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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
june-november 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
Appearance: White crystaline solid
Storage Temperature : Ambient condition

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Healthy, young adult rats (Rattus norvegicus) of Wistar (RccHan: WIST) strain obtained from the JRF Animal Breeding Facility (ABF) were used for the study. Female rats selected for the study were nulliparous and non-pregnant. At initiation of dosing, rats were 7 to 9 weeks old and the body weight variation among the rats were within ±20% of the mean body weight for each sex.
Sex:
male/female
Details on test animals and environmental conditions:
Rats were maintained in an environmentally controlled room. The photoperiod was 12 h light and 12 h darkness; light hours were 06.00 - 18.00 h which was maintained by fluorescent tube lights using autotimer. the relative humidity ranged from 65-67% and air change was done 16-17 per hour.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Dose formulation was administered once daily by oral gavage, using an intubation cannula attachedto a graduated syringe. A constant dose volume of 10 mL/kg b. wt. was used and individual doses were adjusted according to the most recently recorded body weight of each rat. The vehicle control group rats received the corn oil.
Vehicle:
corn oil
Details on oral exposure:
Rats were treated with test item for a period of 90 consecutive days. The first day of dose administration was designated as day 1 for each rat.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Based on results from a prestudy stability study, the test item was found to be stable for up to 24 hours (h) in dose formulation using corn oil as the vehicle. Three samples of dose formulation (upper, middle, and lower layers) from each concentration and one sample from the vehicle control (middle layer) were collected on days 1, 29, 57, and 85 of dosing for homogeneity and active ingredient (a.i.) concentration analysis.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Rats were treated with test item for a period of 90 consecutive days
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
120 mg/kg bw/day (actual dose received)
Dose / conc.:
480 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 animals per group
Control animals:
yes, concurrent vehicle
Details on study design:
Each rat was observed, twice daily for clinical signs, morbidity, and mortality during the treatment period. Body weights were recorded weekly for all rats. Body weight change and food consumption were calculated weekly. Neurobehavioural observation (NBO) was performed on all rats prior to the initiation of treatment and weekly, thereafter. Ophthalmological examination was performed on all rats from each group prior to the initiation of treatment and on surviving rats prior to terminal sacrifice. A Functional Observational Battery (FOB) was performed on all rats during the 12th week of treatment. Haematological and clinical chemistry analyses were performed at the end of the treatment. Urine was collected from all surviving rats, at the end of the treatment for urine analysis.


All surviving rats were sacrificed by carbon dioxide asphyxiation at end of the treatment period. All surviving and found dead rats were subjected to a full gross necropsy under the direct supervision of a veterinary pathologist. Absolute organ weights were recorded, and relative organ weights were calculated for the adrenals, testes, ovaries, epididymides, uterus with cervix, kidneys, brain, heart, spleen, liver, thymus, thyroid and parathyroid. Detailed histopathological examination was carried out in all rats from the control and high dose groups. Adrenals from both sexes, liver from female rats, and kidneys from male rats in the mid and low dose groups were also processed and evaluated as treatment related lesions were observed in these tissues from high dose group rats.

