4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with m-phenylenebis(methylamine)

Endpoint summary

Description of key information

90 d NOAEL = 10 mg/kg bw/d (rat, oral: gavage; 0, 10, 100, 300 mg/kg bw/day): death of one female treated with 300 mg/kg bw/day, early termination of two females and one male treated with 300 mg/kg bw/day, clinical signs of toxicity, reduced body weight development and food consumption (300 mg/kg bw/day males only), changes in hematology parameters (300 mg/kg bw/day males only) and microscopic changes in the mesenteric lymph nodes. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 April 2016 to 03 November 2016

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:

no

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:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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: 204 to 255 g (males), 152 to 195 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: 6 d

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

IN-LIFE DATES: 11 May 2016 (first day of treatment) to 07 September 2016 (final day of necropsy)

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

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

VEHICLE
- Concentration in vehicle: 1.67, 16.67, 50 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:

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-one days.
Samples of test item formulations were taken on six occasions and analyzed for concentration. The results indicate that the prepared formulations were within ± 10% of the nominal concentration.

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.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on previous dose range finding study (Envigo Research Limited Study Number QB13VN; 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).
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Incidences of increased salivation were evident in all animals of both sexes treated with 300 mg/kg bw/day from Day 5 (males) and Day 8 (females) until the termination of treatment. Episodes of noisy respiration were also evident in all animals of both sexes treated with 300 mg/kg bw/day from Day 1 (males) and Day 8 (females) onwards. Incidences of labored respiration, decreased respiratory rate, hunched posture and lethargy were also evident in some males treated with 300 mg/kg bw/day and a decreased respiratory rate was evident in one female from this treatment group. Throughout the treatment-free, twenty-eight day period, four males and four females that were previously given 300 mg/kg bw/day continued to show episodes of noisy respiration. The females that were sacrificed in extremis also showed labored respiration, decreased respiratory rate, pilo-erection and hunched posture and the female that was terminated on Day 27 also had gasping respiration and a distended abdomen. The male that was sacrificed in extremis also showed gasping 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 300 mg/kg bw/day. One male from this treatment group also showed hunched posture on Day 10 only.
No such effects were detected in males treated with 10 mg/kg bw/day however one female from this treatment group had noisy respiration on Day 26 only.
One control female had generalized fur loss between Days 81 and 91. This was considered to be incidental.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two females and one male treated with 300 mg/kg bw/day were sacrificed in extremis on Days 8, 27 and 85. A further female treated with 300 mg/kg bw/day was found dead on Day 82. There were no further unscheduled deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males treated with 300 mg/kg bw/day showed lower body weight gain throughout the treatment period (excluding Weeks 3 and 10). Statistical significance (p<0.05-0.01) was achieved during Weeks 1, 2, 4, 5 and 13. Consequently overall body weight gain was lower than controls. Significant improvement in body weight gain was evident in males previous treated with 300 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 toxicologically significant effects were detected in treated females or in males treated with 100 or 10 mg/kg bw/day.
A statistically significant increase (p<0.05) in body weight gain during Week 10 and a statistically significant reduction (p<0.05) in body weight gain during Week 13 was evident in males treated with 100 and 10 mg/kg bw/day. A true dose related response was not evident and no adverse effect on overall body weight gain was evident in these males. Therefore the intergroup differences were considered not to be of toxicological importance. Females treated with 300 mg/kg bw/day showed a statistically significant reduction (p<0.05) in body weight gain during Week 6. No adverse effect on overall body weight gain was evident in these females. Therefore, in isolation, the intergroup difference was considered not to be of toxicological significance.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males treated with 300 mg/kg bw/day showed a reduction in food consumption throughout the treatment period. Females from this treatment group showed a reduction in food consumption from Week 10 onwards. Incidences of reduced food conversion efficiency were also evident in these animals 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 300 mg/kg bw/day.
No such effects were detected in animals of either sex treated with 100 or 10 mg/kg bw/day.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Incidences of reduced food conversion efficiency were evident in high dose animals 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 300 mg/kg bw/day.

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):

Opthalmoscopic examination of animals of both sexes from the non-recovery control and 300 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):

Males treated with 300 and 100 mg/kg bw/day showed a statistically significant increase (p<0.05-0.01) in neutrophils and a statistically significant reduction (p<0.05) in lymphocytes. A statistically significant increase (p<0.05) in neutrophils was also present in males previously treated with 300 mg/kg bw/day at the end of the twenty-eight day treatment-free period. The majority of individual values for both parameters at 300 mg/kg bw/day were outside of historical control ranges and some individual neutrophil values at 100 mg/kg bw/day and in the recovery animals were also outside of the historical control range. All individual lymphocyte values at 100 mg/kg bw/day were within historical control range.
No toxicologically significant effects were detected in treated females or in males treated with 10 mg/kg bw/day.
Males treated with 300 mg/kg bw/day showed statistically significant increases (p<0.05-0.01) in hemoglobin, hematocrit and mean corpuscular volume. Recovery males that were previously given 300 mg/kg bw/day also showed a statistically significant increase (p<0.01) in hematocrit at the end of the treatment-free period. The majority of individual values for all parameters were within historical control ranges and in the absence of any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.
Females treated with 100 mg/kg bw/day showed a statistically significant increase (p<0.05) in monocytes. The majority of individual values were within historical control range and in the absence of a similar effect in 300 mg/kg bw/day females or any associated histopathological correlates the intergroup difference was considered not to be of toxicological significance.
Following the treatment-free period, males that were previously given 300 mg/kg bw/day showed a statistically significant increase (p<0.05) in erythrocyte count. The majority of individual values were within historical control range and in the absence of a similar effect in 300 mg/kg bw/day males 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 300 and 100 mg/kg bw/day showed a statistically significant reduction (p<0.05-0.01) in bile acids. Males treated with 300 mg/kg bw/day and recovery males that were previously given 300 mg/kg bw/day showed a statistically significant reduction (P<0.05-0.01) in triglycerides. Females treated with 300 mg/kg bw/day showed statistically significant increases (p<0.05-0.01) in urea, total protein, albumin, albumin/globulin ratio, creatinine and bilirubin and statistically significant reductions (p<0.01) in cholesterol and chloride concentration. The effect on urea, total protein, albumin and chloride concentration also extended to females treated with 100 mg/kg bw/day with females from the 10 mg/kg bw/day dose group also showing a statistically significant increase in albumin. The majority of individual values for these parameters for both sexes were within historical control ranges and although 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 300 and 100 mg/kg bw/day showed a statistically significant increase (p<0.05-0.01) in alanine aminotransferase and males treated with 300 mg/kg bw/day also showed a statistically significant increase (p<0.01) in aspartate aminotransferase. These intergroup differences may again indicate minor perturbation 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.
Females from all treatment groups showed a statistically significant increase (p<0.05-0.01) in calcium concentration. Individual values for all treated females were actually below the historical control range and all individual values for control females were also below the historical control range which may have contributed to these differences. Therefore the intergroup differences were considered to reflect biological variation rather than a true effect of treatment.
Following the treatment-free period, males that were previously given 300 mg/kg bw/day showed a statistically significant reduction (p<0.05) in total protein whilst females that were previously given 300 mg/kg bw/day showed a statistically significant reduction (p<0.05) in phosphorus. The majority of individual values were within historical control ranges and in the absence of a similar effect in 300 mg/kg bw/day animals 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 treatment-related changes in functional performance. Statistical analysis of the data did not reveal any significant intergroup differences.

