Octadecanoic acid, 12-hydroxy-, reaction products with ethylenediamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

other: range-finding study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 14 August 2012 to 19 November 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

reproductive toxicity, other

Remarks:

range-finding study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 14 August 2012 to 19 November 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

The objective of this study was to evaluate the potential toxicity of the test substance following daily oral administration (gavage) to Crl:CD(SD) rats for 14 days, in order to select the dose-levels for an OECD 421 reproductive/developmental toxicity screening study.

GLP compliance:

no

Remarks:

Range -finding toxicity study

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 10 weeks
- Weight at study initiation: 353 to 394 g (males) and 234 to 263 (females)
- Fasting period before study:no
- Housing: Four of one sex per cage. The cages were made of a polycarbonate body with a stainless steel mesh lid. S
- Diet: Ad libitum, standard rodent diet (SDS VRF1 Certified). This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent
- Water: Ad libitum, potable water taken from the public supply.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 23°C
- Humidity: 40 to 70%
- Air changes (per hr): Each animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated
- Photoperiod (hrs dark / hrs light): 12 h continuous light and 12 h continuous dark/24 h.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): homogeneous and stable suspensions were obtained with corn oil as a vehicle
- Concentration in vehicle: 20, 60 and 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg bw

Details on mating procedure:

Mating procedure was not carried out as it was a range-finding study.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

No test substance formulation analysis was performed in this study. However, the homogeneity and stability of the formulation were determined. Formulations were confirmed stable for up to 24 hours at ambient temperature (nominally 21°C) and fifteen days when refrigerated (nominally 4°C).

Duration of treatment / exposure:

14 days

Frequency of treatment:

7 days/week

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The high dose (1000 mg/kg bw/day) for this study was the maximum dose level required for the subsequent main study (reproductive/developmental toxicity screening study). The low (100 mg/kg bw/day) and intermediate (300 mg/kg bw/day) dose levels were chosen to allow determination of a dose response.

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- A detailed physical examination was performed on Days 1, 4, 8, 11 and 15 for each animal to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded during acclimatization, on Days -3, 1, 4, 8, 11 and 15 (before necropsy).

FOOD CONSUMPTION :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The weight of the food supplied to each cage, food remaining and an estimate of any spillage was recorded at Day -3 to -1, 1 to 3, 4 to 7, 8 to 10 and 11 to 14. From these records the mean daily consumption per animal (g/animal/d) was calculated for each cage.

WATER CONSUMPTION: Yes
-Daily by visual observation.

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

not examined

Litter observations:

not applicable

Postmortem examinations (parental animals):

SACRIFICE
- Male and female animals: All surviving animals were killed by carbon dioxide asphyxiation on day 15.

GROSS PATHOLOGY:
All animals were subject to a detailed necropsy. All external features and orifices were examined visually. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded.

HISTOPATHOLOGY/ORGAN WEIGHTS
Testes were fixed in modified Davidson's fluid. Epididymides, ovaries, kidneys, spleen and liver from all animals were preserved in 10% neutral buffered formalin but no microscopic examinations of these tissues were performed.
The following organs were dissected free of adjacent fat and other contiguous tissue and the weights were recorded: epididymides, ovaries, kidneys, spleen, liver, testes, bilateral organs were weighed individually.

Postmortem examinations (offspring):

Not applicable

Statistics:

None

Reproductive indices:

Not applicable

Offspring viability indices:

Not applicable

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

CLINICAL SIGNS AND MORTALITY:
There were no premature deaths and no clinical signs.

BODY WEIGHT AND WEIGHT GAIN:
Mean bodyweight gain for females at 1,000 mg/kg bw/day was markedly lower than those at 100 or 300 mg/kg bw/day over the 2 week treatment period. However, this difference was mainly due to one female which showed overall slight bodyweight loss.The weight gain of the other 3 females was unremarkable.The overall bodyweight gain in males was similar in all groups.

FOOD CONSUMPTION:
Food consumption in males was unaffected by treatment and there was no conclusive effect on the food intake of females.

WATER CONSUMPTION:
The visual assessment of water intake did not reveal any dose-related effect up to the end of treatment.

