	4.64*
Total protein (g/dl)	6.3 - 6.6	6.5	6.3	6.0*	5.7
Albumin (g/dl)	4.5 - 4.6	4.6	4.4	4.3	4.0*
Cholesterol (mg/dl)	69 - 74	70	65	62*	45*
Triglycerides (mg/dl)	48 - 62	41	42	37*	44
Urea Nitrogen (mg/dl)	15 - 18	19	17	17*	14*
Calcium (mg/dl)	12.6 - 13.1	12.6	12.4	12.2	12.0*

@Historical control data obtained from four 28-day gavage studies conducted in this laboratory in 2012.

* Statistically different from control mean, males and females analyzed together, by Dunnett’s test, alpha = 0.05.

Bold type indicates the effects were interpreted to be treatment related.

Text Table 9. Urinalysis Differences

Sex	Males
Dose (mg/kg/day)	Historical Control^@	0	30	100	300
Urine Volume (ml)	2.4 - 5.4	5.2	4.3	5.1	5.3*
Specific Gravity	1.048 - 1.074	1.052	1.053	1.048	1.047*
Sex	Females
Dose (mg/kg/day)	Historical Control^@	0	30	100	300
Urine Volume (ml)	1.7 - 2.5	2.8	3.3	3.6	6.3*
Specific Gravity	1.069 - 1.085	1.062	1.059	1.057	1.035*

@Historical control data obtained from four 28-day gavage studies conducted in this laboratory in 2012.

* Statistically different from control mean, males and females analyzed together, by Dunnett’s test, alpha = 0.05.

Bold type indicates the effects were interpreted to be treatment related.

Text Table 10. Final Body Weight and Organ Weight Differences

Sex	Males
Dose (mg/kg/day)	Historical Control^@	0	30	100	300
Final Body wt (g)	198.5 - 216.4	207.0	201.7	208.3	185.8*
Adrenal (g/100)	0.018 - 0.024	0.019	0.020	0.018	0.022*
Liver (g)	5.876 - 6.999	6.501	6.353	6.256	5.113^a
Liver (g/100)	2.957 - 3.228	3.139	3.141	3.000	2.752^a
Spleen (g/100)	0.222 - 0.235	0.225	0.227	0.225	0.241*
Heart (g)	0.646 - 0.737	0.668	0.631	0.644	0.548*
Heart (g/100)	0.305 - 0.341	0.322	0.313	0.309	0.294*
Brain (g)	1.757 - 1.802	1.782	1.738	1.769	1.668*
Thymus (g)	0.317 - 0.342	0.331	0.288*	0.320	0.258*
Thymus (g/100)	0.149 - 0.164	0.160	0.144	0.154	0.139*
Sex	Females
Dose (mg/kg/day)	Historical Control^@	0	30	100	300
Final Body wt (g)	129.6 - 140.3	133.7	133.2	136.3	132.2*
Adrenals (g/100)	0.031 - 0.034	0.031	0.033	0.032	0.039*
Spleen (g)	0.354 - 0.376	0.357	0.361	0.371	0.385^b
Spleen (g/100)	0.268 - 0.277	0.267	0.271	0.272	0.292*
Heart (g)	0.472 - 0.522	0.498	0.476	0.489	0.425*
Heart (g/100)	0.336 - 0.403	0.372	0.358	0.359	0.322*
Brain (g)	1.615 - 1.666	1.667	1.648	1.681	1.640*
Thymus (g)	0.300 - 0.309	0.293	0.282*	0.302	0.241*
Thymus (g/100)	0.219 - 0.237	0.220	0.212	0.222	0.182*

@Historical control data obtained from four 28-day gavage studies conducted in this laboratory in 2012.

* Statistically different from control mean, males and females analyzed together, by Dunnett’s test, alpha = 0.05.

^aStatistically different from control mean, males analyzed separately, by Dunnett’s test, alpha = 0.05.

^bStatistically different from control mean, females analyzed separately, by Dunnett’s test, alpha = 0.05.