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 treated with 300 mg/kg bw/day showed a statistically significant reduction (p<0.05) in spleen weight both absolute and relative to terminal body weight. The majority of individual values for absolute weights were within the historical control range; however, the majority of individual values for relative weights were outside of the historical control range. No associated microscopic spleen changes were evident in these animals, therefore, the intergroup difference was considered not to be of toxicological significance. Males treated with 100 mg/kg bw/day showed a statistically significant reduction (p<0.01) in absolute and relative liver weight and a statistically significant increase (p<0.05) in absolute and relative heart weight. Females treated with 10 mg/kg bw/day showed a statistically significant increase (p<0.05) in heart weight both absolute and relative to terminal body weight. The majority of individual values for all parameters were within historical control ranges and in the absence of similar effects in 300 mg/kg bw/day animals 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 300 mg/kg bw/day that was sacrificed in extremis on Day 85 had gaseous distension in the stomach and ileum, pale and enlarged lungs and a reddened glandular region in the stomach. This animal had tracheal ulceration and bronchopneumonia, indicative of test item irritancy through reflux, and this is considered to be the cause of death.
The female treated with 300 mg/kg bw/day that was sacrificed in extremis on Day 8 had a dark liver and reddened lungs with clear fluid present. This female had edema and inflammation in the lungs and ulceration of the trachea, also indicative of test item irritancy through reflux, and this is considered to be the cause of death.
The female treated with 300 mg/kg bw/day that was sacrificed in extremis on Day 27 had gaseous distention in the intestines and reddened and enlarged lungs. This female had hemorrhage in the lungs as well as minimal inflammatory change, possibly through technical error and this is considered to be the cause of death.
The female treated with 300 mg/kg bw/day that was found dead on Day 82 had gaseous distension in the duodenum, ileum and jejunum. Histological examination did not reveal any obvious cause of death. Some tissues were notably autolysed.
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 non-recovery control male had an enlarged urinary bladder which contained brown fluid. Two non-recovery females treated with 300 mg/kg bw/day and one female treated with 100 mg/kg bw/day had increased pelvic space in one or both kidneys. The left kidney for one of the females treated with 300 mg/kg bw/day was also fluid filled. Following the treatment-free period, one control female had increased pelvic space in the left kidney with this kidney also being fluid-filled, one male that was previously given 300 mg/kg bw/day had small testes and one male that was previously given 300 mg/kg bw/day had a small and dark spleen. In the absence of any associated treatment-related microscopic changes, these findings were considered to be incidental.

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 Node:
Histiocytosis was present in the mesenteric lymph node of all non-recovery animals of both sexes treated with 300 mg/kg bw/day, varying from minimal to moderate in severity. It was also present in all animals of both sexes treated with 100 mg/kg bw/day at minimal or mild severity. No such effects were detected in animals of both sexes treated with 10 mg/kg bw/day. Following the twenty-eight day recovery period, histiocytosis at minimal or mild severity was present in all animals of both sexes that were previously treated with 300 mg/kg bw/day.

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

Spleen:
Increased hemosiderin was present in the spleen of one non-recovery control female and two non-recovery females treated with 300 mg/kg bw/day at a mild severity after evaluation and blind reading of all animals. This was considered not to indicate an effect of treatment and reflects individual variation.

Thymus:
Minimal thymic atrophy was present in two non-recovery males treated with 300 mg/kg bw/day. Whilst thymic atrophy is often considered to be related to stress, without other obvious signs in these animals (adrenal cortical hypertrophy for example) or a corresponding reduction in organ weight, it was considered to be of equivocal significance and therefore cannot be attributed to treatment.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

10 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

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	8	15	22	27	29	36	43	50	57
1(M)	Mean	229.7	259.5	288.6	309.5	.	327.7	343.7	359	373.2	383.4
	S.D.	11.3	15.6	22.2	24.8	.	29.1	30	32.8	35.3	36.8
	N	20	20	20	20	0	20	20	20	20	20
2(M)	Mean	232.8	264.6	294.9	319.3	.	340.7	359.5	374.9	388.6	399.7
	S.D.	11.8	18.5	21.9	24.1	.	27.1	32.7	36.7	39.8	42.4
	N	10	10	10	10	0	10	10	10	10	10
3(M)	Mean	237.9	270.2	296.1	320.7	.	339.3	354.3	369.9	379.2	390.9
	S.D.	10	14.4	15.3	16.8	.	20.7	23.2	25	28.2	29.5
	N	10	10	10	10	0	10	10	10	10	10
4(M)	Mean	229.1	248.9	266.7	289.9	.	306.3	315.3	327.5	335.1	343.5
	S.D.	8.2	10.8	16.5	18.5	.	22.4	25.7	22.7	25.3	29
	N	20	20	20	20	0	20	20	20	20	20

 

Group (sex)		Day Numbers Relative to Start Date
		64	71	78	85	91	98	105	112	119
1(M)	Mean	395.5	403.8	408.8	415.8	422.9	419.6	426.1	431.4	430.3
	S.D.	38.6	39.1	41.3	42.6	44.8	45.7	44.8	43.7	47.1
	N	20	20	20	20	20	10	10	10	10
2(M)	Mean	414.2	425	434	441.1	446.4	.	.	.	.
	S.D.	46.3	47.3	49.5	52.5	54.7	.	.	.	.
	N	10	10	10	10	10	0	0	0	0
3(M)	Mean	406	417.9	426	433.1	436.8	.	.	.	.
	S.D.	30	28.7	27.8	30.1	28.6	.	.	.	.
	N	10	10	10	10	10	0	0	0	0
4(M)	Mean	355.3	365.5	369.2	374.6	373.5	385.5	400.2	409.9	415.3
	S.D.	30.3	33.7	31.3	30.7	32	33.9	32.4	31.3	32.4
	N	20	20	20	20	19	10	10	10	10

 

