Diphenylacetonitrile

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Weight of evidence approach based on the available data of the read-across chemicals.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crj: CD, Nippon Charles River Co., Ltd.

Sex:

male/female

Details on test animals or test system and environmental conditions:

The body weight at the start of administration was 215.2-241.6 g for males and 144.7-171.9 g for females. Animals are placed in a stainless steel hanger cage in a barrier system breeding room set at a temperature of 24 ± 2 ° C, humidity of 55 ± 10%, lighting time from 7 to 19 o'clock, and ventilation rate of 13 times / hour. Grouping was performed by stratified randomization based on body weight the day before the start of treatment.

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Details on oral exposure:

The dose volume was 5 ml / kg, and it was calculated based on the latest body weight for each individual.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 days.

Frequency of treatment:

Once per day.

Remarks:

0 (vehicle), 8, 40 and 200 mg/kg bw/day

No. of animals per sex per dose:

12 rats per sex per dose level at 0 and 200 mg/kg bw/day
6 rats per sex per dose level at 8 and 40 mg/kg bw/day.

Details on study design:

The chemical was given by oral gavage to 6 rats per sex per dose level at 0, 8, 40 and 200 mg/kg bw/day (main study). The study also included recovery groups of male and female rats (6 per sex per dose level) treated at 0 and 200 mg/kg bw/day. These animals were allowed to recover 2 weeks after the last dose was given. Dose levels were selected based on the results of dose range-finding study in which male and female rats were treated at 0, 30, 100 and 300 mg/kg bw/day for two weeks. In this study, treatment at ≥100 mg/kg resulted in growth inhibition and changes in blood biochemistry and organ weights.

Positive control:

Not used.

Observations and examinations performed and frequency:

The animals were examined three times per day for mortality and clinical signs. Body weight and food intake were recorded twice a week during treatment and recovery. Urinalysis was performed at 28 days of study (all animals) and at 14 days of recovery (all animals in the recovery groups). Blood was collected at ≥18 hours of fasting after treatment (all animals) and at ≥18 hours of fasting after recovery (all animals in the recovery groups).

Urinalysis parameters: urine volume, osmotic pressure, specific gravity, pH, color, protein levels, glucose levels, ketone bodies, bilirubin levels, occult blood, urobilinogen levels.
Blood chemistry parameters: total protein, albumin, A/G ratio, total bilirubin, GOT, GPT, gamma-GTP, ALP, total cholesterol, triglycerides, phospholipids, glucose, BUN, creatinine, inorganic phosphorus, Ca, Na, K and Cl.

Sacrifice and pathology:

All animals were sacrificed for necropsy. The absolute and relative weight of the brain, submandibular gland, heart, lungs (including bronchi), thymus, liver, spleen, kidneys, adrenal gland, testis and ovaries were measured. In addition to these organs, the pituitary gland, spinal cord, eyeball, salivary gland, thyroid gland, pancreases, stomach, bladder, femur (including bone marrow) and gross abnormal sites were collected.

Statistics:

For body weight, food intake, urinalysis (excluding qualitative reactions), hematology, blood biochemistry, organ weight, and body weight ratio organ weight, average values and standard deviations were determined for each group, and variance was determined by the Bartlett method. Uniformity was tested. If the variance was uniform, a one-way analysis of variance was performed. If there was a significant difference between groups in this analysis, a paired comparison test was performed for each group using the Dunnett method. If the variance was not uniform, a rank test was performed by the Kruskal-Wallis method. If a significant difference was found between groups, a Dunnett paired comparison test was performed. If there was no significant difference between groups in the above analysis of variance or rank test by the Kruskal-Wallis method, multiple comparisons between groups were not performed. In addition, Fisher's exact test was performed on the findings of histopathological examination. In all cases, the significance level was 5%.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There we no clinical signs at 8 or 40 mg/kg bw/day. Clinical signs at 200 mg/kg included excessive salivation, perinasal contamination, lacrimation, hypoactivity, slow breathing and hair loss. These symptoms except hair loss were short-lived during the treatment period, and after 14 days of recovery no clinical signs other than a few cases of hair loss were observed at 200 mg/kg.