Bolded values interpreted to be treatment related.

Italics values interpreted to be secondary to the body weight decrements.

Text Table 11. Gross Pathological Effects

Sex	Males
Dose (mg/kg/day)	0	30	100	300
Cecum, increased size	0	0	5	5
Cecal lymph node, increased size	0	0	1	3
Mesenteric lymph node, increased size	0	0	1	3
Rectum, absence of formed fecal pellets	0	0	1	2
Sex	Females
Dose (mg/kg/day)	0	30	100	300
Cecum increased size	0	0	1	5
Cecal lymph nodes, increased size	0	0	0	4
Decreased abdominal fat	0	0	1	4
Mesenteric lymph node, increased size	0	0	0	3
Rectum, absence of formed fecal pellets	0	0	0	2

Bolded values interpreted to be treatment related.

Italics values interpreted to be secondary to the body weight decrements.

Text Table 12. Treatment-related Histopathological Effects - Males

Sex	Males
Dose (mg/kg/day)	0	30	100	300
Liver; Aggregates of macrophages-histiocytes; multifocal; Very Slight	5	5	5	1
Slight	0	0	0	4
Liver; Individual cells; necrosis; hepatocyte; with accompanying inflammation; multifocal; Very Slight	0	0	0	2
Slight	0	0	0	1
Moderate	0	0	0	2
Lymph Nodes; Cecal-Sinus histiocytosis Very Slight	1	0	0	1
Slight	0	0	2	1
Moderate	0	0	0	2
Lymph Nodes; Mesenteric-Sinus histiocytosis; Very Slight	0	0	4	0
Slight	0	0	1	4
Moderate	0	0	0	1
Gastrointestinal Tract; Cecum-epithelium; hyperplasia; diffuse; Very Slight	1	1	1	2
Slight	0	0	0	1
Gastrointestinal Tract; Cecum-lamina propria; infiltration; mixed leukocytes; diffuse Very Slight	1	0	0	2
Gastrointestinal Tract; Colon-epithelium; hyperplasia; diffuse Very Slight	0	0	0	1
Slight	0	0	0	2
Gastrointestinal Tract; Colon-lamina propria infiltration; mixed leukocytes; diffuse Very Slight	0	0	0	2
Gastrointestinal Tract; Rectum-lamina propria; infiltration; mixed leukocytes; diffuse; Very Slight	0	0	0	2
Gastrointestinal Tract; Stomach-mucosa; atrophy; nonglandular; diffuse; Very Slight	0	0	0	5
Mesenteric Tissue; Adipose tissue; atrophy; diffuse; Very Slight	0	0	0	3
Slight	0	0	0	3
Prostate: Decreased secretory material; Very Slight	0	0	0	1
Slight	0	0	0	2

Bolded values interpreted to be treatment related.

Italics values interpreted to be secondary to the body weight decrements.

Text Table 13. Treatment-related Histopathological Effects - Females

Sex	Females
Dose (mg/kg/day)	0	30	100	300
Liver; Aggregates of macrophages-histiocytes; multifocal; Very Slight	4	4	5	0
Slight	1	1	0	5
Liver; Periportal; vacuolization; hepatocyte; consistant with fatty change; Very Slight	0	0	4	5
Liver; Individual cells; necrosis; hepatocyte; with accompanying inflammation; multifocal; Very Slight	0	0	0	2
Slight	0	0	0	3
Lymph nodes; Cecal-Sinus histiocytosis; Very Slight	0	0	0	1
Slight	0	0	0	2
Moderate	0	0	0	3
Lymph Nodes; Mesenteric-Sinus histiocytosis; Very Slight	0	0	5	1
Moderate	0	0	0	4
Gastrointestinal Tract; Cecum-epithelium; hyperplasia; diffuse; Very Slight	0	0	0	2
Slight	0	0	0	2
Moderate	0	0	0	1
Gastrointestinal Tract; Cecum-lamina propria; infiltration; mixed leukocytes; diffuse; Very Slight	1	0	0	2
Slight	0	0	0	1
Gastrointestinal Tract; Colon-lamina propria; infiltration; mixed leukocytes; diffuse; Very Slight	0	0	0	3
Gastrointestinal Tract; Rectum-lamina propria; infiltration; mixed leukocytes; diffuse; Very Slight	0	0	0	1
Mesenteric Tissue; Adipose tissue; atrophy; diffuse; Very Slight	0	0	0	1
Slight	0	0	0	4
Adrenal glands; Zona fasciculate; hypertrophy; cortex; diffuse; Very Slight	0	0	0	5
Salivary Gland; Acinus; hypertrophy; difuse; Very Slight	0	0	0	4

