Hexane-1,3,6-tricarbonitrile

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022.06.13-2023.01.16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2023

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 (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Hexane-1,3,6-tricarbonitrile

EC Number:

217-199-7

EC Name:

Hexane-1,3,6-tricarbonitrile

Cas Number:

1772-25-4

Molecular formula:

C9H11N3

IUPAC Name:

hexane-1,3,6-tricarbonitrile

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Charles River, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation:7-8 weeks
- Weight at study initiation: male 174-196g/ female 143-157g
- Fasting period before study:not specified
- Housing:
Full barrier in an air-conditioned room
Temperature: 22  3 °C
Relative humidity: 40–70%
Artificial light, sequence being 12 hours light, 12 hours dark
Air change: 10 x / hour

- Diet (e.g. ad libitum):ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period:4days

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

castor oil

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 days

Frequency of treatment:

The animals were treated with the test item or vehicle on 7 days per week for a period of 28 days.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

25 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

75 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

150 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

Sacrifice and pathology:

One day after the last administration (study day 29) all surviving animals of the treatment period were sacrificed using anaesthesia (ketamine/ xylazine) and subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

slight salivation and moving the bedding at post dose observation was observed in the MD group and salivation (slight, moderate and severe) including moving the bedding at post dose in HD groups were observed during the treatment period. All these clinical signs of moving the bedding and salivation were observed at post dose administration and therefore were considered to be signs of a local reaction to the test item administration.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant increase in the relative liver weights was observed in the HD females. It is considered to be associated with centrilobular hepatocellular hypertrophy that was observed microscopically. Centrilobular hepatocellular hypertrophy was also observed microscopically in males, however, no toxicologically relevant differences in the liver weights was observed in males.
The kidney weights in both sexes were increased in a dose-dependent manner, although no statistically significant differences were observed. The increased kidney weights were likely to be associated with renal lesions recorded microscopically in males (i.e., male rat α2u-globulin nephropathy). Meanwhile, there was no treatment-related microscopic finding in females, and therefore, increased kidney weights in females were deemed to be of no toxicological significance.
Microscopic findings attributed to treatment with the test item were observed in the liver of both sexes and the kidneys of males.
In the liver, at MD group ≥ 75 mg/kg bw/day, minimal centrilobular hepatocellular hypertrophy were observed in both sexes. This was considered to have contributed to increased relative liver weight that was observed in the HD group (150 mg/kg bw/day) females in the organ weight determinations. There were no microscopic findings suggestive of accompanying liver injury (e.g. necrosis, fibrosis, enhancement of inflammatory changes, or hyperplasia) associated with hepatocellular hypertrophy in any animals. Thus, hepatocellular hypertrophy observed in this study is considered to be of metabolic nature and of adaptive character (like as enzyme induction) and was deemed not adverse.
In males of the kidneys, at ≥75 mg/kg bw/day, there was an excessive accumulation of hyaline droplets in the proximal tubular epithelia. Hyaline droplets were not observed in any females. Therefore, excessive hyaline droplet accumulation found in males only was considered to be induced by overload of synthetic protein, that is specific in male rats like as α2u-globulin, derived from hyperfunction of the liver. In addition, under the conditions of this study, excessive hyaline droplet accumulation was accompanied by additional microscopic findings indicative of cell/tissue injury, such as proximal tubular cell necrosis, granular casts, peritubular fibrosis, increased incidence and severity of tubular basophilia, and/or increased incidence of interstitial mononuclear cell infiltrates. These findings were consistent with the lesions known as male rat α2u-globulin nephropathy and the renal lesions were considered to be adverse in the rat-model. However, this type of nephropathy is believed not relevant to human.
Hyaline droplet accumulation that was observed at a minimal severity in the LD group (25 mg/kg bw/day) males was not accompanied by additional findings indicative of cell and tissue injury. The protein α2u in itself is present physiologically in the proximal tubular epithelia of normal or untreated male rats, especially in young adult males. Therefore, minimal accumulation without any accompanying findings indicative of cell/tissue injury which was observed in the LD group was within the range of normal background findings that may be observed in male rats and was deemed not to be treatment-related.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Details on results:

No test item related mortality was observed and all animals survived till the end of study period.
No test item related systemic toxicity was observed in any of the treated groups, however slight salivation and moving the bedding at post dose observation was observed in the MD group and salivation (slight, moderate and severe) including moving the bedding at post dose in HD groups were observed during the treatment period. All these clinical signs of moving the bedding and salivation were observed at post dose administration and therefore were considered to be signs of a local reaction to the test item administration.
No test item related clinical signs or effects on detailed clinical examination and Functional observation battery parameters were observed during the study period.
No test item related effect on mean body weight and food consumption were observed in any of the treated groups when compared to control in both genders.
No test item related effects on blood coagulation or haematology or clinical biochemistry and urine parameters were observed in any of the treated groups.
No test item related macroscopic changes were observed in any of the treated groups.
Statistically significant increase in the relative liver weights was observed in the HD females. It is considered to be associated with centrilobular hepatocellular hypertrophy that was observed microscopically. Centrilobular hepatocellular hypertrophy was also observed microscopically in males, however, no toxicologically relevant differences in the liver weights was observed in males.
The kidney weights in both sexes were increased in a dose-dependent manner, although no statistically significant differences were observed. The increased kidney weights were likely to be associated with renal lesions recorded microscopically in males (i.e., male rat α2u-globulin nephropathy). Meanwhile, there was no treatment-related microscopic finding in females, and therefore, increased kidney weights in females were deemed to be of no toxicological significance.
Microscopic findings attributed to treatment with the test item were observed in the liver of both sexes and the kidneys of males.
In the liver, at MD group ≥ 75 mg/kg bw/day, minimal centrilobular hepatocellular hypertrophy were observed in both sexes. This was considered to have contributed to increased relative liver weight that was observed in the HD group (150 mg/kg bw/day) females in the organ weight determinations. There were no microscopic findings suggestive of accompanying liver injury (e.g. necrosis, fibrosis, enhancement of inflammatory changes, or hyperplasia) associated with hepatocellular hypertrophy in any animals. Thus, hepatocellular hypertrophy observed in this study is considered to be of metabolic nature and of adaptive character (like as enzyme induction) and was deemed not adverse.
In males of the kidneys, at ≥75 mg/kg bw/day, there was an excessive accumulation of hyaline droplets in the proximal tubular epithelia. Hyaline droplets were not observed in any females. Therefore, excessive hyaline droplet accumulation found in males only was considered to be induced by overload of synthetic protein, that is specific in male rats like as α2u-globulin, derived from hyperfunction of the liver. In addition, under the conditions of this study, excessive hyaline droplet accumulation was accompanied by additional microscopic findings indicative of cell/tissue injury, such as proximal tubular cell necrosis, granular casts, peritubular fibrosis, increased incidence and severity of tubular basophilia, and/or increased incidence of interstitial mononuclear cell infiltrates. These findings were consistent with the lesions known as male rat α2u-globulin nephropathy and the renal lesions were considered to be adverse in the rat-model. However, this type of nephropathy is believed not relevant to human.
Hyaline droplet accumulation that was observed at a minimal severity in the LD group (25 mg/kg bw/day) males was not accompanied by additional findings indicative of cell and tissue injury. The protein α2u in itself is present physiologically in the proximal tubular epithelia of normal or untreated male rats, especially in young adult males. Therefore, minimal accumulation without any accompanying findings indicative of cell/tissue injury which was observed in the LD group was within the range of normal background findings that may be observed in male rats and was deemed not to be treatment-related.
The remaining microscopic findings recorded in this study were not test item-related as they were within the range of normal background lesions or physiological alterations that may be observed in this study type and in animals of this strain, age and sex.
Nominal concentrations were confirmed for all dose groups as measured mean recoveries were within ±15 % of nominal values on week 1 and 4 of treatment period.