Group (sex)		Day Numbers Relative to Start Date
		1	8	15	22	27	29	36	43	50	57
1(F)	Mean	167.6	183.5	200.4	212.8	.	225.6	232.9	242.7	249.9	253.5
	S.D.	8.3	9.3	9.4	9.9	.	14.2	14	14.6	15.8	17.8
	N	20	20	20	20	0	20	20	20	20	20
2(F)	Mean	172.6	190	210.3	221.3	.	232.8	241	250.2	256	263.4
	S.D.	6.1	11.3	11.6	13.3	.	19.8	20.1	20.6	22.1	22.3
	N	10	10	10	10	0	10	10	10	10	10
3(F)	Mean	168.8	183.6	203	212.8	.	220.7	228.4	237.7	244.9	248.4
	S.D.	8.4	11	12.2	15.4	.	19.3	19.9	19.4	21.9	26.3
	N	10	10	10	10	0	10	10	10	10	10
4(F)	Mean	171.4	186.9	203.2	211.7	184	227.9	236.2	242	249.8	254.8
	S.D.	10.2	12.1	15.6	17.3	.	16.4	16.9	18.6	19	20.9
	N	20	20	19	19	1	18	18	18	18	18

  

Group (sex)		Day Numbers Relative to Start Date
		64	71	78	85	91	98	105	112	119
1(F)	Mean	256.2	262.6	266.4	265.6	270.6	274.7	273.6	274.5	274.7
	S.D.	19.4	18.2	18.7	19	17.6	17.7	19.4	20.5	19.4
	N	20	20	20	20	20	10	10	10	10
2(F)	Mean	266.4	269.3	276	279.7	281.7	.	.	.	.
	S.D.	21.8	25.5	19.7	22.9	21.5	.	.	.	.
	N	10	10	10	10	10	0	0	0	0
3(F)	Mean	251.2	258	261.3	259.8	264.4	.	.	.	.
	S.D.	24.2	23	24.5	27.3	23.6	.	.	.	.
	N	10	10	10	10	10	0	0	0	0
4(F)	Mean	259.3	265.2	264.2	266.9	267.5	269.5	269.5	273.1	275
	S.D.	20.5	20.6	18.4	18.9	22.6	30.5	27.9	28.8	29.8
	N	18	18	18	17	17	8	8	8	8

 

Group Mean Body Weight Gains

Group (sex)		Day Numbers Relative to Start Date
		1 – 8	8-15	15-22	22-27	22-29	29-36	36-43	43-50	50-57
1(M)	Mean	29.8	29.1	20.9	.	18.3	16	15.4	14.2	10.3
	S.D.	6.6	11.3	4	.	5.7	4.3	6	4.8	3.9
	N	20	20	20	.	20	20	20	20	20
2(M)	Mean	31.8	30.3	24.4	.	21.4	18.8	15.4	13.7	11.1
	S.D.	7.4	4.8	4.4	.	4.3	6.7	5.1	5	4.1
	N	10	10	10	.	10	10	10	10	10
3(M)	Mean	32.3	25.9	24.6	.	18.6	15	15.6	9.3	11.7
	S.D.	5	4.1	5.4	.	5.6	5.6	2.4	5.9	5.4
	N	10	10	10	.	10	10	10	10	10
4(M)	Mean	19.9**	17.8**	23.3	.	16.4**	9.1*	12.2	7.6	8.4
	S.D.	7	10.9	8.1	.	15.1	9.7	7.4	11.1	7.5
	N	20	20	20	.	20	20	20	20	20

 

Group (sex)		Day Numbers Relative to Start Date
		57-64	64-71	71-78	78-85	85-91	91-98	98-105	105-112	112-119
1(M)	Mean	12.1	8.3	5	7	7.1	3.5	6.5	5.3	1.1
	S.D.	4.1	3.5	5.3	2.7	4.6	3.1	4.9	3.2	8.9
	N	20	20	20	20	20	10	10	10	10
2(M)	Mean	14.5	10.8*	9	7.1	5.3*	.	.	.	.
	S.D.	5.5	3.3	2.5	4.8	4.7	.	.	.	.
	N	10	10	10	10	10	.	.	.	.
3(M)	Mean	15.1	11.9*	8.1	7.1	3.7*	.	.	.	.
	S.D.	4.7	3	5.7	7.7	2.8	.	.	.	.
	N	10	10	10	10	10	.	.	.	.
4(M)	Mean	11.8	10.3*	3.7	5.5	2.1*	7.6*	14.7**	9.7**	5.4*
	S.D.	6.3	7.6	9.6	11.2	10.4	5	5	3.4	4
	N	20	20	20	20	19	10	10	10	10

 

Group (sex)		Day Numbers Relative to Start Date
		1 – 8	8-15	15-22	22-27	22-29	29-36	36-43	43-50	50-57
1(F)	Mean	15.9	16.9	12.5	.	12.8	7.3	9.9	7.2	3.6
	S.D.	5.9	5.7	4.5	.	7.2	7.4	5.2	4.9	6.1
	N	20	20	20	.	20	20	20	20	20
2(F)	Mean	17.4	20.3	11	.	11.5	8.2	9.2	5.8	7.4
	S.D.	6.4	7.8	4.5	.	9.4	6.5	6.2	6.3	4.9
	N	10	10	10	.	10	10	10	10	10
3(F)	Mean	14.8	19.4	9.8	.	7.9	7.7	9.3	7.2	3.5
	S.D.	5.2	6	6.9	.	6.2	6	5.9	7.2	6.9
	N	10	10	10	.	10	10	10	10	10
4(F)	Mean	15.5	15.9	8.5	4.0	14.9	8.2	5.8*	7.8	5
	S.D.	4.5	7.2	9.3	.	10.8	5.7	6.2	7.9	9.7
	N	20	19	19	1	18	18	18	18	18

 

Group (sex)		Day Numbers Relative to Start Date
		57-64	64-71	71-78	78-85	85-91	91-98	98-105	105-112	112-119
1(F)	Mean	2.8	6.4	3.8	0.8	5.1	1.8	1.1	0.9	0.2
	S.D.	6.8	6	5.2	6.2	5.8	4.8	3.6	3.3	4
	N	20	20	20	20	20	10	10	10	10
2(F)	Mean	3	2.9	6.7	3.7	2	.	.	.	.
	S.D.	5.7	9.6	10.5	6.3	7.2	.	.	.	.
	N	10	10	10	10	10	.	.	.	.
3(F)	Mean	2.8	6.8	3.3	1.5	4.6	.	.	.	.
	S.D.	6.1	5.1	7	4.5	7.4	.	.	.	.
	N	10	10	10	10	10	.	.	.	.
4(F)	Mean	4.5	5.8	1.0	2.7	0.6	2.9	0	3.6	1.9
	S.D.	9.6	7.3	9.6	5.3	8.5	6.4	4.4	4.8	2.6
	N	18	18	18	17	17	8	8	8	8

 