Bolded values interpreted to be treatment related.

Italics values interpreted to be secondary to the body weight decrements.

Conclusions:

Under the conditions of this study, the no-observed-effect level (NOEL) was 30 mg/kg/day BADGE IPD (#33) in male and female F344/DuCrl rats.

Executive summary:

Five male and five female F344/DuCrl rats per group were administered 0, 30, 100, or 300 milligrams BADGE IPD (#33) per kilogram body weight per day (mg/kg/day, mkd) in propylene glycol (PG) via oral gavage for 28 days to evaluate the potential for toxicity. Parameters evaluated were daily cage-side and clinical observations, weekly detailed clinical observations, ophthalmic examinations, body weights, feed consumption, hematology, prothrombin time, urinalysis, clinical chemistry, selected organ weights, and gross and histopathologic examinations.

Treatment-related effects on cage-side, clinical, and detailed clinical observations were limited to two males and one female given 300 mg/kg/day with soft feces and/or fecal soiling. There were no treatment-related effects in ophthalmic observations and prothrombin time at any dose level.

Males given 300 mg/kg/day had treatment-related decreases in body weight and body weight gains that were 11.1 and 22.7% less than controls, respectively by test day (TD) 28. Treatment-related decreases in feed consumption were observed in 300 mg/kg/day males from TD 1-15. Females given 300 mg/kg/day and males given 100 mg/kg/day had treatment-related decreases in feed consumption from TD 1-4.

Males and females given 100 or 300 mg/kg/day had treatment-related alterations in hematology parameters including: decreased hemoglobin (100 and 300 mg/kg/day), decreased hematocrit (300 mg/kg/day), increased white blood cell counts (300 mg/kg/day), increased reticulocytes (100 and 300 mg/kg/day), and a very slight increase in polychromasia of red blood cells (males given 100 mg/kg/day, and both sexes given 300 mg/kg/day).

Males and females given 100 or 300 mg/kg/day had treatment-related alterations in serum chemistry analytes including: increased alanine aminotransferase (300 mg/kg/day) and aspartate aminotransferase (300 mg/kg/day) activities and decreases in total protein (100 and 300 mg/kg/day), albumin (300 mg/kg/day), cholesterol (100 and 300 mg/kg/day), blood urea nitrogen (100 and 300 mg/kg/day), and bile acids (300 mg/kg/day). Females given 300 mg/kg/day had a treatment-related increase in urine volume with decreased specific gravity. At necropsy, males and females given 300 mg/kg/day had treatment-related decreases in final (fasting) body weights and multiple organ weights, which were secondary to body weight decrements. Organs affected included: liver (relative and absolute weights in males), spleen (relative weight in males), heart (relative and absolute weights in males and females), brain (absolute weight in males and females), and thymus (relative and absolute weights in males and females) weights. Female rats given 300 mg/kg/day had treatment-related increased spleen and adrenal weights (relative and absolute weights). Males and females given 100 or 300 mg/kg/day had treatment-related gross-pathological alterations including: increase in the size of the cecum and cecal lymph nodes, the absence of formed fecal pellets within the rectum, and decreased body fat reserves (females only) which were more prevalent in the high-dose group. Males and females given 100 or 300 mg/kg/day had treatment-related histological changes of the liver and lymph nodes (cecal and mesenteric), which were more prevalent in the high-dose group. In the liver, males and females given 300 mg/kg/day had multifocal individual hepatocyte necrosis (very slight to moderate) with accompanying inflammation. Background levels of multifocal aggregates of histiocytes were increased in severity from very slight to slight in both sexes. Females given 100 or 300 mg/kg/day had very slight periportal hepatocyte vacuolization, consistent with fatty change. In the mesenteric and cecal lymph nodes, males and females given 100 or 300 mg/kg/day had treatment-related very slight to moderate sinus histiocytosis. Males and females given 300 mg/kg/day had treatment-related very slight to moderate epithelial hyperplasia of the cecum and colon (males only), as well as a very slight to slight, diffuse infiltration of mixed leukocytes within the lamina propria of the cecum, colon, and rectum. Males and females given 300 mg/kg/day also had treatment-related histological alterations secondary to body weight changes, including: atrophy of adipose tissue and decreases in the amounts of secretory material in the prostate. Females given 300 mg/kg/day had treatment-related histopathological changes secondary to stress, including: hypertrophy of the adrenal zona fasciculata cells, and acinar cell hypertrophy of the submandibular salivary glands. Under the conditions of this study, the no-observed-effect level (NOEL) was 30 mg/kg/day BADGE IPD (#33) in male and female F344/DuCrl rats.

