Tricyclo[8.2.2.24,7]hexadeca-1(12),4,6,10,13,15-hexaene

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Remarks:

sub-acute oral repeated dose toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

1

Reference substance name:

Dichlorotricyclo[8.2.2.24,7]hexadeca-1(12),4,6,10,13,15-hexaene, mixed isomers

EC Number:

249-236-8

EC Name:

Dichlorotricyclo[8.2.2.24,7]hexadeca-1(12),4,6,10,13,15-hexaene, mixed isomers

Cas Number:

28804-46-8

Molecular formula:

C16H14Cl2

IUPAC Name:

Unknown constituents

Test material form:

solid: granular

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 days

Frequency of treatment:

once a day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

450 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 animals/sex/group, total six groups

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

Each rat was observed twice daily for clinical signs, morbidity and mortality during the study period. Body weight was recorded on day 1 (prior to treatment) and at weekly intervals thereafter during the treatment and recovery periods. Feed consumption was calculated at weekly intervals until terminal and recovery sacrifices. Detailed clinical/Neurobehavioral observations (NBO) were conducted on each rat once prior to treatment initiation and at weekly intervals thereafter. Ophthalmological examination was made on all animals from each group prior to the initiation of treatment and pre-sacrifice. Functional observational battery (FOB) test was performed on all animals (G1 to G4) during the 4th week of treatment and 2nd week of the recovery period (G5 and G6). Haematological and clinical chemistry analyses were performed at the end of the treatment (G1 to G4) and recovery periods (G5 and G6). Urine samples were collected from all animals at the end of the treatment (G1 to G4) and recovery periods (G5 and G6) for urine analysis.

All the rats were sacrificed by carbon dioxide asphyxiation and subjected to gross pathological examination at the end of the treatment (G1 to G4) and recovery (G5 and G6) periods. Absolute organ weights were recorded and organ weights relative to terminal body weight were calculated for the organs viz., adrenals, brain, ovaries, testes, uterus with cervix, prostate + seminal vesicles and coagulating glands, epididymides, heart, kidneys, liver, spleen, thymus, thyroid and parathyroid in all the rats. Detailed histopathological examination was carried out in the control and high dose groups while the examination was extended for liver, thyroid of low, mid and recovery groups of both sexes while kidney from low, mid and recovery group of males.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No sign of toxicity was observed in animals treated from low group, while, salivation was observed in mid dose from day 21 to 28 [male (2/5) and female (3/5)] and high dose from day 18 to 28 [male (5/5) and female (5/5)]. Salivation could be considered due to test item treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically, significant decrease in terminal body weight was observed in male rats of mid and high dose groups when compared with vehicle control group. Similarly, inspite of statistical significant decrease in terminal body weight was biologically significant in female rats of high dose group when compared with vehicle control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean feed consumption male and female rats of treated groups was comparable with vehicle control group.

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically, significant increase in RBC and monocyte were observed male rats of high dose group when compared with vehicle control group. Variation in RBC and monocyte could be considered as treatment related effect.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically, significant increase in cholesterol, triglyceride, ALT and calcium were observed in either sex of high dose group when compared with vehicle control group except in female rats increase in triglyceride was statistically non significant (353% of control). Statistically, significant increase in creatinine level was observed in male rats of high dose group when compared with vehicle control group while in female, inspite of statistical significant, creatinine was biologically increase i.e. 10% of vehicle control. Statistically, significant increase in ALP was observed in female rats of high dose group when compared with vehicle control group. Statistically, significant decrease in chloride and albumin: globulin ratio was observed in female rats of high dose group when compared with vehicle control group. These variations were co-related with findings of dose range finding study, hence, they could be considered as treatment related effects.