Examinations

Statistics:
Raw data were recorded in standard formats and processed to get group means and standard deviations with significance among the vehicle control and treatment groups using an in-house validated statistical software. Data were summarised in tabular form. All parameters characterised by continuous data - body weight, body weight change, food consumption, urination, defaecation, rearing, organ weight, relative organ weight, haematology, and clinical chemistry -were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s test (Gad, S.C. and Weil, C.S., 1994). Where the data do not meet the homogeneity of variance, Student’s t-test was performed to calculate significance.
All analyses and comparisons were evaluated at the 5% (P≤0.05) and 1% (P≤0.01) levels.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs for the 120 mg/kg group were limited to salivation seen post dosing only. Mild to moderate salivation and mild tonic clonic convulsions were noted in the high dose treated group.
Salivation started approximately 10 to 15 minutes post dosing and lasted until approximately at 50 to 60 minutes post dosing. Salivation observed in treatment groups was considered a response to the dose solution and toxicologically irrelevant. Tonic clonic convulsions were observed only at high dose treated dose group animals. Tonic clonic convulsions for high dose animals were seen approximately 10 to 15 minutes post dosing and convulsions were observed for less than 30 minutes. Tonic clonic convulsion began to be observed at different intervals in treatment. Tonic clonic convulsions seen in high dose group rats were considered as effect of test item treatment and adverse in nature. Additionally, lethargy and weakness were observed on days 84 to 87 in a male rat N° 62 of high dose. Rat N° 62 was found dead after showing lethargy and weakness 4 days prior. Hence, these changes were considered as treatment related adverse effect.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two male rats from the high dose group (rat N° 62 on day 88 and rat N° 68 on day 89) died during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weight and mean body weight change of male and female rats of treatment groups were comparable with those of the vehicle control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean food consumption of rats from treatment groups were comparable with that of the vehicle control group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmological examination did not reveal any abnormalities
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In high dose male rats, statistically significant decreases were observed in haemoglobin, haematocrit. Similarly statistically significant increases were noted for WBC and monocytes in female rats of high dose group. These alterations were considered as an effect of test item treatment. As decrease observed in haemoglobin and haematocrit was below 7%, it was considered as non-adverse . Similarly, in absence of lesions indicating inflammatory reactions, increase noted in WBC and monocyte was not considered as adverse. Statistically signficant decrease was observed in prothrombin time in high dose male. As difference between control and high dose group was less than 2 seconds it was not considered as toxicological significant.Statistically significant increase was noted in platelets in mid dose male rats, which was not considered as effect test item treatment due to lack of dose dependency.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In high dose group, statistically significant decrease was observed in ALT in male rats and there is no biological relevance of low ALT. In high dose group, statistically significant decrease was observed in chloride in male rats, and albumin:globulin ratio in female rats. These changes were considered as non-adverse in absence of similar effect in sodium (for decrease in chloride) and total protein, albumin and globulin (for decerase in albumin: globulin ratio).
Statistically significant decrease was noted in urea and BUN in low dose male rats which was not related to test item treatment due to lack of dose dependency.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urine analysis parameters did not reveal any alterations related to treatment.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
In the home cage, all rats from the treatment and vehicle control groups showed normal posture, asleep (curled up often asleep), sitting A (sitting but with head hung down), sitting C (sitting or standing alert, watching) and rearing. Clonic and tonic movements were absent in home cage during NBO.

NBO performed during handling of rats did not reveal any abnormality. All rats revealed normal behaviour during removal (very easy - rats sit quietly) and handling (easy - alert, limbs put against the body). No rat showed lacrimation, salivation, and piloerection. Eyelids were wide open in all rats. Eye and skin examination did not reveal any abnormality in any rat
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant increase was noted in the absolute and relative weight of adrenals and relative weight of liver in both sexes of high dose group. A statistically significant increase was also noted in absolute weight of liver and relative weight of kidneys in high dose males. These changes were considered related to the test item treatment. Statistically significant increases observed in absolute weight of the brain in high-dose female rats was considered as related to treatment. However, microscopic examination of brain did not reveal histopathological lesions hence it was not considered as non-adverse.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
External and internal examination of rats of either sex across various groups (G1 to G4) did not reveal any abnormalities except in two high dose rats showed enlarged livers.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic observations revealed test item treatment related changes in adrenal (both sexes), liver (high dose female rats) and in kidneys (high dose male rats).
In adrenals, cortical hypertrophy was observed in few rats of high dose group, which was also noted in mid dose group male rats with lower incidence and severity than high dose. The lesion could be related to stress and considering lower incidence and severity (minimal to mild), it was considered as non-adverse.
In male rats, microscopic examination of kidneys revealed tubular hyaline droplets (a2µ globulin) in few rats of high dose group and 1 rat of mid dose group. Alpha2u-globulin nephropathy is one type of hyaline droplet nephropathy, was confirmed by presence of granular cast and special stain (chromotrope aniline blue). The expression of hyaline droplets in kidneys requires the presence of the α2u-globulin protein, which is synthesized in the liver of male rats. α2u-globulin nephropathy is sex- and species specific and not considered relevant to human.

In female rats, increase in incidence of hepatocellular vacuolation was observed in high dose group. As lesions were also noted in control group, it was considered as non-adverse.Histopathological examination of found dead rats did not reveal any lesion which indicate cause of death
Histopathological findings: neoplastic:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
Remarks on result:
other: the No Observed Adverse Effect Level (NOAEL) of TMPD Glycol is 120 mg/kg b. wt./day when administered through oral gavage up to 90 days in Wistar rat, under conditions and procedure followed in the present study.
Remarks:
the No Observed Adverse Effect Level (NOAEL) of TMPD Glycol is 120 mg/kg b. wt./day when administered through oral gavage up to 90 days in Wistar rat, under conditions and procedure followed in the present study.