Cross-reference 3

Reason / purpose for cross-reference:: data waiving: supporting information

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 August 2016 - 01 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 22 January 2001

Deviations:

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

30 May 2008

Deviations:

GLP compliance:

yes (incl. QA statement)

Limit test:

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Sprague-Dawley Crl:CD (SD) IGS BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Weight at study initiation: 189 to 285 g
- Fasting period before study: no
- Housing: individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes
- Diet (e.g. ad libitum): pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK), ad libitum
- Water (e.g. ad libitum): mains drinking water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 26 August 2016 (first day of treatment) to 15 September 2016 (final day of necropsy)

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The stability and homogeneity of the test item formulations were previously determined by Envigo Research Limited, Shardlow, UK Analytical Services (Envigo Study Number YY43XT). Results showed the formulations to be stable for at least twenty-two days. Formulations were therefore prepared once and stored at approximately +4 °C in the dark and under nitrogen.

VEHICLE
- Concentration in vehicle: 4.17, 16.7, 41.7 mg/mL
- Amount of vehicle (if gavage): 6 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken of the test item formulations and were analyzed for concentration of BADGE-IPD at Envigo Analytical Laboratory, Shardlow. The results indicate that the prepared formulations were within ± 3% of the nominal concentration.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant (Animals were delivered in two batches containing females prior to Day 3 of gestation.)
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

Day 3 to Day 19 of gestation

Frequency of treatment:

daily

Duration of test:

18 d (aminals were sacrificed on day 20 of gestation)

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

24 (control, low and mid dose) / 26 (high dose)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen based on previous toxicity data (Envigo Research Limited, Study Number PM78LM).
- Rationale for animal assignment (if not random): randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period; during the dosing period, observations were recorded immediately before dosing, up to thirty minutes after dosing and one hour after dosing

BODY WEIGHT: Yes
- Time schedule for examinations: Day 3 (before the start of treatment) and on Days 4, 5, 8, 11, 14 and 17 of gestation. Body weights were also recorded for surviving animals at terminal kill (Day 20).

FOOD CONSUMPTION: Yes
Food consumption was recorded for each surviving individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: ovaries and uteri

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- other: placental weight

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: all per litter

Statistics:

The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:
Female body weight change, food consumption and gravid uterus weight: Shapiro Wilk normality test and Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnett’s multiple comparison test or, if unequal variances were observed, on alternative multiple comparison test.
All caesarean necropsy parameters and fetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.
Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis non-parametric analysis of variance and Mann-Whitney ‘U’ test.