ORGAN WEIGHTS:
Liver weights of females at 1000 mg/kg/day were slightly lower than those at 100 or 300 mg/kg bw/day groups. However, due to the absence of any similar finding in the males, the significance of this difference was unclear.

GROSS PATHOLOGY:
No effects

Dose descriptor:

other: Maximum tolerated dose

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: Animals were not mated as it was a range-finding study.

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Conclusions:

Since no major findings were observed in this range-finding toxicity study, the dose levels of 100, 300 and 1,000 mg/kg bw/day were considered suitable for use in an associated main OECD 421 reproductive/developmental toxicity screening study.

Executive summary:

In a dose range-finding study performed in compliance with Good Laboratory Practice, the test substance was administered daily by gavage to CD rats for 14 days. The purpose of this study was to select the dose-levels for an OECD 421 reproductive/developmental toxicity screening study.

Three groups, each comprising four male and four female rats, received the test substance at doses of 100, 300 or 1,000 mg/kg bw/day. During the study, the animals were checked at least twice daily for mortality and clinical condition. Bodyweight and food consumption were recorded twice weekly. Water consumption was assessed by daily visual observation. Animals were sacrificed on completion of the treatment period (day 15) and a complete macroscopic post-mortem examination was performed. The kidneys, liver , spleen, ovaries, testes and epididymides were weighted and preserved although no microscopic examination was performed.

There were no mortalities and the clinical condition of the animals was unaffected by treatment. Three females receiving 1000 mg/kg/day showed unremarkable bodyweight gains, but one female showed overall slight weight loss of uncertain relationship to treatment.  Overall bodyweight gain in males was similar in all groups. Food consumption in males was unaffected by treatment and there was no conclusive effect on the food intake of females. There were no effects of treatment on the organ weights of animals which received 100, 300 or 1000 mg/kg/day except a slightly lower liver weight in females that received 1,000 mg/kg bw/day.

Macroscopic examination at necropsy after 14 days of treatment did not reveal any abnormalities.   There were no findings in this preliminary study that would preclude use of 1000 mg/kg/day as a high dose and it was therefore concluded that dose levels of 100, 300 and 1000 mg/kg/day would be suitable for use in an associated main OECD 421 reproductive/developmental toxicity screening study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

The objective of this study was to evaluate the potential toxicity of the test substance following daily oral administration (gavage) to Crl:CD(SD) rats for 14 days, in order to select the dose-levels for an OECD 421 reproductive/developmental toxicity screening study.

GLP compliance:

no

Remarks:

Range -finding toxicity study

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Octadecanoic acid, 12-hydroxy-, reaction products with ethylenediamine

EC Number:

309-629-8

EC Name:

Octadecanoic acid, 12-hydroxy-, reaction products with ethylenediamine

Cas Number:

100545-48-0

Molecular formula:

No discrete molecular formula available for this UVCB substance

IUPAC Name:

Reaction products of 12-hydroxyoctadecanoic acid with ethane-1,2-diamine

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 10 weeks
- Weight at study initiation: 353 to 394 g (males) and 234 to 263 (females)
- Fasting period before study: no
- Housing: Four of one sex per cage. The cages were made of a polycarbonate body with a stainless steel mesh lid.
- Diet: Ad libitum, standard rodent diet (SDS VRF1 Certified). This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent
- Water: Ad libitum, potable water taken from the public supply.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 23°C
- Humidity: 40 to 70%
- Air changes (per hr): Each animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated
- Photoperiod (hrs dark / hrs light): 12 h continuous light and 12 h continuous dark/24 h.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): homogeneous and stable suspensions were obtained with corn oil as a vehicle
- Concentration in vehicle: 20, 60 and 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg bw

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

No test substance formulation analysis was performed in this study. However, the homogeneity and stability of the formulation were determined. Formulations were confirmed stable for up to 24 hours at ambient temperature (nominally 21°C) and fifteen days when refrigerated (nominally 4°C).