Effect levels

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

On the basis of this 28-day repeated dose oral toxicity study with Hexane-1,3,6-tricarbonitrile in male and female Wistar rats with dose levels of 25, 75 and 150 mg/kg bw/day the following conclusions can be made:
There was no test item related effect observed on mortality, clinical signs, body weight development, food consumption, functional observation battery, weekly detailed clinical observations, haematology and blood coagulation, clinical biochemistry, urinalysis and gross pathological findings in males and females.
Statistically significant increase in the relative liver weights was observed in the HD females, which was associated with centrilobular hepatocellular hypertrophy. Centrilobular hepatocellular hypertrophy was also observed in males but without any changes in the liver weights.
The kidney weights in both sexes were increased in a dose-dependent manner, although no statistically significant differences were observed. The increased kidney weights were likely to be associated with renal lesions recorded microscopically in males (i.e., male rat α2u-globulin nephropathy). Meanwhile, there was no treatment-related microscopic finding in females, and therefore, increased kidney weights in females were deemed to be of no toxicological significance
Histopathologically, no test item related adverse findings were observed in females up to 150 mg/kg bw/day and the no-observed-adverse-effect-level (NOAEL) was established at 150 mg/kg bw/day for females. As for males, due to presence of treatment-related renal lesions (α2u-globulin nephropathy) with accompanying findings indicative of cell/tissue injury at ≥75 mg/kg bw/day, the NOAEL was considered to be at 25 mg/kg bw/day in the rat-model. However, this type of nephropathy is believed not relevant to human. Meanwhile, due to presence of hepatocellular hypertrophy without any further indicators of liver injury at ≥75 mg/kg bw/day, the no-observed-effect-level (NOEL) may be established at 25 mg/kg bw/day for both sexes.

Executive summary:

The objective of this study was to assess the possible health hazards which could arise from repeated exposure of Hexane-1,3,6-tricarbonitrile via oral administration to rats over a period of 28 days.

The test item was administered daily in a graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 28 days. The control group were handled identically as the dose groups but received the vehicle only. The groups comprised 5 male and 5 female Wistar rats each.

The following doses were evaluated:

Control:            0 mg/kg body weight/day

Low Dose:      25 mg/kg body weight/day

Medium Dose: 75 mg/kg body weight/day

High Dose:   150 mg/kg body weight/day

The test item formulation was prepared at least every 10 days (stability time frame as given by Eurofins Agroscience Services Study No. S22-03900). The test item was diluted in aqueous 2 % Kolliphor EL solution and administered daily during a 28-day treatment period to male and female animals. Dose volumes were adjusted individually based on weekly body weight measurement.

During the period of administration, the animals were observed precisely each day for signs of toxicity. Body weight and food consumption were measured weekly.

At the conclusion of the test, all animals were sacrificed and observed macroscopically. The wet weight of a subset of tissues was taken and a set of organs/tissues was preserved. A full histopathological evaluation of the tissues was performed on high dose and control animals. Any gross lesion macroscopically identified was examined microscopically in all animals. Organs which showed test item related histopathological changes were extended to lower dose groups.

 

No test item related mortality was observed and all animals survived till the end of study period.

No test item related systemic toxicity was observed in any of the treated groups, however slight salivation and moving the bedding at post dose observation was observed in the MD group and salivation (slight, moderate and severe) including moving the bedding at post dose in HD groups were observed during the treatment period. All these clinical signs of moving the bedding and salivation were observed at post dose administration and therefore were considered to be signs of a local reaction to the test item administration.

No test item related clinical signs or effects on detailed clinical examination and Functional observation battery parameters were observed during the study period.

No test item related effect on mean body weight and food consumption were observed in any of the treated groups when compared to control in both genders.

No test item related effects on blood coagulation or haematology or clinical biochemistry and urine parameters were observed in any of the treated groups.

No test item related macroscopic changes were observed in any of the treated groups.

Statistically significant increase in the relative liver weights was observed in the HD females. It is considered to be associated with centrilobular hepatocellular hypertrophy that was observed microscopically. Centrilobular hepatocellular hypertrophy was also observed microscopically in males, however, no toxicologically relevant differences in the liver weights was observed in males.

The kidney weights in both sexes were increased in a dose-dependent manner, although no statistically significant differences were observed. The increased kidney weights were likely to be associated with renal lesions recorded microscopically in males (i.e., male rat α2u-globulin nephropathy). Meanwhile, there was no treatment-related microscopic finding in females, and therefore, increased kidney weights in females were deemed to be of no toxicological significance.