Target system / organ toxicity

Key result
Critical effects observed:
no
Organ:
other: Mortality and clinical signs

Applicant's summary and conclusion

Conclusions:
During treatment period, mild tonic clonic convulsion were observed post dosing on multiple days of the study in all rats of high dose treated animals at dose level 480 mg/kg b. wt./day and lead 2 of 10 mortalities in male rats. These effects were considered as treatment related adverse effect.
Based on the mortality and convulsions observed at 480 mg/kg b. wt/day treated animals, it is concluded that TMPD Glycol did not produce any toxicity or adverse effects up to 120 mg/kg b. wt./day when administered repeatedly through oral gavage for 90 consecutive days in Wistar rats. Therefore, the No Observed Adverse Effect Level (NOAEL) of TMPD Glycol is 120 mg/kg b. wt./day when administered through oral gavage up to 90 days in Wistar rat, under conditions and procedure followed in the present study.
Executive summary:

The present study was conducted to determine adverse effects occurring as a result of the repeated daily oral administration of the TMPD Glycol for a period of 90 consecutive days on Wistar rats. Results of A.I. concentration and homogeneity analysis of dose formulation samples collected on days 1, 29, 57, and 85 were within acceptable range of ±15% of nominal concentration.

Mortality was observed in two high dose male rats (rat N° 62 on day 88 and rat N° 68 on day 89).Clinical signs for the 120 mg/kg group were limited to salivation seen post dosing only. The salivation was observed erratically in male (2 to 5) and female (1) at 120 mg/kg dose treated animals. In the 480 mg/kg tested group, mild to moderate salivation and mildtonic clonic convulsion were observed post dosing in all animals on multiple days of the study. However, all clinical signs were resolved by the following morning.No treatment related change was observed during ophthalmological examination of treated rats.

No treatment related change was observed during neurobehavioural observation of treated rats except rearing counts for high dose males and females were either numerically or statistically significantly higher than control starting at week 6 and through the remainder of the study. These findings were stand alone findings and were not supported by any other neurobehavioural parameters and motor function findings in the high dose group. Hence, it could not be considered as adverse in nature.No treatment related change was observed in functional observation battery parameters of treated rats. Mean body weight, mean body weight change, and food consumption of treatment groups were comparable with that of the vehicle control group.

In haematology, test item treatment at high dose group lead to statistically significant decreases in haemoglobin and haematocrit in males, and statistically significant increase in WBC and monocytes in females. Thedecrease in haemoglobin and haematocrit percentages were below 7% and further can be considered as non-toxicology significant (Hall and Everds, 2013). Similarly, in the absence of lesions in histopathology indicating inflammatory reactions, increase in WBC and monocyte was not considered as adverse.

In clinical chemistry, test item treatment at high dose level caused statistically significant decrease chloride in male rats and decrease in albumin: globulin ratio in female rats. These changes were discrete and considered as non-adverse in absence of similar effect in sodium (for decrease in chloride) and total protein, albumin and globulin (for decerase in albumin: globulin ratio) and further were not correlated with any other toxicology effects.

A statistically significant increase in the weight of liver and adrenals in both sexes, and kidneys in male rats from the high dose group was noted when compared to the vehicle control group. Microscopic evaluation of these tissues showed histologic changes in the adrenals of animals of both sexes, in the kidney of male rats and the liver of female rats. These finding are considered treatment related. However, the lesions in adrenal could be related to stress and considering lower incidence and severity (minimal to mild), it was can be considered as non-adverse.Alpha2u-globulin nephropathy in kidney is one type of hyaline droplet nephropathy, which wasconfirmed by presence of granular cast and special stain (chromotrope aniline blue). α2u-globulin nephropathy is sex- and species specific and not considered relevant to human. Further hepatocellular vacuolation in liver were also noted in control group and therefore these can be considered as non-adverse. External and internal examination of terminally sacrificed rats of either sex did not reveal any treatment related lesions for any of the treated groups except in two rats from high dose showed enlarged liver.

Based on the mortality and convulsions, clinical signs observed at 480 mg/kg b. wt/day in treated animals, a NOAEL of 120 mg/kg/d was considered for this study.