3-{3-[(3,5,5-trimethylhexanoyl)oxy]-2,2-bis({[(3,5,5-trimethylhexanoyl)oxy]methyl})propoxy}-2,2-bis({[(3,5,5-trimethylhexanoyl)oxy]methyl})propyl 3,5,5-trimethylhexanoate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The study design was based on OECD 421 of 27 July 1995. The objective was to evaluate the portential toxic effects of an analogue of the registered substance following daily oral (gavage) administration to male and female rats from before mating, through mating and, for females, through gestation until day 5 post-partum. On completion of the treatment period, additional dams and litters were held for a two week treatment-free period in the control and high dose groups in order to evaluate the reversibility of any findings. The study provided initial information on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of the conceptus and partrutition, and on reversibility of potential findings on development of the offspring or gonadal weight and morphology.

Four groups of 10 male and 1o female (groups 2 to 4) or 15 male and 15 female (group 5) Sprague-Dawley rats received the test item daily by oral (gavage) administration before mating, through mating and, for females, through gestation until day 5 post-partum. The test item was administered as a solution in the vehicle, corn oil, at dose levels of 100, 250, 500 and 1000 mg/kg bw/day. Another group of 15 males and 15 females received the vehicle alone, under the same experimental conditions, and acted as the control group. A constant dose volume of 4 mL/kg bw/day was used. On completion of the treatment period, the animals in each group were sacrificed, except for five animals per sex in the control and high dose groups which were kept for a 2 -week recovery period. The concentrations of the dose formulations were checked in study weeks 1, 3 and 6.

The animals were checked twice daily for mortality and morbidity and once daily for clinical signs during the treatment and treatment-free periods. Body weight and food consumption were recorded once a week during pre-mating and mating periods (food consumption not during mating), and during gestation on days 0, 7, 14 and 20 post-coitum plus lactation on days 1 and 5 post-partum. These data were also recorded on days 12 and 19 post-coitum for treatment-free females and weekly after the mating period for treatment-free males.

Animals were paired for mating after 2 weeks of treatment and the females were allowed to litter and rear their progeny until day 5 post-partum or until day 19 post-partum for treatment-free females. The total litter sizes and number of pups of each sex were recorded after birth. Pups were observed daily for clinical signs, abnormal behaviour and external abnormalities and weighed on days 1 and 5 post-partum. Pups of treatment-free females were also weighed on days 12 and 19 post-partum.

Males were sacrificed after at least 5 weeks of treatment and females on day 6 post-partum except for treatment-free animals, which were sacrificed 2 weeks after the end of treatment (thus on day 20 post-partum for females). Final body weights and selected organ weights (epididymides, testes, kidneys, liver, spleen) were recorded and a macroscopic post-mortem examination of the prinicpal thoracic and abdominal organs was performed. Particular attention was paid to the reproductive organs. A microscopic examination was performed on ovaries(with oviducts) from all females sacrificed on day 6 post-partum in the control and high dose groups and epididymides and/or testes from all males sacrificed at the end of the treatment period in the control and high dose groups. A microscopic examination was also performed on the kidneys and liver from all males sacrificed at the end of the treatment or treatment-free period in all groups and on all macroscopic lesions. Pups were sacrificed on day 5 post-partum (or day 19 post-coitum for treatment-free pups) and submitted for post-mortem examination of the principal thoracic and abdominal organs.

Chemical analysis: No test item was detectable above the limit of quantification in the control dose formulation and the test item concentrations in the analysed dose formulations were within an acceptable range.

The following findings were reported for F0 animals:

- No premature deaths in the test-item treated groups during treatment and treatment-free periods.

- No toxicologically significant clinical signs during treatment and treatment-free periods.

- No toxicologically significant effects on mean body weights, mean body weight changes and mean food consumption during treatment and treatment-free periods.

- No effects on mean pairing, mating, fertility and delivery data.

- Test item-related kidney and liver mean weight increases were seen in males treated with 100 mg/kg bw/day or above.

- Test item-related irregular colour and or tan discolouration of kidneys and increased incidence of accentuated lobular pattern together with tan discolouration was observed in males treated with 100 mg/kg bw/day or above. At the end of the treatment-free period, there were no test item-related gross findings in either the liver or the kidneys.

- A series of test-item related microscopic findings was found in the kidneys and liver of males treated with 100 mg/kg bw/day or above. These findings were degradation/necrosis of tubules (adverse) in 2/10 males treated at 1000 mg/kg bw/day, hyaline droplets in tubular epithelium, tubular dilation with or without eosinophilic content in kidneys, and hepatocellular hypertrophy plus increased periportal vacuolation in the liver. At the end of the treatment-free period, the reversibility of the liver changes was complete whilst reversibility of kidney changes was incomplete.