Mortality:

no mortality observed

Description (incidence):

All animals survived until the time of planned death.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No significant changes in mean body weight were observed at 8 or 40 mg/kg bw/day. Male rats treated at 200 mg/kg showed markedly lower body weights from day 3 to 28 of treatment (mean, 17.5% decrease) and from day 1 to 10 of recovery (mean, 14.3% decrease) compared to the control data. During the recovery period, male rats treated at 200 mg/kg bw/day gained more weight than the control group and no significant difference in mean body weight was observed after 14 days of recovery. Female rats treated at 200 mg/kg bw/day showed less marked decreases in mean body weight compared to the control group and only at day 3, 7 and 10 of treatment (mean, 10.3% decrease).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food intake was unaffected by treatment up to 40 mg/kg. Male rats treated at 200 mg/kg showed reduced food intake at day 3 (by 66%) and 14 (by 20%) of study when compared to the control group. During recovery, male rats treated at 200 mg/kg showed increased food intake at day 3 and 14 when compared to the control group. Female rats treated at 200 mg/kg showed reduced food intake only at day 3 (by 75%) of study when compared to the control group. The influence of body weight changes on food intake were not investigated as food intake was expressed as g/day/rat.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Changes in blood biochemistry at 200 mg/kg at 28 days of study included increased protein levels, increased albumin levels, increased total cholesterol levels, increased phospholipid levels, increased calcium levels, and decreased chlorine levels. In addition, male rats treated at 200 mg/kg showed a decrease in alkaline phosphatase whereas female rats treated at 200 mg/kg showed an increase in alanine aminotransferase, an increase in triglyceride levels, an increase in glucose, and a decrease in sodium. These effects were not considered to be of toxicological significance because none of the effects were observed after 14 days of recovery and because there were no histological correlates other than hepatocyte hypertrophy.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Changes in blood biochemistry at 200 mg/kg at 28 days of study included increased protein levels, increased albumin levels, increased total cholesterol levels, increased phospholipid levels, increased calcium levels, and decreased chlorine levels. In addition, male rats treated at 200 mg/kg showed a decrease in alkaline phosphatase whereas female rats treated at 200 mg/kg showed an increase in alanine aminotransferase, an increase in triglyceride levels, an increase in glucose, and a decrease in sodium. These effects were not considered to be of toxicological significance because none of the effects were observed after 14 days of recovery and because there were no histological correlates other than hepatocyte hypertrophy.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Urinalysis data at 28 days of study showed increased urine volume (at 200 mg/kg), decreased osmotic pressure (at ≥40 mg/kg), decrease in specific gravity (at ≥40 mg/kg), decreased pH (at 200 mg/kg), and decreased protein levels (at 200 mg/kg) in both sexes when compared to the control data. These effects were considered to be of doubtful toxicological significance because no changes in urinalysis were observed after 14 days of recovery and because the microscopic examination of the kidneys showed renal damage only in 1 of 24 animals at 200 mg/kg.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Changes in organ weights included increased liver and kidney weights at 200 mg/kg in both sexes when compared to the control data.

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The microscopic examination of the animals not allowed to recover showed a significant increase in the incidence of hepatocyte hypertrophy in males at ≥40 mg/kg and in females at 200 mg/kg as well as a dose-dependent increase in the incidence of hyaline droplets in the proximal tubular epithelium in males at ≥8 mg/kg. The increase of hyaline droplets in the proximal tubular epithelium was not considered to be of toxicological significance. One male treated at 200 mg/kg showed increased infiltration of basophils in the proximal tubule epithelium, dilatation of the distal tubule, papillary necrosis, and dilatation of the pelvic cavity. No other histopathological findings were made.

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Effect level:

>= 40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

Dose descriptor:

LOAEL

Effect level:

<= 200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Critical effects observed:

no

Conclusions:

NOAEL for the test chemical was considered at 40 mg/kg bw/day in both sexes in this 28-days repeated dose toxicity study. The observed effects at 200 mg/kg bw/day showed full recovery or partial recovery and were therefore considered to be reversible. No signs of signifiant target organ toxicity were observed at necropsy.

Executive summary:

The chemical was given by oral gavage to 6 rats per sex per dose level at 0, 8, 40 and 200 mg/kg bw/day (main study). The study also included recovery groups of male and female rats (6 per sex per dose level) treated at 0 and 200 mg/kg bw/day. These animals were allowed to recover 2 weeks after the last dose was given. All animals survived until the time of planned death and there we no clinical signs at 8 or 40 mg/kg bw/day. Clinical signs at 200 mg/kg included excessive salivation, perinasal contamination, lacrimation, hypoactivity, slow breathing and hair loss. These symptoms except hair loss were short-lived during the treatment period, and after 14 days of recovery no clinical signs other than a few cases of hair loss were observed at 200 mg/kg. No significant changes in mean body weight were observed at 8 or 40 mg/kg bw/day. Male rats treated at 200 mg/kg showed markedly lower body weights from day 3 to 28 of treatment (mean, 17.5% decrease) and from day 1 to 10 of recovery (mean, 14.3% decrease) compared to the control data. During the recovery period, male rats treated at 200 mg/kg bw/day gained more weight than the control group and no significant difference in mean body weight was observed after 14 days of recovery. Female rats treated at 200 mg/kg bw/day showed less marked decreases in mean body weight compared to the control group and only at day 3, 7 and 10 of treatment (mean, 10.3% decrease). Food intake was unaffected by treatment up to 40 mg/kg. Male rats treated at 200 mg/kg showed reduced food intake at day 3 (by 66%) and 14 (by 20%) of study when compared to the control group. During recovery, male rats treated at 200 mg/kg showed increased food intake at day 3 and 14 when compared to the control group. Female rats treated at 200 mg/kg showed reduced food intake only at day 3 (by 75%) of study when compared to the control group. The influence of body weight changes on food intake were not investigated as food intake was expressed as g/day/rat. Urinalysis data at 28 days of study showed increased urine volume (at 200 mg/kg), decreased osmotic pressure (at ≥40 mg/kg), decrease in specific gravity (at ≥40 mg/kg), decreased pH (at 200 mg/kg), and decreased protein levels (at 200 mg/kg) in both sexes when compared to the control data. These effects were considered to be of doubtful toxicological significance because no changes in urinalysis were observed after 14 days of recovery and because the microscopic examination of the kidneys showed renal damage only in 1 of 24 animals at 200 mg/kg. Changes in blood biochemistry at 200 mg/kg at 28 days of study included increased protein levels, increased albumin levels, increased total cholesterol levels, increased phospholipid levels, increased calcium levels, and decreased chlorine levels. In addition, male rats treated at 200 mg/kg showed a decrease in alkaline phosphatase whereas female rats treated at 200 mg/kg showed an increase in alanine aminotransferase, an increase in triglyceride levels, an increase in glucose, and a decrease in sodium. These effects were not considered to be of toxicological significance because none of the effects were observed after 14 days of recovery and because there were no histological correlates other than hepatocyte hypertrophy. Changes in organ weights included increased liver and kidney weights at 200 mg/kg in both sexes when compared to the control data. The microscopic examination of the animals not allowed to recover showed a significant increase in the incidence of hepatocyte hypertrophy in males at ≥40 mg/kg and in females at 200 mg/kg as well as a dose-dependent increase in the incidence of hyaline droplets in the proximal tubular epithelium in males at ≥8 mg/kg. The increase of hyaline droplets in the proximal tubular epithelium was not considered to be of toxicological significance. One male treated at 200 mg/kg showed increased infiltration of basophils in the proximal tubule epithelium, dilatation of the distal tubule, papillary necrosis, and dilatation of the pelvic cavity. No other histopathological findings were made. NOAEL for the test chemical was considered at 40 mg/kg bw/day in both sexes in this 28-days repeated dose toxicity study. The observed effects at 200 mg/kg bw/day showed full recovery or partial recovery and were therefore considered to be reversible. No signs of signifiant target organ toxicity were observed at necropsy.

Cross-reference 2

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Data is from a secondary literature.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

other: Feed: Rodent Chow Diet.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted periodically for homogeneity and test article concentration levels.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily by dietary exposure

Remarks:

0, 200, 1000, 5000 ppm

No. of animals per sex per dose:

20 rats per sex per dose level.

Control animals:

yes, plain diet

Details on study design:

Four groups of Sprague Dawley albino rats (10/sex) were exposed to graded concentrations of 0, 200, 1,000, or 5,000 ppm test chemical in the diet for 13 weeks. An additional 10 rats/sex/group were designated as recovery rats and were retained for 4 weeks after the 13-week feeding period and received untreated rodent chow during that latter interval. Test article was prepared neat in a premix and subsequent diets prepared weekly.

Positive control:

Not used.

Observations and examinations performed and frequency:

Daily physical exams and clinical observations were performed on each animal. Body weights and food consumption were recorded weekly for each animal. Ophthalmoscopic exams were performed at study start and after 13 weeks on test for all animals. The following clinical exams were performed on each animal prior to necropsy: GLU (Glutamate), CK (Creatinine Kinase), ALT (Alanine Amino Transferase), AST (Aspartate Amino Transferase),ALKP (Alkaline phosphatase), GGT (gamma- glutamyl transferase) , BUN (blood urea nitrogen), CREA (Creatinine), Na (Sodium), K (Potassium), Ca (Calcium), Cl (Chloride), Phos (Phosphate), TPRO, ALB (Albumin), TBIL (T. Bilirubin), CHOL (Cholesterol), RBC (Red Blood Corpuscles), HGB (Hemoglobin), MCV (Mean Corpuscular Volume), WBC (White Blood Cells), PLAT (Platelets), GLOB (Globulin), A:G ratio (Albumin to Globulin ratio), HCT (Hematocrit), MHC (Mean Hematocrit Value), MCHC (Mean Corpuscular Hematorit Count), urine appearance, volume, Spec. grav., occult blood, protein, pH, ketones, urobilinogen, GLU (Glutamine), BILI (Bilirubin), sediments.