During recovery period, the alterations observed in ALT, triglyceride, creatinine, ALP, chloride and albumin: globulin ratio were recovered. Also the effects of increase in cholesterol and calcium were diminished in males while continued in females.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Significant increase was observed in volume of urine in high dose (G4) males and females though statistical significance was registerd for males only. High dose females revealed statistically significant decrease in specific gravity and increase in pH. These effects could be related to test item treatment.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistically, significant increase relative weight of liver were observed in mid and high dose group of both sexes and low dose of females when compared with vehicle control group. Statistically, significant increase relative weight of thyroid and parathyroid in high dose group of both sexes when compared with vehicle control group. This effect was also observed in mid dose and low dose groups (without statistical support) in dose dependent manner.
In absolute weights of kidneys significant increase was registered in high dose males. It was supported by treatment related histopathological lesions in kidneys of only male animals. Increase in absolute weights was also noticed in mid dose males though it was devoid of statistical significance.
There was significant decrease in absolute weight of heart in high dose females. It was also observed in high dose males though it lacked statistical significance. Significant decrease in absolute weight was also observed in brain of G4 males. Decrease in absolute weight (without statistical support) was also observed in weight of prostate plus seminal vesicles in high dose. These effects were considered as related to test item treatment.
Some of the effects observed at the end of treatment (increase in relative weight of thyroid in females and kidneys in males; and decrease in weight of prostate and terminal body weight in males) were continued with reduced severity after recovery period. Other effects were almost completely recovered after ceasing the treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

External examination of terminally and recovery sacrificed animals of either sex across various groups (G1 to G6) did not reveal any abnormality.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Internal examination of animals revealed enlargement of liver in all animal of high dose group of both sexes while in 2 animals (Animal No 21 and 26) from mid dose group
Test item treatment was associated with hypertrophy of hepatocyte (centriolobular and/or diffuse) in mid dose and high dose group. Severity and incidence was more in mid dose males than mid dose females. The lesion was also observed in low dose males.
Test item treatment was also associated with follicular hypertrophy (thyroid) in all 3 treated groups. Treatments also lead to hyaline droplets accumulation in cortical tubules in kidneys of male rats of all 3 treated groups. This effect was not observed in female animals. In treated males, all dose groups showed minimal to mild severity of basophilic/ regenerating tubules in the cortex.
In treated females, hepatocellular hypertrophy was recovered completely while in males thought it was continued, reduction in incidence and severity was observed after recovery period. Though follicular hypertrophy was observed in one treated female, marked recovery was observed at the end of recovery period.
For the lesion of hyaline droplets in kidneys, complete recovery was observed while basophilic/regenerative tubules were continued in treated male animal.

Histopathological findings: neoplastic:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Basis for effect level:

body weight and weight gain

clinical biochemistry

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

urinalysis

Target system / organ toxicity

1

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

50 mg/kg bw/day (nominal)

2

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

oral cavity

Treatment related:

yes

3

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

System:

musculoskeletal system

Organ:

other: body weight

Treatment related:

yes

4

Critical effects observed:

yes

Lowest effective dose / conc.:

450 mg/kg bw/day (nominal)

System:

cardiovascular

Organ:

blood

heart

Treatment related:

yes

5

Critical effects observed:

yes

Lowest effective dose / conc.:

450 mg/kg bw/day (nominal)

System:

urinary

Organ:

other: urine

Treatment related:

yes

6

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

7

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

System:

endocrine system

Organ:

parathyroid gland

thyroid gland

Treatment related:

yes

8

Critical effects observed:

yes

Lowest effective dose / conc.:

450 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

9

Critical effects observed:

yes

Lowest effective dose / conc.:

450 mg/kg bw/day (nominal)

System:

nervous system

Organ:

brain

Treatment related:

yes

10

Critical effects observed:

yes

Lowest effective dose / conc.:

450 mg/kg bw/day (nominal)

System:

male reproductive system

Organ:

seminal vesicle

other: prostate

Treatment related:

yes

Applicant's summary and conclusion

Conclusions:

Based on the above findings, it is concluded that the DPX-C (Di-Cloro-Di-p-Xililene) did not produce any toxicity or adverse effect at dose level 50 mg/kg b. wt./day when administered orally through gavage for 28 consecutive days. Therefore, the No Observed Adverse Effect Level (NOAEL) of DPX-C (Di-Cloro-Di-p-Xililene) is 50 mg/kg b. wt./day when administered through oral gavage up to 28 days in Wistar rats under the conditions and procedure followed in the present study.