 

The following findings were reported for pups:

- No test item-related effects on pup viability during treatment and treatment-free periods.

- No test-item related clinical signs during treatment and treatment-free periods.

- No toxicologically significant effects on mean body weight and mean body weight gains during treatment and treatment-free periods.

- No clear indication of a test item treatment effect on percentage of male pups at birth.

- No test item-related necropsy findings during treatment and treatment-free periods.

 

The No Observed Adverse Effect Level (NOAEL) for parental systemic toxicity was considered to be 500 mg/kg bw/day for males based on the adverse microscopic effects in kidneys seen at the higher dose and 1000 mg/kg bw/day for females based on the absence of adverse effects in females. However, the renal effects seen in male rats are not relevant to human exposure. The No Observed Effect Level (NOEL) for parental reproductive performance was considered to be 1000 mg/kg bw/day based on the absence of effects in mean reproductive data at all dose levels. The No Observed Adverse Effect Level (NOAEL) for toxic effects on progency was considered to be 1000 mg/kg bw/day based on absence of adverse effects in pups at all dose levels.

Short description of key information:
The No Observed Effect Level (NOEL) for parental reproductive performance was considered to be 1000 mg/kg bw/day based on the absence of effects in mean reproductive data at all dose levels (OECD 421).

Justification for selection of Effect on fertility via oral route:
GLP guideline study

Effects on developmental toxicity

Description of key information

The No Observed Adverse Effect Level (NOAEL) for toxic effects on progency was considered to be 1000 mg/kg bw/day based on absence of adverse effects in pups at all dose levels (OECD 421).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Supporting data from study conducted on analogue substance and reported in literature.

Additional information

Key study

The study design was based on OECD 421 of 27 July 1995. The objective was to evaluate the portential toxic effects of the test item following daily oral (gavage) administration to male and female rats from before mating, through mating and, for females, through gestation until day 5 post-partum. On completion of the treatment period, additional dams and litters were held for a two week treatment-free period in the control and high dose groups in order to evaluate the reversibility of any findings. The study provided initial information on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of the conceptus and partrutition, and on reversibility of potential findings on development of the offspring or gonadal weight and morphology.

Four groups of 10 male and 10 female (groups 2 to 4) or 15 male and 15 female (group 5) Sprague-Dawley rats received the test item daily by oral (gavage) administration before mating, through mating and, for females, through gestation until day 5 post-partum. The test item was administered as a solution in the vehicle, corn oil, at dose levels of 100, 250, 500 and 1000 mg/kg bw/day. Another group of 15 males and 15 females received the vehicle alone, under the same experimental conditions, and acted as the control group. A constant dose volume of 4 mL/kg bw/day was used. On completion of the treatment period, the animals in each group were sacrificed, except for five animals per sex in the control and high dose groups which were kept for a 2 -week recovery period. The concentrations of the dose formulations were checked in study weeks 1, 3 and 6.

The animals were checked twice daily for mortality and morbidity and once daily for clinical signs during the treatment and treatment-free periods. Body weight and food consumption were recorded once a week during pre-mating and mating periods (food consumption not during mating), and during gestation on days 0, 7, 14 and 20 post-coitum plus lactation on days 1 and 5 post-partum. These data were also recorded on days 12 and 19 post-coitum for treatment-free females and weekly after the mating period for treatment-free males.

Animals were paired for mating after 2 weeks of treatment and the females were allowed to litter and rear their progeny until day 5 post-partum or until day 19 post-partum for treatment-free females. The total litter sizes and number of pups of each sex were recorded after birth. Pups were observed daily for clinical signs, abnormal behaviour and external abnormalities and weighed on days 1 and 5 post-partum. Pups of treatment-free females were also weighed on days 12 and 19 post-partum.

Males were sacrificed after at least 5 weeks of treatment and females on day 6 post-partum except for treatment-free animals, which were sacrificed 2 weeks after the end of treatment (thus on day 20 post-partum for females). Final body weights and selected organ weights (epididymides, testes, kidneys, liver, spleen) were recorded and a macroscopic post-mortem examination of the prinicpal thoracic and abdominal organs was performed. Particular attention was paid to the reproductive organs. A microscopic examination was performed on ovaries(with oviducts) from all females sacrificed on day 6 post-partum in the control and high dose groups and epididymides and/or testes from all males sacrificed at the end of the treatment period in the control and high dose groups. A microscopic examination was also performed on the kidneys and liver from all males sacrificed at the end of the treatment or treatment-free period in all groups and on all macroscopic lesions. Pups were sacrificed on day 5 post-partum (or day 19 post-coitum for treatment-free pups) and submitted for post-mortem examination of the principal thoracic and abdominal organs.