Microscopic findings attributed to treatment with the test item were observed in the liver of both sexes and the kidneys of males.

In the liver, at MD group ≥ 75 mg/kg bw/day, minimal centrilobular hepatocellular hypertrophy were observed in both sexes. This was considered to have contributed to increased relative liver weight that was observed in the HD group (150 mg/kg bw/day) females in the organ weight determinations. There were no microscopic findings suggestive of accompanying liver injury (e.g. necrosis, fibrosis, enhancement of inflammatory changes, or hyperplasia) associated with hepatocellular hypertrophy in any animals. Thus, hepatocellular hypertrophy observed in this study is considered to be of metabolic nature and of adaptive character (like as enzyme induction) and was deemed not adverse.

In males of the kidneys, at ≥75 mg/kg bw/day, there was an excessive accumulation of hyaline droplets in the proximal tubular epithelia. Hyaline droplets were not observed in any females. Therefore, excessive hyaline droplet accumulation found in males only was considered to be induced by overload of synthetic protein, that is specific in male rats like as α2u-globulin, derived from hyperfunction of the liver. In addition, under the conditions of this study, excessive hyaline droplet accumulation was accompanied by additional microscopic findings indicative of cell/tissue injury, such as proximal tubular cell necrosis, granular casts, peritubular fibrosis, increased incidence and severity of tubular basophilia, and/or increased incidence of interstitial mononuclear cell infiltrates. These findings were consistent with the lesions known as male rat α2u-globulin nephropathy and the renal lesions were considered to be adverse in the rat-model. However, this type of nephropathy is believed not relevant to human.

Hyaline droplet accumulation that was observed at a minimal severity in the LD group (25 mg/kg bw/day) males was not accompanied by additional findings indicative of cell and tissue injury. The protein α2u in itself is present physiologically in the proximal tubular epithelia of normal or untreated male rats, especially in young adult males. Therefore, minimal accumulation without any accompanying findings indicative of cell/tissue injury which was observed in the LD group was within the range of normal background findings that may be observed in male rats and was deemed not to be treatment-related.

The remaining microscopic findings recorded in this study were not test item-related as they were within the range of normal background lesions or physiological alterations that may be observed in this study type and in animals of this strain, age and sex.

Nominal concentrations were confirmed for all dose groups as measured mean recoveries were within ±15 % of nominal values on week 1 and 4 of treatment period.

 

On the basis of this 28-day repeated dose oral toxicity study with Hexane-1,3,6-tricarbonitrile in male and female Wistar rats with dose levels of 25, 75 and 150 mg/kg bw/day the following conclusions can be made:

There was no test item related effect observed on mortality, clinical signs, body weight development, food consumption, functional observation battery, weekly detailed clinical observations, haematology and blood coagulation, clinical biochemistry, urinalysis and gross pathological findings in males and females.

Statistically significant increase in the relative liver weights was observed in the HD females, which was associated with centrilobular hepatocellular hypertrophy. Centrilobular hepatocellular hypertrophy was also observed in males but without any changes in the liver weights.

The kidney weights in both sexes were increased in a dose-dependent manner, although no statistically significant differences were observed. The increased kidney weights were likely to be associated with renal lesions recorded microscopically in males (i.e., male rat α2u-globulin nephropathy). Meanwhile, there was no treatment-related microscopic finding in females, and therefore, increased kidney weights in females were deemed to be of no toxicological significance

Histopathologically, no test item related adverse findings were observed in females up to 150 mg/kg bw/day and the no-observed-adverse-effect-level (NOAEL) was established at 150 mg/kg bw/day for females. As for males, due to presence of treatment-related renal lesions (α2u-globulin nephropathy) with accompanying findings indicative of cell/tissue injury at ≥75 mg/kg bw/day, the NOAEL was considered to be at 25 mg/kg bw/day in the rat-model. However, this type of nephropathy is believed not relevant to human. Meanwhile, due to presence of hepatocellular hypertrophy without any further indicators of liver injury at ≥75 mg/kg bw/day, the no-observed-effect-level (NOEL) may be established at 25 mg/kg bw/day for both sexes.