Executive summary:

EXECUTIVE SUMMARY:This study was conducted to evaluate the potential toxicity of DPX-C (Di-Cloro-Di-p-Xililene) in Wistar rats following daily administration through oral gavage for at least 28 days.

METHOD:

Test System Route Doses Duration of Treatment N° of animals Vehicle

Wistar Rat Oral gavage 0, 50, 150 and 450 mg/kg b. wt./day Treatment period: 28 days; Recovery period: 14 days 5 animals/sex/group, total six groups Corn oil

 

The dose formulation was prepared fresh everyday prior to dosing by mixing the test item separately with vehicle. Three samples of dose formulation from each concentration and one from control were collected on days 1 and 28 of dosing for homogeneity and active ingredient concentration analysis.

Each rat was observed twice daily for clinical signs, morbidity and mortality during the study period. Body weight was recorded on day 1 (prior to treatment) and at weekly intervals thereafter during the treatment and recovery periods. Feed consumption was calculated at weekly intervals until terminal and recovery sacrifices. Detailed clinical/Neurobehavioral observations (NBO) were conducted on each rat once prior to treatment initiation and at weekly intervals thereafter.Ophthalmological examination was made on all animals from each group prior to the initiation of treatment and pre-sacrifice.Functional observational battery (FOB) test was performed on all animals (G1 to G4) during the 4^thweek of treatment and 2^ndweek of the recovery period (G5 and G6). Haematological and clinical chemistry analyses were performed at the end of the treatment (G1 to G4) and recovery periods (G5 and G6). Urine samples were collected from all animals at the end of the treatment (G1 to G4) and recovery periods (G5 and G6) for urine analysis.

All the rats were sacrificed by carbon dioxide asphyxiation and subjected to gross pathological examination at the end of the treatment (G1 to G4) and recovery (G5 and G6) periods. Absolute organ weights were recorded and organ weights relative to terminal body weight were calculated for the organs viz.,adrenals, brain, ovaries, testes, uterus with cervix, prostate + seminal vesicles and coagulating glands, epididymides, heart, kidneys, liver, spleen, thymus, thyroid and parathyroidin all the rats. Detailed histopathological examination was carried out in the control and high dose groups while the examination was extended forliver, thyroid of low, mid and recovery groups of both sexes while kidney from low, mid and recovery group of males.

RESULTS:

The results of dose formulation analyses were within acceptable range of ± 15 % of nominal concentration.

No mortality and morbidity was observed during study period.

No sign of toxicity was observed in animals treated from low group, while, salivation was observed in mid dose from day 21 to 28 [male (2/5) and female (3/5)] and high dose from day 18 to 28 [male (5/5) and female (5/5)]. Salivation could be considered due to test item treatment.

No treatment related changes were observed in neurobehavioral observations and functional observation battery of treated group animals.

Ophthalmological examination did not reveal any abnormality in vehicle control and treatment groups.

Treatment related decrease in body weight and body weight change were observed in treated groups when compared with vehicle control group except in low dose group the reduction was very low i.e. 1 to 8% of vehicle control.

Mean feed consumption male and female rats of treated groups was comparable with vehicle control group.

Statistically, significant increase in RBC and monocyte were observed male rats of high dose group when compared with vehicle control group. Variation in RBC and monocyte could be considered as treatment related effect.