Group Mean Hematological Values

Non-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	15.7	9.151	47.64	17.17	52.06	32.98	7.62	1.098
	S.D.	0.58	0.394	2.15	0.48	1.09	0.48	1.16	0.375
	N	10	10	10	10	10	10	10	10
2(M)	Mean	15.71	9.086	47.56	17.33	52.44	32.99	7.66	1.284
	S.D.	0.31	0.365	0.72	0.97	2.67	0.3	1.45	0.401
	N	10	10	10	10	10	10	10	10
3(M)	Mean	15.68	9.136	47.87	17.18	52.44	32.77	6.49	1.442*
	S.D.	0.58	0.441	2.04	0.68	1.81	0.48	0.97	0.269
	N	10	10	10	10	10	10	10	10
4(M)	Mean	16.42**	9.239	49.86*	17.8	54.01*	32.93	8.17	3.326**
	S.D.	0.54	0.364	1.83	0.77	2.13	0.29	1.72	1.531
	N	9	9	9	9	9	9	9	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	6.441	0.006n	0.076	0.000n	9.53	585	16.01
	S.D.	1.026	0.019	0.065	0	0.46	79.6	1.52
	N	10	10	10	10	10	10	10
2(M)	Mean	6.302	0.000n	0.074	0.000n	9.65	575	16.29
	S.D.	1.259	0	0.089	0	0.76	90	1.14
	N	10	10	10	10	10	10	10
3(M)	Mean	4.983*	0.007n	0.059	0.000n	9.71	579.9	16.67
	S.D.	0.94	0.022	0.058	0	0.89	75	1.11
	N	10	10	10	10	10	10	10
4(M)	Mean	4.838*	0.009n	0.09	0.000n	9.91	581.9	16.34
	S.D.	2.089	0.027	0.075	0	1.05	43.7	1.8
	N	9	9	9	9	9	9	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	14.94	8.267	44.72	18.09	54.1	33.44	4.56	1.088
	S.D.	0.45	0.262	1.43	0.44	1.19	0.31	1.35	1.371
	N	10	10	10	10	10	10	10	10
2(F)	Mean	14.83	8.116	44.34	18.3	54.71	33.45	4.49	0.681
	S.D.	0.56	0.448	1.63	0.47	1.48	0.31	0.77	0.174
	N	10	10	10	10	10	10	10	10
3(F)	Mean	15.32	8.215	45.85	18.67	55.89	33.4	4.69	1.073
	S.D.	0.43	0.428	1.28	0.91	2.48	0.24	1.35	0.479
	N	10	10	10	10	10	10	10	10
4(F)	Mean	15.06	8.368	45.4	18.02	54.27	33.2	4.64	1.046
	S.D.	0.63	0.454	2.19	0.38	1.06	0.34	1.36	0.513
	N	9	9	9	9	9	9	9	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(F)	Mean	3.419	0.000n	0.057	0.000n	9.06	621.4	14.69
	S.D.	1.019	0	0.055	0	0.94	49.5	1.66
	N	10	10	10	10	10	10	10
2(F)	Mean	3.762	0.000n	0.048	0.000n	8.86	617.4	15.24
	S.D.	0.686	0	0.059	0	0.69	103.6	2.04
	N	10	10	10	10	10	10	10
3(F)	Mean	3.583	0.014*,n	0.022	0.000n	8.88	598.7	15.79
	S.D.	1.443	0.024	0.029	0	0.49	47	1.02
	N	10	10	10	10	10	10	10
4(F)	Mean	3.563	0.000n	0.034	0.000n	8.61	650.3	14.51
	S.D.	1.014	0	0.051	0	0.34	52.7	1.6
	N	9	9	9	9	9	9	9

            

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	16.01	9.047	47.84	21.46	52.9	33.45	7.45	1.222
	S.D.	0.53	0.442	1.51	12.52	2.81	0.44	1.15	0.283
	N	10	10	10	10	10	10	10	10
4(M)	Mean	16.51	9.410*	49.81**	17.57	53.1	33.14	7.53	1.726*
	S.D.	0.54	0.318	1.26	1.01	2.51	0.47	0.98	0.575
	N	10	10	10	10	10	10	10	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(M)	Mean	6.168	0.000n	0.059	0.000n	9.92	591.4	15.19
	S.D.	0.972	0	0.069	0	0.61	101.7	2.02
	N	10	10	10	10	10	10	10
4(M)	Mean	5.743	0.000n	0.061	0.000n	10.06	612.9	14.61
	S.D.	0.824	0	0.064	0	0.57	70.3	0.84
	N	10	10	10	10	10	10	10

       

		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	15.56	8.466	45.72	18.41	54.02	34.06	4.24	0.678
	S.D.	0.62	0.383	1.77	0.68	1.68	0.35	0.65	0.151
	N	10	10	10	10	10	10	10	10
4(F)	Mean	15.7	8.371	46.1	18.81	55.15	34.09	4.68	0.931
	S.D.	0.47	0.453	1.39	0.96	2.06	0.57	1.2	0.362
	N	8	8	8	8	8	8	8	8

       

		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	3.524	0.000n	0.04	0.000n	8.58	584.9	13.88
	S.D.	0.67	0	0.045	0	0.7	66.1	1.48
	N	10	10	10	10	10	10	10
4(F)	Mean	3.718	0.000n	0.028	0.000n	8.65	589.5	14.29
	S.D.	1.061	0	0.04	0	0.41	98.7	1.48
	N	8	8	8	8	8	8	8

 