Duration of treatment / exposure:

14 days

Frequency of treatment:

7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The high dose (1000 mg/kg bw/day) for this study was the maximum dose level required for the subsequent main study (reproductive/developmental toxicity screening study). The low (100 mg/kg bw/day) and intermediate (300 mg/kg bw/day) dose levels were chosen to allow determination of a dose response.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- A detailed physical examination was performed on Days 1, 4, 8, 11 and 15 for each animal to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded during acclimatization, on Days -3, 1, 4, 8, 11 and 15 (before necropsy).

FOOD CONSUMPTION :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The weight of the food supplied to each cage, food remaining and an estimate of any spillage was recorded at Day -3 to -1, 1 to 3, 4 to 7, 8 to 10 and 11 to 14. From these records the mean daily consumption per animal (g/animal/d) was calculated for each cage.

WATER CONSUMPTION: Yes
-Daily by visual observation.

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were subject to a detailed necropsy. All external features and orifices were examined visually. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded.

HISTOPATHOLOGY:
Testes were fixed in modified Davidson's fluid. Epididymides, ovaries, kidneys, spleen and liver from all animals were preserved in 10% neutral buffered formalin but no microscopic examinations of these tissues were performed.

Other examinations:

Yes
The following organs were dissected free of adjacent fat and other contiguous tissue and the weights were recorded: epididymides, ovaries, kidneys, spleen, liver, testes, bilateral organs were weighed individually.

Statistics:

none

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):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY:
There were no premature deaths and no clinical signs.

BODY WEIGHT AND WEIGHT GAIN:
Mean bodyweight gain for females at 1,000 mg/kg bw/day was markedly lower than those at 100 or 300 mg/kg bw/day over the 2 week treatment period. However, this difference was mainly due to one female which showed overall slight bodyweight loss.The weight gain of the other 3 females was unremarkable.The overall bodyweight gain in males was similar in all groups.

FOOD CONSUMPTION:
Food consumption in males was unaffected by treatment and there was no conclusive effect on the food intake of females.

WATER CONSUMPTION:
The visual assessment of water intake did not reveal any dose-related effect up to the end of treatment.

ORGAN WEIGHTS:
Liver weights of females at 1000 mg/kg/day were slightly lower than those at 100 or 300 mg/kg bw/day groups. However, due to the absence of any similar finding in the males, the significance of this difference was unclear.

GROSS PATHOLOGY:
No effects

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: Except a slightly lower liver weight in females at 1,000 mg/kg bw/day when compared to controls, no other effects were observed.

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

With the exception of a slightly lower liver weight in females at 1,000 mg/kg bw/day when compared to controls, no other effects were observed when the test substance was administered by gavage for fourteen days to male and female rats.

Executive summary:

In a dose range-finding study performed in compliance with Good Laboratory Practice, the test substance was administered daily by gavage to CD rats for 14 days.

Three groups, each comprising four male and four female rats, received the test substance at doses of 100, 300 or 1,000 mg/kg bw/day. During the study, the animals were checked at least twice daily for mortality and clinical condition. Bodyweight and food consumption were recorded twice weekly. Water consumption was assessed by daily visual observation. Animals were sacrificed on completion of the treatment period (day 15) and a complete macroscopic post-mortem examination was performed. The kidneys, liver , spleen, ovaries, testes and epididymides were weighed and preserved although no microscopic examination was performed.

There were no mortalities and the clinical condition of the animals was unaffected by treatment. Three females receiving 1000 mg/kg/day showed unremarkable bodyweight gains, but one female showed overall slight weight loss of uncertain relationship to the treatment. Overall bodyweight gain in males was similar in all groups. Food consumption in males was unaffected by treatment and there was no conclusive effect on the food intake of females. There were no effects of treatment on the organ weights of animals which received 100, 300 or 1000 mg/kg/day except for a slightly lower liver weight in females that received 1,000 mg/kg bw/day. Macroscopic examination at necropsy after 14 days of treatment did not reveal any abnormalities.  

Based on these results, it could be concluded that the NOAEL for the test substance was 1000 mg/kg bw/day and dose levels of 100, 300 and 1000 mg/kg/day would be suitable for use in an associated main OECD 421 reproductive/developmental toxicity screening study.