Chemical analysis: No test item was detectable above the limit of quantification in the control dose formulation and the test item concentrations in the analysed dose formulations were within an acceptable range.

The following findings were reported for F0 animals:

- No premature deaths in the test-item treated groups during treatment and treatment-free periods.

- No toxicologically significant clinical signs during treatment and treatment-free periods.

- No toxicologically significant effects on mean body weights, mean body weight changes and mean food consumption during treatment and treatment-free periods.

- No effects on mean pairing, mating, fertility and delivery data.

- Test item-related kidney and liver mean weight increases were seen in males treated with 100 mg/kg bw/day or above.

- Test item-related irregular colour and or tan discolouration of kidneys and increased incidence of accentuated lobular pattern together with tan discolouration was observed in males treated with 100 mg/kg bw/day or above. At the end of the treatment-free period, there were no test item-related gross findings in either the liver or the kidneys.

- A series of test-item related microscopic findings was found in the kidneys and liver of males treated with 100 mg/kg bw/day or above. These findings were degradation/necrosis of tubules (adverse) in 2/10 males treated at 1000 mg/kg bw/day, hyaline droplets in tubular epithelium, tubular dilation with or without eosinophilic content in kidneys, and hepatocellular hypertrophy plus increased periportal vacuolation in the liver. At the end of the treatment-free period, the reversibility of the liver changes was complete whilst reversibility of kidney changes was incomplete.

 

The following findings were reported for pups:

- No test item-related effects on pup viability during treatment and treatment-free periods.

- No test-item related clinical signs during treatment and treatment-free periods.

- No toxicologically significant effects on mean body weight and mean body weight gains during treatment and treatment-free periods.

- No clear indication of a test item treatment effect on percentage of male pups at birth.

- No test item-related necropsy findings during treatment and treatment-free periods.

 

The No Observed Adverse Effect Level (NOAEL) for parental systemic toxicity was considered to be 500 mg/kg bw/day for males based on the adverse microscopic effects in kidneys seen at the higher dose and 1000 mg/kg bw/day for females based on the absence of adverse effects in females. However, the renal effects seen in male rats are not relevant to human exposure. The No Observed Effect Level (NOEL) for parental reproductive performance was considered to be 1000 mg/kg bw/day based on the absence of effects in mean reproductive data at all dose levels. The No Observed Adverse Effect Level (NOAEL) for toxic effects on progency was considered to be 1000 mg/kg bw/day based on absence of adverse effects in pups at all dose levels.

Supporting study

In a supporting literature study, the effect of an analogue substance on developmental toxicity was investigated via the dermal route. The two highest dose levels caused some local irritation at the site of application, but no decreases in maternal weight gain or feed consumption. Three dead animals in the control group and one dead animal in the high dosage group occurring within 6 h after first application were not considered to be treatment related and were replaced. One dam having a litter consisting of seven early resorptions was pointed out as single non-dosage dependent event and to be within the ranges observed historically at the test facility. Necropsy findings were limited to skin flaking and scabbing first identified in life and observations related to wearing the Elizabethan collar (local alopecia, chromorhinorrhea, and neck lesions). The NOAEL was reported as 2000 mg/kg bw/day.

Toxicity to reproduction: other studies

Additional information

With respect to systemic toxicity, the presence of hyaline droplets in kidneys of males correlated with increases in mean kidney weights at necropsy. The hyaline droplets were consistent with what is described in the literature as a2u-globulin nephropathy, which is known to increase after treatment with a wide range of drugs or chemicals (Greaves, 2012). These hyaline droplets were observed with a dose related trend and were associated with a higher incidence and/or severity of tubular basophilia. The prolonged accumulation of hyaline droplets is associated with chronic cell damage and increased cell turnover, which may explain the tubular basophilia seen in all test item-treated groups and the degeneration/necrosis seen at 1000 mg/kg bw/day. These findings were therefore considered to be adverse at 1000 mg/kg bw/day. However, humans excrete proteins of a similar nature in only trace amounts and these findings are therefore considered not to be relevant to humans (Hard et al, 1993; Swenberg 1993).

Justification for classification or non-classification

No treatment-related effects on mating, fertility, gestation length or litter size in the 100, 250, 500 or 1000 mg/kg bw/day groups and no adverse effect on offspring development from maternal treatment at 100, 250, 500 or 1000 mg/kg bw/day.