Group Mean Blood Chemical Values

Non-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	33.5	175.6	6.432	3.62	1.294	146.2	4.75	102.7	1.62	1.39
	S.D.	6.9	17.6	0.225	0.13	0.097	2.1	0.968	1.9	0.097	0.27
	N	10	10	10	10	10	10	10	10	10	10
2(M)	Mean	32.2	170.7	6.192	3.44	1.23	146.3	4.704	103.4	1.581	1.28
	S.D.	6.6	27.6	0.947	0.63	0.128	1.7	0.423	1.3	0.158	0.29
	N	10	10	10	10	10	10	10	10	10	10
3(M)	Mean	32.2	170.1	6.276	3.51	1.275	145.5	4.627	103.3	1.643	1.28
	S.D.	5.7	16	0.282	0.2	0.074	2.1	0.315	1.7	0.081	0.15
	N	10	10	10	10	10	10	10	10	10	10
4(M)	Mean	34.1	166.7	6.161	3.49	1.307	148	5.213	101.9	1.617	1.54
	S.D.	7	28.7	0.727	0.42	0.088	2.5	1.65	2.2	0.22	0.41
	N	9	9	9	9	9	9	9	9	9	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	0.00n	85.1	63.6	113.4	0.783	272.9	77.4	0.102	10.42
	S.D.	0	23.3	8.9	26.9	0.057	130.5	9.9	0.041	3.86
	N	10	10	10	10	10	10	10	10	10
2(M)	Mean	0.00n	74.9	58.9	119.9	0.823	267.9	84.6	0.117	8.18
	S.D.	0	15.1	14.4	32.8	0.2	173.2	20.2	0.022	2.78
	N	10	10	10	10	10	10	10	10	10
3(M)	Mean	0.00n	102.4	86.5*	109.3	0.797	165.6	78.3	0.118	7.24*
	S.D.	0	22.9	25.5	15.4	0.071	43.7	17.2	0.018	4.62
	N	10	10	10	10	10	10	10	10	10
4(M)	Mean	0.00n	128.9**	89.3*	88.1	0.773	135.7**	64	0.11	4.27**
	S.D.	0	39.4	27.4	32.9	0.112	72	14	0.045	0.94
	N	8	9	9	9	9	9	9	9	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	38.3	149	7.239	4.31	1.48	148	4.953	105.3	1.784	1.58
	S.D.	3.9	16.3	0.377	0.36	0.185	2.1	1.156	1.8	0.143	0.27
	N	10	10	10	10	10	10	10	10	10	10
2(F)	Mean	33.7	153	7.457	4.56*	1.578	148	4.41	105.5	1.881*	1.48
	S.D.	3.5	13.3	0.517	0.31	0.07	2.3	0.289	1.8	0.067	0.23
	N	10	10	10	10	10	10	10	10	10	10
3(F)	Mean	43.0*	149	7.837**	4.77**	1.556	147.6	4.838	103.6*	1.934**	1.76
	S.D.	4.8	13.7	0.401	0.24	0.12	2.2	0.459	1.5	0.079	0.45
	N	10	10	10	10	10	10	10	10	10	10
4(F)	Mean	51.7**	155.9	7.918**	4.92**	1.644*	146	5.576	103.1**	1.942**	1.68
	S.D.	6.8	17.7	0.54	0.25	0.14	2.6	3.729	1.6	0.09	0.47
	N	9	9	9	9	9	9	9	9	9	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(F)	Mean	0.00n	102.1	63.9	66.2	0.799	141	74.4	0.077	11.63
	S.D.	0	39.9	13.4	43.9	0.074	68.7	9.4	0.044	9.05
	N	10	10	10	10	10	10	10	10	10
2(F)	Mean	0.10n	86.9	63.2	72.4	0.818	159.4	71.8	0.104	15.38
	S.D.	0.32	15.6	11.4	16.5	0.046	41.7	13.3	0.02	10.3
	N	10	10	10	10	10	10	10	10	10
3(F)	Mean	0.00n	101.9	102.8**	55.4	0.826	161.7	69.4	0.098	12.83
	S.D.	0	50.3	15.9	9.9	0.042	52.5	11.9	0.037	12.52
	N	10	10	10	10	10	10	10	10	10
4(F)	Mean	0.00n	124	111.1**	50.2	0.862*	127.3	59.4**	0.116*	8.88
	S.D.	0	64.7	36.4	19.8	0.062	46.5	8.8	0.047	5.03
	N	9	9	9	9	9	9	9	9	9

 

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	46.3	158.7	6.98	3.95	1.297	147.4	5.013	103.8	2.805	1.81
	S.D.	6.9	19.4	0.16	0.1	0.089	2.1	0.865	2.6	0.262	0.13
	N	10	10	10	10	10	10	10	10	10	10
4(M)	Mean	43.7	160.4	6.766*	3.89	1.367	145.9	4.744	102.9	2.811	1.79
	S.D.	4	7.8	0.25	0.18	0.12	1.5	0.347	1.1	0.087	0.21
	N	10	10	10	10	10	10	10	10	10	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(M)	Mean	0.00n	86.7	66.2	135	0.789	237.1	94.5	0.116	13.78
	S.D.	0	21.9	15.3	34.2	0.115	55.8	18.7	0.018	8.3
	N	10	10	10	10	10	10	10	10	10
4(M)	Mean	0.00n	111.6	84.8	121	0.783	164.5*	87.2	0.104	15.68
	S.D.	0	47	46.9	20	0.065	67.1	12.8	0.015	10.32
	N	10	10	10	10	10	10	10	10	10

 

		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	51.2	159.6	7.116	4.35	1.568	145.9	4.716	103.5	2.672	1.35
	S.D.	15.1	15.7	0.576	0.38	0.081	3.5	0.925	2.1	0.102	0.22
	N	10	10	10	10	10	10	10	10	10	10
4(F)	Mean	57.9	163.8	7.559	4.61	1.565	144.8	4.335	102.9	2.699	1.14*
	S.D.	9.2	12.8	0.423	0.29	0.066	1.5	0.514	2.2	0.088	0.2
	N	8	8	8	8	8	8	8	8	8	8

 

		yGT	ASAT	ALAT	AP	Creat	Tri	Chol	Bili	Bile acids
Group (sex)
1(F)	Mean	0.00n	139.5	88.9	50	0.776	175.3	89.8	0.112	16.31
	S.D.	0	82.4	36.1	11.5	0.191	62.8	14.2	0.027	17.81
	N	10	10	10	10	10	10	10	10	10
4(F)	Mean	0.00n	100.6	65.9	52.4	0.87	156.4	91.6	0.101	15
	S.D.	0	28.7	20.1	11	0.121	73.1	10.9	0.016	9.01
	N	8	8	8	8	8	8	8	8	8

 

Histopathology

Non-recovery animals

	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg bw/d)	0	10	100	300	0	10	100	300
Mesenteric lymph node Histiocytosis
Minimal	0	0	5	0	0	0	3	0
Slight	0	0	5	7	0	0	7	6
Moderate	0	0	0	2	0	0	0	3
Total	0	0	10	9	0	0	10	9
Plasmacytosis
Mild	0	0	0	0	0	0	0	1
Spleen Hematopoiesis
Mild	1	.	.	0	2	1	4	1
Hemosiderin increased
mild	0	.	.	0	1	0	0	3
Thymus Atrophy
Minimal	0	.	.	2	0	.	.	0
N	10	10	10	9	10	10	10	9

 

Recovery animals:                

	1M	4M	1F	4F
Dose (mg/kg bw/d)
Mesenteric lymph node Histiocytosis
Minimal	0	2	2	1
Mild	0	6	0	6
Moderate	0	2	0	1
Total	0	10	0	8
Ectasia
mild	0	1	0	0
Spleen Hematopoiesis
Mild	.	.	0	1
N	10	10	10	8

BADGE-MXDA fertility parameters

 