Historical control data:

attached as .pdf file

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Noisy respiration and increased salivation were evident in the majority of females treated with 250 mg/kg bw/day throughout the treatment period. One of these females also showed decreased respiratory rate whilst another two females were observed with pilo erection on Day 17 of gestation. Of the females that were sacrificed in extremis at this level, three showed increased salivation and all of them showed respiratory pattern changes (noisy respiration, gasping/labored respiration and/or decreased respiratory rate). In addition, the female that was sacrificed in extremis on Day 10 had hunched posture and lethargy and the female that was sacrificed in extremis on Day 19 had hunched posture, pilo-erection, dehydration, red/brown stained snout and tiptoe gait.
Instances of noisy respiration were evident in eight females treated with 100 mg/kg bw/day on Days 5-10, 12, 13 and/or 16-20 and two females treated with 100 mg/kg bw/day had increased salivation on Days 4, 6 and/or 10. The female found dead on Day 16 of gestation showed no clinical signs before death.
At 25 mg/kg bw/day, three females had noisy respiration on Days 15 or 17 and one female had increased salivation on Day 15 only.

Mortality:

mortality observed, treatment-related

Description (incidence):

Four females treated with 250 mg/kg bw/day were sacrificed in extremis on Days 10, 15 and 19 of gestation due to adverse clinical signs and in three of the females, excessive body weight losses were also noted.
One female treated with 100 mg/kg bw/day was found dead on Day 16 of gestation. Macroscopic examinations of this female revealed a hole in the esophagus, therefore this death was considered to be the result of a dosing trauma rather than a direct effect of the test item.
There were no further unscheduled deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain for females treated with 250 mg/kg bw/day between Days 3 and 8 of gestation was generally comparable to controls. Cumulative body weight gain from Day 11 onwards was lower and statistical significance (p<0.05) was achieved between Days 3 and 11, 3 and 14 and 3 and 17. Incidences of actual body weight losses were also evident in surviving females, sporadically throughout the treatment period. Three out of the four females that were sacrificed in extremis showed significant body weight loss prior to termination. Body weight gain for surviving females when adjusted for gravid uterus weight was also statistically significantly (p<0.01) reduced when compared to controls.
Body weight gain during gestation, including after adjustment for the contribution of the gravid uterus, was considered to be unaffected by treatment at 100 or 25 mg/kg bw/day.
Females treated with 25 mg/kg bw/day showed a statistically significant (p<0.05) increase in body weight gain between Days 5 and 8 of gestation. An increase in body weight gain is considered not to represent an adverse effect of treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Females treated with 250 mg/kg bw/day showed a statistically significant reduction (p<0.05-0.001) in food consumption between Days 3 and 17 of gestation. Food consumption between Days 17 and 20 was also lower, although statistical significance was not achieved.
No differences as compared to the control group were detected on food consumption in females treated with 100 or 25 mg/kg bw/day.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

Daily visual inspection of water bottles did not reveal any overt intergroup differences.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Three of the females treated with 250 mg/kg bw/day that were sacrificed in extremis had gaseous distension in the stomach and/or gastro-intestinal tract. One of the females treated with 250 mg/kg bw/day that was sacrificed in extremis on Day 15 also had a dark liver and dark patches on all lobes of the lungs.
The female treated with 100 mg/kg bw/day that was found dead on Day 16 of gestation had gaseous distension in the stomach and gastro-intestinal tract, a dark liver, dark red patches on the lungs and a hole in the esophagus (approximately 3mm x 3mm). Due to the observations evident in the esophagus, this death was most likely to be the result of a dosing trauma rather than a direct effect of the test item.
No toxicologically significant macroscopic abnormalities were detected in the surviving females treated with 250 mg/kg bw/day or in females treated with 100 or 25 mg/kg bw/day.
One female treated with 100 mg/kg bw/day had a small left adrenal and an enlarged right adrenal. A further female from this treatment group had patchy fur loss on both hindlimbs. One female treated with 25 mg/kg bw/day had gaseous distension in the duodenum and a control female had pale adrenals. These observations were considered to be low incidental findings and of no toxicological significance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