Statistically, significant increase in cholesterol, triglyceride, ALT and calcium were observed in either sex of high dose group when compared with vehicle control group except in female rats increase in triglyceride was statistically non significant (353% of control). Statistically, significant increase in creatinine level was observed in male rats of high dose group when compared with vehicle control group while in female, inspite of statistical significant, creatinine was biologically increase i.e. 10% of vehicle control. Statistically, significant increase in ALP was observed in female rats of high dose group when compared with vehicle control group. Statistically, significant decrease in chloride and albumin: globulin ratio was observed in female rats of high dose group when compared with vehicle control group. These variations were co-related with findings of dose range finding study, hence, they could be considered as treatment related effects.

During recovery period, the alterations observed in ALT, triglyceride, creatinine, ALP, chloride and albumin: globulin ratio were recovered. Also the effects of increase in cholesterol and calcium were diminished in males while continued in females.

Significant increase was observed in volume of urine in high dose (G4) males and females though statistical significance was registerd for males only. High dose females revealed statistically significant decrease in specific gravity and increase in pH. These effects could be related to test item treatment.

Statistically, significant decrease in terminal body weight was observed in male rats of mid and high dose groups when compared with vehicle control group. Similarly, inspite of statistical significant decrease in terminal body weight was biologically significant in female rats of high dose group when compared with vehicle control group.

Statistically, significant increase relative weight of liver were observed in mid and high dose group of both sexes and low dose of females when compared with vehicle control group. Statistically, significant increase relative weight of thyroid and parathyroid in high dose group of both sexes when compared with vehicle control group. This effect was also observed in mid dose and low dose groups (without statistical support) in dose dependent manner.

In absolute weights of kidneys significant increase was registered in high dose males. It was supported by treatment related histopathological lesions in kidneys of only male animals. Increase in absolute weights was also noticed in mid dose males though it was devoid of statistical significance.

There was significant decrease in absolute weight of heart in high dose females. It was also observed in high dose males though it lacked statistical significance. Significant decrease in absolute weight was also observed in brain of G4 males. Decrease in absolute weight (without statistical support) was also observed in weight of prostate plus seminal vesicles in high dose. These effects were considered as related to test item treatment.

Some of the effects observed at the end of treatment (increase in relative weight of thyroid in females and kidneys in males; and decrease in weight of prostate and terminal body weight in males) were continued with reduced severity after recovery period. Other effects were almost completely recovered after ceasing the treatment.

External examination of terminally and recovery sacrificed animals of either sex across various groups (G1 to G6) did not reveal any abnormality.

Internal examination of animals revealed enlargement of liver in all animal of high dose group of both sexes while in 2 animals (Animal No 21 and 26) from mid dose group

Test item treatment was associated with hypertrophy of hepatocyte (centriolobular and/or diffuse) in mid dose and high dose group. Severity and incidence was more in mid dose males than mid dose females. The lesion was also observed in low dose males.

Test item treatment was also associated with follicular hypertrophy (thyroid) in all 3 treated groups. Treatments also lead to hyaline droplets accumulation in cortical tubules in kidneys of male rats of all 3 treated groups. This effect was not observed in female animals. In treated males, all dose groups showed minimal to mild severity of basophilic/ regenerating tubules in the cortex.

In treated females, hepatocellular hypertrophy was recovered completely while in males thought it was continued, reduction in incidence and severity was observed after recovery period. Though follicular hypertrophy was observed in one treated female, marked recovery was observed at the end of recovery period.

For the lesion of hyaline droplets in kidneys, complete recovery was observed while basophilic/regenerative tubules were continued in treated male animal.

CONCLUSION:

Based on the above findings, it is concluded that the DPX-C (Di-Cloro-Di-p-Xililene) did not produce any toxicity or adverse effect at dose level 50 mg/kg b. wt./day when administered orally through gavage for 28 consecutive days. Therefore, the No Observed Adverse Effect Level (NOAEL) of DPX-C (Di-Cloro-Di-p-Xililene) is 50 mg/kg b. wt./day when administered through oral gavage up to 28 days in Wistar rats under the conditions and procedure followed in the present study.