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
Testes
Submitted	20	10	10	19
No Visible Lesions	20	10	10	18
small	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	1.62830 0.18488       10	1.67464    0.15323    10	1.74595    0.21200    10	1.53051                                                                            0.20489                                                                            9	-	-	-	-
	Mean (%) S.D.     N	0.380      0.031      10	0.380      0.060      10	0.401      0.052      10	0.418                                                                           0.064                                                                           9	-	-	-	-
Testes	Mean (g) S.D.     N	3.77051    0.42495    10	3.81649    0.19625    10	3.93235    0.29053    10	3.75139                                                                            0.37542                                                                            9	-	-	-	-
	Mean (%) S.D.     N	0.883      0.098      10	0.866 0.106 10	0.903 0.076 10	1.021 0.107 9	-	-	-	-
Ovaries	Mean (g) S.D.     N	-	-	-	-	0.12624    0.02200    9	0.10666    0.02534    10	0.11020    0.02475    10	0.12373                                0.02204                                9
	Mean (%) S.D.     N	-	-	-	-	0.046      0.008      9	0.038      0.008      10	0.042      0.009      10	0.047                               0.007                               9
Uterus And Cervix	Mean (g) S.D.     N	-	-	-	-	0.99179    0.37925 10	0.96745 0.39192 10	0.88045    0.27004    10	0.83181                                0.46430 9
	Mean (%) S.D.     N	-	-	-	-	0.371      0.141      10	0.344      0.136      10	0.332      0.096      10	0.316                               0.175                               9

 

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	1.52527  0.18201  10	1.50451                                                                                                    0.24307 10	-	-
	Mean (%) S.D.     N	0.356 0.040 10	0.364 0.065 10	-	-
Testes	Mean (g) S.D.     N	3.70899  0.40210  10	3.63139 0.53509 10	-	-
	Mean (%) S.D.     N	0.865    0.072    10	0.878 0.147 10	-	-
Ovaries	Mean (g) S.D.     N	-	-	0.09885  0.02070  10	0.08621                                                                                  0.01204 8
	Mean (%) S.D.     N	-	-	0.036    0.008    10	0.032 0.005 8
Uterus And Cervix	Mean (g) S.D.     N	-	-	0.88128  0.21986  10	0.82868 0.31206 8
	Mean (%) S.D.     N	-	-	0.321    0.073    10	0.300 0.113 8

 

 

 

 

 

 

Conclusions:

The oral (gavage) administration of BADGE-MXDA for up to ninety consecutive days, to Wistar rats of both sexes at dose levels of 10, 100 or 300 mg/kg bw/day resulted in treatment-related effects in animals of both sexes treated with 300 and 100 mg/kg bw/day. These findings included the death of one female treated with 300 mg/kg bw/day, the necessary early termination of two females and one male treated with 300 mg/kg bw/day, clinical signs of toxicity, reduced body weight development and food consumption (300 mg/kg bw/day males only), changes in the hematology parameters measured (300 mg/kg bw/day males only) and microscopic changes in the mesenteric lymph nodes. These changes were considered to represent an adverse effect of treatment. No toxicologically significant effects were evident in animals of both sexes treated with 10 mg/kg bw/day, therefore, 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-MXDA 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 300 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 (300 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 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.

Results

Mortality

Two females and one male treated with 300 mg/kg bw/day were sacrificedin extremison Days 8, 27 and 85. A further female treated with 300 mg/kg bw/day was found dead on Day 82. There were no further unscheduled deaths.

Clinical Observations

Increased salivation and noisy respiration was evident in animals of both sexes treated with 300 mg/kg bw/day and to a lesser extent in animals of both sexes treated with 100 mg/kg bw/day throughout the treatment period. Incidences of labored respiration, decreased respiratory rate, hunched posture and lethargy were also evident in males treated with 300 mg/kg bw/day and a decreased respiratory rate was evident in one female from this treatment group. The females that were sacrificedin extremisalso showed labored respiration, decreased respiratory rate, pilo-erection and hunched posture and one of the females also had gasping respiration and a distended abdomen. The male that was sacrificedin extremisalso showed gasping respiration. No such effects were detected in males treated with 10 mg/kg bw/day, however, one female from this treatment group had noisy respiration on Day 26 only.

Behavioral Assessment

Weekly behavioral assessments revealed instances of noisy respiration in some animals of both sexes treated with 300 mg/kg bw/day, in some females treated with 100 mg/kg bw/day and in one male and one female treated with 10 mg/kg bw/day. One of the males treated with 300 mg/kg bw/day also showed an isolated incidence of a decreased respiratory rate and pilo-erection.

Functional Performance Tests

There were no treatment-related changes in functional performance.

Sensory Reactivity Assessments

There were no treatment-related changes in sensory reactivity.

Body Weight

Males treated with 300 mg/kg bw/day showed lower body weight gain throughout the treatment period, consequently overall body weight gain was lower than controls. Significant improvement in body weight gain was evident in males previously treated with 300 mg/kg bw/day during the twenty-eight day treatment-free period. No such adverse effects were detected in treated females or in males treated with 100 or 10 mg/kg bw/day. 

Food Consumption

Males treated with 300 mg/kg bw/day showed a reduction in food consumption throughout the treatment period. Females from this treatment group showed a reduction in food consumption from Week 10 onwards. Incidences of reduced food conversion efficiency were also evident in these animals during the treatment period. Improvement in food consumption and food conversion efficiency was evident during the twenty-eight day treatment-free period. No such effects were detected in animals of both sexes treated with 100 or 10 mg/kg bw/day.

Water Consumption

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

Ophthalmoscopy

Opthalmoscopic examination of animals of both sexes from the non-recovery control and 300 mg/kg bw/day dose groups during Week 12 of the treatment period did not indicate any treatment-related

differences.

Hematology

Males treated with 300 and 100 mg/kg bw/day showed an increase in neutrophils and a reduction in lymphocytes. The effect on neutrophils was also present in males previously treated with 300 mg/kg bw/day at the end of the twenty-eight day treatment-free period. No such effects were detected in treated females or in males 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 300 mg/kg bw/day that was sacrificedin extremison Day 85 had gaseous distension in the stomach and ileum, pale and enlarged lungs and a reddened glandular region in the stomach. This animal had tracheal ulceration and bronchopneumonia, indicative of test item irritancy through gastric reflux, and this is considered to be the cause of death.

The female treated with 300 mg/kg bw/day that was sacrificedin extremison Day 8 had a dark liver and reddened lungs with clear fluid present. This female had edema and inflammation in the lungs and ulceration of the trachea, also indicative of test item irritancy through gastric reflux, and this is considered to be the cause of death.

The female treated with 300 mg/kg bw/day that was sacrificedin extremison Day 27 had gaseous distention in the intestines and reddened and enlarged lungs. This female had hemorrhage in the lungs as well as minimal inflammatory change, possibly through technical error and this is considered to be the cause of death.

The female treated with 300 mg/kg bw/day that was found dead on Day 82 had gaseous distension in the duodenum, ileum and jejunum. Histological examination did not reveal any obvious cause of death. Some tissues were notably autolysed.