Sacrifice and pathology:

Complete necropsies were performed on all rats at study termination and a set of 46 tissues collected for microscopic exam. Histopathologic examinations were performed on all animals from the control and HD groups after 13 weeks, as well as lungs, liver, kidneys, and gross lesions from 200 ppm and 1000 ppm animals after 13 weeks. Absolute and relative organ (brain, gonads, heart, kidneys, liver and spleen) weights were recorded at necropsy. Gonads of all high dose and control animals were examined microscopically.

Other examinations:

No Data Available

Statistics:

Body weights and gains and food consumption and ratio data were evaluated using multivariate repeated-measure analysis of variance while other data were log-transformed and statistically analyzed using both multivariate and univariate two-factor fixed-effect analysis of variance (ANOVA). All comparisons for combined data of sexes were conducted using the Dunnett's test for multiple comparisons. A minimum significance level of p<0.05 was used throughout.

Clinical signs:

no effects observed

Description (incidence and severity):

No signs of test article-related clinical toxicity were observed during the 13-week treatment period, nor were any adverse signs noted during the recovery period.

Mortality:

no mortality observed

Description (incidence):

None of the test or recovery animals died during the 13-week feeding or 4-week treatment/recovery periods.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Male rats treated at 5000 ppm and female rats treated at ≥1000 ppm showed lower mean weekly body weights during the 13-week treatment period when compared to the control data. These effects were accompanied by reduced food intake. During recovery, increased food intake and/or body weight gain and increased food conversion ratios were observed during one or several weeks of recovery. The observed effects on mean weekly body weights were therefore attributed to poor palatability of the feeding materials.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Male rats treated at 5000 ppm and female rats treated at ≥1000 ppm showed lower mean weekly body weights during the 13-week treatment period when compared to the control data. These effects were accompanied by reduced food intake. During recovery, increased food intake and/or body weight gain and increased food conversion ratios were observed during one or several weeks of recovery. The observed effects on mean weekly body weights were therefore attributed to poor palatability of the feeding materials.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ocular manifestations of toxicity.

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment-related effects observed.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment-related effects observed.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No treatment-related effects observed.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No absolute organ weight changes attributable to treatment were observed, nor were there any gross lesions or histopathological effects related to treatment, including male and female gonads. The few statistically significant differences in relative weights observed in both sexes in the high dose group and mid dose females were attributed to their substantive decreased body weights seen at termination of treatment and not direct target organ toxicity.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related gross lesions were observed in this study.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related effects observed.

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

Critical effects observed:

no

Conclusions:

NOAEL for the test chemical was concluded at 5000 ppm in both sexes in this 90-days feeding study. By conversion for adult rats, 5000 ppm corresponds to 250 mg/kg bw/day.

Executive summary:

The chemical was given to 10 rats per sex per dose level at the dietary concentrations of 0, 200, 1000 and 5000 ppm for a total of 90 days. The study also included recovery groups of male and female rats (10 per sex per dose level) that were allowed to recover for 4 weeks after 90 days of treatment. No mortality and no clinical signs of toxicity were observed during the study period. Male rats treated at 5000 ppm and female rats treated at ≥1000 ppm showed lower mean weekly body weights during the 13-week treatment period when compared to the control data. These effects were accompanied by reduced food intake. During recovery, increased food intake and/or body weight gain and increased food conversion ratios were observed during one or several weeks of recovery. The observed effects on mean weekly body weights were therefore attributed to poor palatability of the feeding materials. No treatment-related effects on haematology, clinical chemistry, or urinalysis were observed, nor were there any ocular manifestations of toxicity. The few statistically significant differences noted in the above parameters lacked dose dependency, were within the historical control range or occurred only in recovery animals. No absolute organ weight changes attributed to the test chemical were observed. The few statistically significant differences in relative organ weights were attributed to decreased terminal body weights at ≥1000 ppm. No treatment-related gross pathology or histopathology were observed. NOAEL for the test chemical was concluded at 5000 ppm in both sexes in this 90-days feeding study. By conversion for adult rats, 5000 ppm corresponds to 250 mg/kg bw/day.

Data source

Reference

Reference Type:

other: secondary literature

Title:

WoE, Repeated dose toxicity of the test chemical

Author:

Sustainability Support Services (Europe) AB

Year:

2019

Bibliographic source:

SSS_RepeatDose_86293_2019

Materials and methods

Principles of method if other than guideline:

Study 2: Equivalent or similar to OECD 407
Study 3: According to OECD 408 (adopted 1981).