There was no obvious effect of maternal treatment on litter data as assessed by numbers of implantations, in-utero offspring survival (as assessed by the mean numbers of early or late resorptions), live litter size, sex ratio or pre- or post-implantation losses at 25, 100 or 250 mg/kg bw/day.
Intergroup differences for mean fetal, litter or placental weights did not indicate any obvious effects of maternal treatment at 25, 100 or 250 mg/kg bw/day.
Statistical analysis of the data did not reveal any significant intergroup differences.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Basis for effect level:

clinical signs

gross pathology

mortality

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant reduction (p<0.01) in the number of fetuses/litters showing unossified areas of the occipital (supra-occipital) was evident at 100 mg/kg bw/day. The group mean value was within historical control range and the observation of one variant at a lower incidence compared with controls is not significant when evaluated in isolation. In the absence of a similar effect at the high dose group or any particular pattern of abnormal skeletal development of skeletal structures affecting treated fetuses, the observation of one affected skeletal structure can be considered unlikely to represent true developmental abnormality. This takes account of this finding being seen regularly on this study type amongst control group fetuses.

Visceral malformations:

no effects observed

Description (incidence and severity):

Neither the type, incidence nor the distribution of findings observed during external examination of the fetuses at necropsy on gestation Day 20 and subsequent detailed visceral and skeletal examination indicated any adverse effect of maternal exposure on fetal development.
Statistical analysis did not reveal any significant intergroup differences.

Dose descriptor:

NOEL

Effect level:

>= 250 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no effects observed

Abnormalities:

no effects observed

Developmental effects observed:

results tables are attached as .pdf file below

Conclusions:

Treatment at 250 mg/kg bw/day was associated with increased salivation and respiratory pattern changes in the majority of females, the early termination of four females on Days 10, 15 and 19 and treatment-related effects on body weight performance and food consumption in the remaining females. The resulting lower overall body weight gain in surviving females remained lower than controls even when adjusted for the contribution of the gravid uterus.
No macroscopic abnormalities were detected in the surviving females of the high dose group however three of the females from the high dose group that were sacrificed in extremis had gaseous distension in the stomach and/or gastrointestinal tract. One of the females that was sacrificed in extremis on Day 15 also had a dark liver and dark patches on all lobes of the lungs. Despite the observed maternal effects, there were no obvious effects of maternal treatment on the survival, growth or morphological development of the offspring and therefore this dosage is considered to represent a No Observed Effect Level (NOEL) for the developing conceptus.
At 100 and 25 mg/kg bw/day, treatment was associated with instances of noisy respiration and increased salivation (at a lesser extent than at 250 mg/kg bw/day). There were no obvious effects on body weight or food intake during gestation and no treatment-related macroscopic necropsy findings were apparent for adult animals at these dosage. Given the transient nature of the clinical signs evident, 100 mg/kg bw/day is considered to represent a ‘No Observed Adverse Effect Level’ (NOAEL) for the pregnant female.

Executive summary:

The study was performed to investigate the effects of the test item BADGE-IPD on embryonic and fetal development following repeated administration by gavage to the pregnant female during gestation including the period of organogenesis.

The study was designed to comply with the following guidelines:

· US EPA Health Effects Test Guideline OPPTS 870.3700, ‘Prenatal Developmental Toxicity Study’ (August 1998)

· Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, (24 November 2000)

· OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001)

· Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Methods

The test item was administered by gavage, between Days 3 and 19 of gestation, to three groups at dose levels of 25, 100, and 250 mg/kg bw/day. The low and intermediate dose groups contained twenty-four time mated Sprague-Dawley Crl:CD^®(SD) IGS BR strain rats, and the high dose group contained twenty-six time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats. A further group of twenty-four time mated females was exposed to the vehicle only (Propylene Glycol) to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the study.

All surviving females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weights, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

Results…….

Mortality

One female treated with 100 mg/kg bw/day was found dead on Day 16 of gestation. Macroscopic examinations of this female revealed a hole in the esophagus, therefore this death was considered to be the result of a dosing trauma rather than a direct effect of the test item.