Organ Weights

No toxicologically significant effects were detected in the organ weights measured

Histopathology

The following treatment-related microscopic abnormalities were detected:

Mesenteric Lymph Node: Histiocytosis was present in the mesenteric lymph nodes of all non-recovery animals of both sexes treated with 300 mg/kg bw/day, varying from minimal to moderate in severity. It was also present in all animals of both sexes treated with 100 mg/kg bw/day at minimal or mild severity. No such effects were detected in animals of both sexes treated with 10 mg/kg bw/day. Following the twenty-eight day recovery period, histiocytosis at minimal or mild severity was present in all animals of both sexes that were previously treated with 300 mg/kg bw/day.

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 or an equivocal finding rather than an effect of treatment:

Spleen: Increased hemosiderin was present in the spleen of one non-recovery control female and two non-recovery females treated with 300 mg/kg bw/day at a mild severity after evaluation and blinded reading of all animals. This was considered not to indicate an effect of treatment and reflects individual variation.

Thymus: Minimal thymic atrophy was present in two non-recovery males treated with 300 mg/kg bw/day. Whilst thymic atrophy is often considered to be related to stress, without other obvious signs in these animals (adrenal cortical hypertrophy for example) or a corresponding reduction in organ weight, it was considered to be of equivocal significance and therefore cannot be attributed to treatment.

Conclusion

The oral (gavage) administration of BADGE-MXDA for up to ninety consecutive days, to Wistar rats of both sexes at dose levels of 10, 100 or 300 mg/kg bw/day resulted in treatment-related effects in animals of both sexes treated with 300 and 100 mg/kg bw/day. These findings included the death of one female treated with 300 mg/kg bw/day, the necessary early termination of two females and one male treated with 300 mg/kg bw/day, clinical signs of toxicity, reduced body weight development and food consumption (300 mg/kg bw/day males only), changes in the hematology parameters measured (300 mg/kg bw/day males only) and microscopic changes in the mesenteric lymph nodes. These changes were considered to represent an adverse effect of treatment. No toxicologically significant effects were evident in animals of both sexes treated with 10 mg/kg bw/day, therefore, the 'No Observed Adverse Effect Level (NOAEL) was considered to be 10 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP/Guideline 90 day study

System:

immune system

Organ:

mesenteric lymph node

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

For the assessment of repeated dose toxicity of BADGE-MXDA, a subacute (28 day) and a subchronic (90 day) study is available.

 

Subacute study

The potential toxicity of BADGE MXDA (#42), an industrial chemical, in rats following oral gavage administration for 28 days was studied in a GLP/guideline study. Five male and five female F344/DuCrl rats per group were administered 0, 25, 100, or 400 milligrams BADGE MXDA (#42) per kilogram body weight per day (mg/kg/day, mkd) in propylene glycol (PG) via gavage. At the end of the study on test day (TD) 28, males and females given 400 mg/kg/day had treatment-related 7% and 4% decreases in body weight, respectively as compared to controls. Males and females given 400 mg/kg/day had decreased (26.6 and 25.5%, respectively) body weight gains on TD 8; subsequently, body weight gains improved and at study termination the final body weight gains were decreased by 14.4 and 11.0%, respectively, compared to controls. There were no treatment-related effects on body weight, body weight gain, or feed consumption for males or females in the 25 or 100 mg/kg/day dose groups.

 

Urinalysis showed treatment-related higher urine volumes with concomitant decrease in specific gravity in males and females of the high-dose group.

 

At necropsy, males and females in the 400 mg/kg/day group had treatment-related lower final (fasting) body weights. Males and females given 400 mg/kg/day had treatment-related decreases in absolute and relative weights of liver, heart, thymus and absolute kidney and spleen weights which were interpreted to be reflective of the treatment-related decrements in body weights/body weight gains and feed consumption. Treatment-related gross pathological observations consisted of enlarged cecum and enlarged cecal lymph nodes in males and females given 400 mg/kg/day.

 

Treatment-related histopathological changes consisted of very slight or slight diffuse mucosal hyperplasia and a very slight or slight diffuse subacute to chronic inflammation of the mucosa of cecum in males and females given 400 mg/kg/day. A very slight treatment-related cecal mucosal hyperplasia and mucosal inflammation were also observed in 4/5 or 5/5 males given 100 mg/kg/day, respectively and in 1/5 or 2/5 males given 25 mg/kg/day, respectively. A very slight treatment-related inflammation was also noted in the cecal mucosa of 4/5 females given 100 mg/kg/day. The colon and rectum of males and females given 400 mg/kg/day had treatment-related very slight diffuse mucosal hyperplasia and very slight or slight diffuse subacute to chronic inflammation. Treatment-related lymphoid hyperplasia was noted in the cecal lymph nodes of males given 100 or 400 mg/kg/day and in females given 400 mg/kg/day. In addition, dose-related increased sinus histiocytosis (macrophage hyperplasia) in the cecal lymph node was observed in 1/5, 4/5, or 5/5 males given 25, 100, or 400 mg/kg/day, respectively and in 5/5 females given 400 mg/kg/day. Treatment-related, increased sinus histiocytosis was also noted in the mesenteric lymph nodes of males and females in the 400 mg/kg/day group. Females given 400 mg/kg/day had a very slight treatment-related hyperplasia of the epithelium lining the limiting ridge of the forestomach. All these treatment-related histopathological changes in the gastrointestinal tract and associated lymph nodes were interpreted to be localized, point of contact effects associated with the potential irritancy of the test material at the affected sites. A very slight diffuse acinar hypertrophy of the submandibular salivary gland was noted in females given 400 mg/kg/day and was interpreted to be an adaptive physiologic response to the repeated oral administration of the test material. Other treatment-related effects attributed to lower body weights/body weight gains in the high-dose group included 1) very slight decreases in the amounts of secretory material within the seminal vesicles of males, and 2) a slight to moderate atrophy of the adipose tissue of the mesentery in males and females consistent with decreased fat reserves.

 

Under the conditions of this study, based on the microscopic effects in the cecum and cecal lymph node of low-dose males interpreted to be due to point of contact, localized irritant effects of the test material, a no-observed-effect-level (NOEL) for BADGE MXDA (#42) in male F344/DuCrl rats could not be determined, and the NOEL for female rats was 25 mg/kg/day. Based on the absence of systemic effects at 100 mg/kg/day, the NOEL for systemic toxicity was determined to be 100 mg/kg/day in both sexes.

 

Subchronic study

In a subchronic toxicity study according to OECD Guideline 407 BADGE-MXDA 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 300 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 (300 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.

Two females and one male treated with 300 mg/kg bw/day were sacrificedin extremison Days 8, 27 and 85. A further female treated with 300 mg/kg bw/day was found dead on Day 82. There were no further unscheduled deaths.