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Diphenylacetonitrile

EC Number:

201-662-5

EC Name:

Diphenylacetonitrile

Cas Number:

86-29-3

Molecular formula:

C14H11N

IUPAC Name:

2,2-diphenylacetonitrile

Test material form:

not specified

Details on test material:

Name: Diphenylacetonitrile
CAS No. 86-29-3
Molecular Weight: 193.248 g/mol
Molecular Formula: C14-H11-N
SMILES: C(c1ccccc1)(c1ccccc1)C#N

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Study 2: Crj: CD, Nippon Charles River Co., Ltd. Study 3: Sprague-Dawley (no further details)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Study 2: The body weight at the start of administration was 215.2-241.6 g for males and 144.7-171.9 g for females. Animals are placed in a stainless steel hanger cage in a barrier system breeding room set at a temperature of 24 ± 2 ° C, humidity of 55 ± 10%, lighting time from 7 to 19 o'clock, and ventilation rate of 13 times / hour. Grouping was performed by stratified randomization based on body weight the day before the start of treatment.

Study 3: No details.

Administration / exposure

Route of administration:

other: Study 2: Oral gavage. Study 3: Oral feed

Vehicle:

other: Study 2: methylcellulose; Study 3: Feed: Rodent Chow Diet.

Details on oral exposure:

Study 2: The dose volume was 5 ml / kg, and it was calculated based on the latest body weight for each individual.
Study 3: No further details

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Study 2: Not specified
Study 3: Analyses were conducted periodically for homogeneity and test article concentration levels.

Duration of treatment / exposure:

Study 2: 28 days
Study 3: 13 weeks

Frequency of treatment:

Study 2: Once Daily
Study 3: Daily by dietary exposure

Doses / concentrations

Doses / concentrations 1

Remarks:

0 (vehicle), 8, 40 and 200 mg/kg bw/day (Study 2)

Doses / concentrations 2

Remarks:

0, 200, 1000, 5000 ppm (Study 3)

No. of animals per sex per dose:

Study 2: 12 rats per sex per dose level at 0 and 200 mg/kg bw/day and 6 rats per sex per dose level at 8 and 40 mg/kg bw/day.
Study 3: 20 rats per sex per dose level.

Control animals:

yes, concurrent vehicle

yes, plain diet

Details on study design:

Study 2: The chemical was given by oral gavage to 6 rats per sex per dose level at 0, 8, 40 and 200 mg/kg bw/day (main study). The study also included recovery groups of male and female rats (6 per sex per dose level) treated at 0 and 200 mg/kg bw/day. These animals were allowed to recover 2 weeks after the last dose was given. Dose levels were selected based on the results of dose range-finding study in which male and female rats were treated at 0, 30, 100 and 300 mg/kg bw/day for two weeks. In this study, treatment at ≥100 mg/kg resulted in growth inhibition and changes in blood biochemistry and
organ weights.

Study 3: Four groups of Sprague Dawley albino rats (10/sex) were exposed to graded concentrations of 0, 200, 1,000, or 5,000 ppm test chemical in the diet for 13 weeks. An additional 10 rats/sex/group were designated as recovery rats and were retained for 4 weeks after the 13-week feeding period and
received untreated rodent chow during that latter interval. Test article was prepared neat in a premix and subsequent diets prepared weekly.

Positive control:

Study 2 and 3: No positive control used.

Examinations

Observations and examinations performed and frequency:

Study 2: The animals were examined three times per day for mortality and clinical signs. Body weight and food intake were recorded twice a week during treatment and recovery. Urinalysis was performed at 28 days of study (all animals) and at 14 days of recovery (all animals in the recovery groups). Blood was collected at ≥18 hours of fasting after treatment (all animals) and at ≥18 hours of fasting after recovery (all animals in the recovery groups). Urinalysis parameters: urine volume, osmotic pressure, specific gravity, pH, color, protein levels, glucose levels, ketone bodies, bilirubin levels, occult blood, urobilinogen levels. Blood chemistry parameters: total protein, albumin, A/G ratio, total bilirubin, GOT, GPT, gamma-GTP, ALP, total cholesterol, triglycerides, phospholipids, glucose, BUN, creatinine, inorganic phosphorus, Ca, Na, K and Cl.

Study 3: Daily physical exams and clinical observations were performed on each animal. Body weights and food consumption were recorded weekly for each animal. Ophthalmoscopic exams were performed at study start and after 13 weeks on test for all animals. The following clinical exams were performed on each animal prior to necropsy: GLU (Glutamate), CK (Creatinine Kinase), ALT (Alanine Amino Transferase), AST (Aspartate Amino Transferase),ALKP (Alkaline phosphatase), GGT (gamma- glutamyl transferase) , BUN (blood urea nitrogen), CREA (Creatinine), Na (Sodium), K (Potassium), Ca (Calcium), Cl (Chloride), Phos (Phosphate), TPRO, ALB (Albumin), TBIL (T. Bilirubin), CHOL (Cholesterol), RBC (Red Blood Corpuscles), HGB (Hemoglobin), MCV (Mean COrpuscular Volume), WBC (White Blood Cells), PLAT (Platelets), GLOB (Globulin), A:G ratio (Albumin to Globulin ratio), HCT (Hematocrit), MHC (Mean Hematocrit Value), MCHC (Mean Corpuscular Hematorit Count), urine appearance, volume, Spec. grav., occult blood, protein, pH, ketones, urobilinogen, GLU (Glutamine), BILI (Bilirubin), sediments.