There were no further unscheduled deaths.

Clinical Observations

Noisy respiration and increased salivation were evident in the majority of females treated with 250 mg/kg bw/day throughout the treatment period. In addition to increased salivation and respiratory pattern changes, one of the females that were sacrificedin extremisalso showed hunched posture and lethargy and another one of these females showed dehydration, hunched posture, pilo-erection, tiptoe gait and a red/brown stained snout.

Instances of noisy respiration and increased salivation were also evident in some females treated with 100 and 25 mg/kg bw/day albeit to a lesser extent.

Body Weight

Body weight gain for females treated with 250 mg/kg bw/day between Days 3 and 8 of gestation was generally comparable to controls. However cumulative body weight gain from Day 11 onwards was lower and incidences of actual body weight losses were evident in surviving females throughout the treatment period. Three out of the four females that were sacrificedin extremisshowed significant body weight loss prior to termination. Body weight gain for surviving females when adjusted for gravid uterus weight was also reduced when compared to controls.

No such effects were evident in females treated with 100 or 25 mg/kg bw/day.

Food Consumption

Females treated with 250 mg/kg bw/day showed a reduction in food consumption throughout the treatment period.

No differences as compared to the control group were detected on food consumption in females treated with 100 or 25 mg/kg bw/day.

Water Consumption

Daily visual inspection of water bottles did not reveal any overt intergroup differences.

Post Mortem Studies

The female treated with 100 mg/kg bw/day that was found dead had gaseous distension in the stomach and gastro-intestinal tract, a dark liver, dark red patches on the lungs and a hole in the esophagus (approximately 3mm x 3mm). Due to the observations evident in the esophagus, this death was most likely to be the result of a dosing trauma rather than a direct effect of the test item.

No toxicologically significant macroscopic abnormalities were detected in the surviving females treated with 250 mg/kg bw/day or in females treated with 100 or 25 mg/kg bw/day.

Litter Data and Litter Placental and Fetal Weights

The number of implantations, subsequent embryofetal survival, live litter size and sex ratio on Day 20 of gestation were considered to be unaffected by maternal treatment at 25, 100 or 250 mg/kg bw/day. Mean fetal, placental and litter weights were also considered to have been unaffected by maternal treatment at 25, 100 or 250 mg/kg bw/day.

Fetal Examination

External examination of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 25, 100 or 250 mg/kg bw/day. Findings at detailed skeletal and visceral examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 25, 100 or 250 mg/kg bw/day.

Conclusion

The oral (gavage) administration of ,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with 3-aminomethyl-3,5,5-trimethylcyclohexylamine; called BADGE-IPD; CASRN 38294-64-3 to pregnant rats during gestation at dose levels of 25, 100 and 250 mg/kg bw/day, resulted in treatment related effects at 250 mg/kg bw/day. A reduction in cumulative body weight gain and food consumption was evident at this level together with adverse clinical signs detected and the early sacrifice of four females (Days 10, 15 and 19 of gestation).

Although one female treated with 100 mg/kg bw/day was found dead on Day 16, macroscopic observations revealed a hole in the esophagus, therefore this death was most likely to be the result of a dosing trauma rather than a direct effect of the test item. No adverse effects were evident in body weight gain or food consumption at 100 mg/kg bw/day, therefore 100 mg/kg bw/day was considered to represent the ‘No Observed Adverse Effect Level’ (NOAEL) for the pregnant female.

No treatment-related changes were detected in the offspring parameters measured or on embryofetal development. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 250 mg/kg bw/day.

Data source

Materials and methods

Results and discussion

Overall reproductive toxicity

Reproductive effects observed:: not specified

Applicant's summary and conclusion

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Registration Dossier

Registration Dossier

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Diss Factsheets

4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with 3-aminomethyl-3,5,5-trimethylcyclohexylamine

Toxicity to reproduction

Administrative data

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Data source

Materials and methods

Results and discussion

Overall reproductive toxicity

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