Increased salivation and noisy respiration was evident in animals of both sexes treated with 300 mg/kg bw/day and to a lesser extent in animals of both sexes treated with 100 mg/kg bw/day throughout the treatment period. Incidences of labored respiration, decreased respiratory rate, hunched posture and lethargy were also evident in males treated with 300 mg/kg bw/day and a decreased respiratory rate was evident in one female from this treatment group. The females that were sacrificedin extremisalso showed labored respiration, decreased respiratory rate, pilo-erection and hunched posture and one of the females also had gasping respiration and a distended abdomen. The male that was sacrificedin extremisalso showed gasping respiration. No such effects were detected in males treated with 10 mg/kg bw/day, however, one female from this treatment group had noisy respiration on Day 26 only.

Weekly behavioral assessments revealed instances of noisy respiration in some animals of both sexes treated with 300 mg/kg bw/day, in some females treated with 100 mg/kg bw/day and in one male and one female treated with 10 mg/kg bw/day. One of the males treated with 300 mg/kg bw/day also showed an isolated incidence of a decreased respiratory rate and pilo-erection.

There were no treatment-related changes in functional performance and sensory reactivity.

Males treated with 300 mg/kg bw/day showed lower body weight gain throughout the treatment period, consequently overall body weight gain was lower than controls. Significant improvement in body weight gain was evident in males previously treated with 300 mg/kg bw/day during the twenty-eight day treatment-free period. No such adverse effects were detected in treated females or in males treated with 100 or 10 mg/kg bw/day. 

Males treated with 300 mg/kg bw/day showed a reduction in food consumption throughout the treatment period. Females from this treatment group showed a reduction in food consumption from Week 10 onwards. Incidences of reduced food conversion efficiency were also evident in these animals during the treatment period. Improvement in food consumption and food conversion efficiency was evident during the twenty-eight day treatment-free period. No such effects were detected in animals of both sexes treated with 100 or 10 mg/kg bw/day.

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

Opthalmoscopic examination of animals of both sexes from the non-recovery control and 300 mg/kg bw/day dose groups during Week 12 of the treatment period did not indicate any treatment-related

differences.

Males treated with 300 and 100 mg/kg bw/day showed an increase in neutrophils and a reduction in lymphocytes. The effect on neutrophils was also present in males previously treated with 300 mg/kg bw/day at the end of the twenty-eight day treatment-free period. No such effects were detected in treated females or in males treated with 10 mg/kg bw/day.

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

No toxicologically significant macroscopic abnormalities were detected in surviving animals.

The male treated with 300 mg/kg bw/day that was sacrificed in extremis on Day 85 had gaseous distension in the stomach and ileum, pale and enlarged lungs and a reddened glandular region in the stomach. This animal had tracheal ulceration and bronchopneumonia, indicative of test item irritancy through gastric reflux, and this is considered to be the cause of death.

The female treated with 300 mg/kg bw/day that was sacrificed in extremis on Day 8 had a dark liver and reddened lungs with clear fluid present. This female had edema and inflammation in the lungs and ulceration of the trachea, also indicative of test item irritancy through gastric reflux, and this is considered to be the cause of death.

The female treated with 300 mg/kg bw/day that was sacrificed in extremis on Day 27 had gaseous distention in the intestines and reddened and enlarged lungs. This female had hemorrhage in the lungs as well as minimal inflammatory change, possibly through technical error and this is considered to be the cause of death.

The female treated with 300 mg/kg bw/day that was found dead on Day 82 had gaseous distension in the duodenum, ileum and jejunum. Histological examination did not reveal any obvious cause of death. Some tissues were notably autolysed.

No toxicologically significant effects were detected in the organ weights measured

 

The following treatment-related microscopic abnormalities were detected:

Mesenteric Lymph Node: Histiocytosis was present in the mesenteric lymph nodes of all non-recovery animals of both sexes treated with 300 mg/kg bw/day, varying from minimal to moderate in severity. It was also present in all animals of both sexes treated with 100 mg/kg bw/day at minimal or mild severity. No such effects were detected in animals of both sexes treated with 10 mg/kg bw/day. Following the twenty-eight day recovery period, histiocytosis at minimal or mild severity was present in all animals of both sexes that were previously treated with 300 mg/kg bw/day.

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 or an equivocal finding rather than an effect of treatment:

Spleen: Increased hemosiderin was present in the spleen of one non-recovery control female and two non-recovery females treated with 300 mg/kg bw/day at a mild severity after evaluation and blinded reading of all animals. This was considered not to indicate an effect of treatment and reflects individual variation.

Thymus: Minimal thymic atrophy was present in two non-recovery males treated with 300 mg/kg bw/day. Whilst thymic atrophy is often considered to be related to stress, without other obvious signs in these animals (adrenal cortical hypertrophy for example) or a corresponding reduction in organ weight, it was considered to be of equivocal significance and therefore cannot be attributed to treatment.

 

The oral (gavage) administration of BADGE-MXDA for up to ninety consecutive days, to Wistar rats of both sexes at dose levels of 10, 100 or 300 mg/kg bw/day resulted in treatment-related effects in animals of both sexes treated with 300 and 100 mg/kg bw/day. These findings included the death of one female treated with 300 mg/kg bw/day, the necessary early termination of two females and one male treated with 300 mg/kg bw/day, clinical signs of toxicity, reduced body weight development and food consumption (300 mg/kg bw/day males only), changes in the hematology parameters measured (300 mg/kg bw/day males only) and microscopic changes in the mesenteric lymph nodes. These changes were considered to represent an adverse effect of treatment. No toxicologically significant effects were evident in animals of both sexes treated with 10 mg/kg bw/day, therefore, the 'No Observed Adverse Effect Level (NOAEL) was considered to be 10 mg/kg bw/day.

 

Sinus histiocytosis is described as a normal finding in mesenteric lymph nodes; the macrophages may contain endogenous pigment (hemosiderin, lipofuscin) or exogenous material reflecting antigen uptake from the gastrointestinal tract. This is a common effect related to accumulation and delayed clearance of the test item. These changes are consistent with an inert compound. Thus, the effects are not regarded as significant toxic effect, but rather a port-of-entry effect. Additionally, inflammatory cells can be found in lymph nodes draining sites of the administration of an irritating test compound (Elmore, 2007).

Overall, the observed histopathological changes in the mesenteric lymph nodes (histiocytosis and granulomas) were interpreted to be localized, point of contact effects associated with the potential irritancy or delayed clearance of the test material and thus, do not warrant classification for specific organ toxicity after repeated exposure.

 

 

Reference:

Elmore, SA (2007). Histopathology of the Lymph Nodes.Toxicol Pathol. 2006; 34(5): 425–454.

Justification for classification or non-classification

The observed histopathological changes in the mesenteric lymph nodes (histiocytosis and granulomas) were interpreted to be localized, point of contact effects associated with the potential irritancy or delayed clearance of the test material and thus, do not warrant classification for specific organ toxicity after repeated exposure.