Sacrifice and pathology:

Study 2: All animals were sacrificed for necropsy. The absolute and relative weight of the brain, submandibular gland, heart, lungs (including bronchi), thymus, liver, spleen, kidneys, adrenal gland, testis and ovaries were measured. In addition to these organs, the pituitary gland, spinal cord, eyeball, salivary gland, thyroid gland, pancreases, stomach, bladder, femur (including bone marrow) and gross abnormal sites were collected.

Study 3: Complete necropsies were performed on all rats at study termination and a set of 46 tissues collected for microscopic exam. Histopathologic examinations were performed on all animals from the control and HD groups after 13 weeks, as well as lungs, liver, kidneys, and gross lesions from 200 ppm and 1000 ppm animals after 13 weeks. Absolute and relative organ (brain, gonads, heart, kidneys, liver and spleen) weights were recorded at necropsy. Gonads of all high dose and control animals were examined microscopically.

Statistics:

Study 2: For body weight, food intake, urinalysis (excluding qualitative reactions), hematology, blood biochemistry, organ weight, and body weight ratio organ weight, average values and standard deviations were determined for each group, and variance was determined by the Bartlett method. Uniformity was tested. If the variance was uniform, a one-way analysis of variance was performed. If there was a significant difference between groups in this analysis, a paired comparison test was performed for each group using the Dunnett method. If the variance was not uniform, a rank test was performed by the Kruskal-Wallis method. If a significant difference was found between groups, a Dunnett paired comparison test was performed. If there was no significant difference between groups in the above analysis of variance or rank test by the Kruskal-Wallis method, multiple comparisons between groups were not performed. In addition, Fisher's exact test was performed on the findings of histopathological examination. In all cases, the significance level was 5%.

Study 3: Body weights and gains and food consumption and ratio data were evaluated using multivariate repeated-measure analysis of variance while other data were log-transformed and statistically analyzed using both multivariate and univariate two-factor fixed-effect analysis of variance (ANOVA). All comparisons for combined data of sexes were conducted using the Dunnett's test for multiple comparisons. A minimum significance level of p<0.05 was used throughout.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Study 2: There we no clinical signs at 8 or 40 mg/kg bw/day. Clinical signs at 200 mg/kg included excessive salivation, perinasal contamination, lacrimation, hypoactivity, slow breathing and hair loss. These symptoms except hair loss were short-lived during the treatment period, and after 14 days of recovery no clinical signs other than a few cases of hair loss were observed at 200 mg/kg.

Study 3: No signs of test article-related clinical toxicity were observed during the 13-week treatment period, nor were any adverse signs noted during the recovery period.

Mortality:

no mortality observed

Description (incidence):

Study 2 and 3: No mortality was observed until planned death.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Study 2: No significant changes in mean body weight were observed at 8 or 40 mg/kg bw/day. Male rats treated at 200 mg/kg showed markedly lower body weights from day 3 to 28 of treatment (mean, 17.5% decrease) and from day 1 to 10 of recovery (mean, 14.3% decrease) compared to the control data. During the recovery period, male rats treated at 200 mg/kg bw/day gained more weight than the control group and no significant difference in mean body weight was observed after 14 days of recovery. Female rats treated at 200 mg/kg bw/day showed less marked decreases in mean body weight compared to the control group and only at day 3, 7 and 10 of treatment (mean, 10.3% decrease).

Study 3: Male rats treated at 5000 ppm and female rats treated at ≥1000 ppm showed lower mean weekly body weights during the 13-week treatment period when compared to the control data. These effects were accompanied by reduced food intake. During recovery, increased food intake and/or body weight gain andincreased food conversion ratios were observed during one or several weeks of recovery. The observed effects on mean weekly body weights were therefore attributed to poor palatability of the feeding materials.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Study 2: Food intake was unaffected by treatment up to 40 mg/kg. Male rats treated at 200 mg/kg showed reduced food intake at day 3 (by 66%) and 14 (by 20%) of study when compared to the control group. During recovery, male rats treated at 200 mg/kg showed increased food intake at day 3 and 14 when compared to the control group. Female rats treated at 200 mg/kg showed reduced food intake only at day 3 (by 75%) of study when compared to the control group. The influence of body weight changes on food intake were not investigated as food intake was expressed as g/day/rat.

Study 3: Male rats treated at 5000 ppm and female rats treated at ≥1000 ppm showed lower mean weekly body weights during the 13-week treatment period when compared to the control data. These effects were accompanied by reduced food intake. During recovery, increased food intake and/or body weight gain and increased food conversion ratios were observed during one or several weeks of recovery. The observed effects on mean weekly body weights were therefore attributed to poor palatability of the feeding materials.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Study 2: Not examined.
Study 3: There were no ocular manifestations of toxicity.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Study 2: Changes in blood biochemistry at 200 mg/kg at 28 days of study included increased protein levels, increased albumin levels, increased total cholesterol levels, increased phospholipid levels, increased calcium levels, and decreased chlorine levels. In addition, male rats treated at 200 mg/kg showed a decrease in alkaline phosphatase whereas female rats treated at 200 mg/kg showed an increase in alanine aminotransferase, an increase in triglyceride levels, an increase in glucose, and a decrease in sodium. These effects were not considered to be of toxicological significance because none of the effects were observed after 14 days of recovery and because there were no histological correlates other than hepatocyte hypertrophy.

Study 3: No treatment-related effects observed.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Study 2: Changes in blood biochemistry at 200 mg/kg at 28 days of study included increased protein levels, increased albumin levels, increased total cholesterol levels, increased phospholipid levels, increased calcium levels, and decreased chlorine levels. In addition, male rats treated at 200 mg/kg showed a decrease in alkaline phosphatase whereas female rats treated at 200 mg/kg showed an increase in alanine aminotransferase, an increase in triglyceride levels, an increase in glucose, and a decrease in sodium. These effects were not considered to be of toxicological significance because none of the effects were observed after 14 days of recovery and because there were no histological correlates other than hepatocyte hypertrophy.

Study 3: No treatment-related effects observed.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Study 2: Urinalysis data at 28 days of study showed increased urine volume (at 200 mg/kg), decreased osmotic pressure (at ≥40 mg/kg), decrease in specific gravity (at ≥40 mg/kg), decreased pH (at 200 mg/kg), and decreased protein levels (at 200 mg/kg) in both sexes when compared to the control data. These effects were considered to be of doubtful toxicological significance because no changes in urinalysis were observed after 14 days of recovery and because the microscopic examination of the kidneys showed renal damage only in 1 of 24 animals at 200 mg/kg.

Study 3: No treatment-related effects observed.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Study 2: Changes in organ weights included increased liver and kidney weights at 200 mg/kg in both sexes when compared to the control data.

Study 3: No absolute organ weight changes attributable to treatment were observed, nor were there any gross lesions or histopathological effects related to treatment, including male and female gonads. The few statistically significant differences in relative weights observed in both sexes in the high dose group and mid dose females were attributed to their substantive decreased body weights seen at termination of treatment and not direct target organ toxicity.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Study 2: Not specified.

Study 3:No treatment-related gross lesions were observed in this study.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Study 2: The microscopic examination of the animals not allowed to recover showed a significant increase in the incidence of hepatocyte hypertrophy in males at ≥40 mg/kg and in females at 200 mg/kg as well as a dose-dependent increase in the incidence of hyaline droplets in the proximal tubular epithelium in males at ≥8 mg/kg. The increase of hyaline droplets in the proximal tubular epithelium was not considered to be of toxicological significance. One male treated at 200 mg/kg showed increased infiltration of basophils in the proximal tubule epithelium, dilatation of the distal tubule, papillary necrosis, and dilatation of the pelvic cavity. No other histopathological findings were made.

Study 3: No treatment-related effects observed.

Histopathological findings: neoplastic:

no effects observed

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

>= 40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

Remarks on result:

other: Study 2

2

Dose descriptor:

LOAEL

Effect level:

<= 200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Remarks on result:

other: Study 2

3

Dose descriptor:

NOAEL

Effect level:

>= 5 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

Remarks on result:

other: Study 3

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the available read-across data, NOAEL for the target chemical in rats is expected to be in the region of 40 to 250 mg/kg bw/day upon repeated exposure of at least 28 days.

Executive summary:

Repeated Dose Toxicity Study:

Data for read-across chemicals were evaluated for repeated dose toxicity by the oral route. The studies are summarised below.

Study 2:

The read-across chemical was given by oral gavage to 6 rats per sex per dose level at 0, 8, 40 and 200 mg/kg bw/day (main study). The study also included recovery groups of male and female rats (6 per sex per dose level) treated at 0 and 200 mg/kg bw/day. These animals were allowed to recover 2 weeks after the last dose was given.All animals survived until the time of planned death and there we no clinical signs at 8 or 40 mg/kg bw/day. Clinical signs at 200 mg/kg included excessive salivation, perinasal contamination, lacrimation, hypoactivity, slow breathing and hair loss. These symptoms except hair loss were short-lived during the treatment period, and after 14 days of recovery no clinical signs other than a few cases of hair loss were observed at 200 mg/kg. No significant changes in mean body weight were observed at 8 or 40 mg/kg bw/day. Male rats treated at 200 mg/kg showed markedly lower body weights from day 3 to 28 of treatment (mean, 17.5% decrease) and from day 1 to 10 of recovery (mean, 14.3% decrease) compared to the control data. During the recovery period, male rats treated at 200 mg/kg bw/day gained more weight than the control group and no significant difference in mean body weight was observed after 14 days of recovery. Female rats treated at 200 mg/kg bw/day showed less marked decreases in mean body weight compared to the control group and only at day 3, 7 and 10 of treatment (mean, 10.3% decrease). Food intake was unaffected by treatment up to 40 mg/kg. Male rats treated at 200 mg/kg showed reduced food intake at day 3 (by 66%) and 14 (by 20%) of study when compared to the control group. During recovery, male rats treated at 200 mg/kg showed increased food intake at day 3 and 14 when compared to the control group. Female rats treated at 200 mg/kg showed reduced food intake only at day 3 (by 75%) of study when compared to the control group. The influence of body weight changes on food intake were not investigated as food intake was expressed as g/day/rat. Urinalysis data at 28 days of study showed increased urine volume (at 200 mg/kg), decreased osmotic pressure (at ≥40 mg/kg), decrease in specific gravity (at ≥40 mg/kg), decreased pH (at 200 mg/kg), and decreased protein levels (at 200 mg/kg) in both sexes when compared to the control data. These effects were considered to be of doubtful toxicological significance because no changes in urinalysis were observed after 14 days of recovery and because the microscopic examination of the kidneys showed renal damage only in 1 of 24 animals at 200 mg/kg. Changes in blood biochemistry at 200 mg/kg at 28 days of study included increased protein levels, increased albumin levels, increased total cholesterol levels, increased phospholipid levels, increased calcium levels, and decreased chlorine levels. In addition, male rats treated at 200 mg/kg showed a decrease in alkaline phosphatase whereas female rats treated at 200 mg/kg showed an increase in alanine aminotransferase, an increase in triglyceride levels, an increase in glucose, and a decrease in sodium. These effects were not considered to be of toxicological significance because none of the effects were observed after 14 days of recovery and because there were no histological correlates other than hepatocyte hypertrophy. Changes in organ weights included increased liver and kidney weights at 200 mg/kg in both sexes when compared to the control data. The microscopic examination of the animals not allowed to recover showed a significant increase in the incidence of hepatocyte hypertrophy in males at ≥40 mg/kg and in females at 200 mg/kg as well as a dose-dependent increase in the incidence of hyaline droplets in the proximal tubular epithelium in males at ≥8 mg/kg. The increase of hyaline droplets in the proximal tubular epithelium was not considered to be of toxicological significance. One male treated at 200 mg/kg showed increased infiltration of basophils in the proximal tubule epithelium, dilatation of the distal tubule, papillary necrosis, and dilatation of the pelvic cavity. No other histopathological findings were made. NOAEL for the test chemical was considered at 40 mg/kg bw/day in both sexes in this 28-days repeated dose toxicity study. The observed effects at 200 mg/kg bw/day showed full recovery or partial recovery and were therefore considered to be reversible. No signs of signifiant target organ toxicity were observed at necropsy.

Study 3:

The read-across chemical was given to 10 rats per sex per dose level at the dietary concentrations of 0, 200, 1000 and 5000 ppm for a total of 90 days. The study also included recovery groups of male and female rats (10 per sex per dose level) that were allowed to recover for 4 weeks after 90 days of treatment. No mortality and no clinical signs of toxicity were observed during the study period. Male rats treated at 5000 ppm and female rats treated at ≥1000 ppm showed lower mean weekly body weights during the 13-week treatment period when compared to the control data. These effects were accompanied by reduced food intake. During recovery, increased food intake and/or body weight gain and increased food conversion ratios were observed during one or several weeks of recovery. The observed effects on mean weekly body weights were therefore attributed to poor palatability of the feeding materials. No treatment-related effects on haematology, clinical chemistry, or urinalysis were observed, nor were there any ocular manifestations of toxicity. The few statistically significant differences noted in the above parameters lacked dose dependency, were within the historical control range or occurred only in recovery animals. No absolute organ weight changes attributed to the test chemical were observed. The few statistically significant differences in relative organ weights were attributed to decreased terminal body weights at ≥1000 ppm. No treatment-related gross pathology or histopathology were observed. NOAEL for the test chemical was concluded at 5000 ppm in both sexes in this 90-days feeding study. By conversion for adult rats, 5000 ppm corresponds to 250 mg/kg bw/day.

Based on the available read-across data, NOAEL for the target chemical in rats is expected to be in the region of 40 to 250 mg/kg bw/day upon repeated exposure of at least 28 days.