Octadecanoic acid, reaction products with diethylenetriamine and urea, acetates

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

relative humidity exceeded the given range in the guideline (18.4 - 66.2 % instead of 30 - 70 %)

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

relative humidity exceeded the given range in the guideline (18.4 - 66.2 % instead of 30 - 70 %)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Identification: FAT #: 93580/A
CAS Number: 84962-05-0
Active Ingredients: Octadecanoic acid, reaction products with diethylenetriamine and urea, acetates
Description: White-yellowish waxy solid; odour: acetic acid
Batch Number: 0022357000
Purity/Concentration as Supplied: More than 90% of octadecanoic acid, reaction products with diethylenetriamine and urea, acetates
Purity: 94.3%
Correction for Purity: No
Stability of Test Item in Vehicle: At least 8 days if stored at room temperature
Expiry Date (Retest Date) as handled by Harlan Laboratories Ltd: 31-Dec-2018
Storage Conditions: Room temperature (20 ± 5 °C), in the dark
Safety Precautions: Routine hygienic procedures (gloves, goggles, face mask).

Species:

rat

Strain:

other: RccHan: WIST(SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories BV, Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: 339-389 g (males); 223-273 g (females)
- Fasting period before study: no
- Housing: In groups of three to five animals in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding with paper enrichment. During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet (e.g. ad libitum): pelleted standard Harlan Teklad 2018C rodent maintenance diet, ad libitum
- Water (e.g. ad libitum): community tap-water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.9 - 23.6
- Humidity (%): 18.4 - 66.2
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Details on route of administration:

Rationale for Method: Administration by gavage is a common and accepted route of exposure for studies of this type.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
FAT 93580/A was weighed, crushed using a mortar and a small amount of vehicle was added to disperse the test item. The suspension was transferred to a glass beaker and the remaining vehicle was added. Using a magnetic stirrer and/or an ultra turrax, a homogeneous suspension was prepared. Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer. Dose formulations were stored at room temperature (20 ± 5 °C) in glass beakers. Based upon the results of stability analyses, dose formulations were stable for at least 8 days.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 0, 25, 75, 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
- Lot/batch no.: 492194511, 103197718

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. To confirm the stability (8 days) samples of about 2 g of each concentration were taken from the middle of each aliquot used on day 7 of the treatment. During the last week of the treatment, samples were taken from the middle to confirm concentration.

The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to the analytical laboratory and stored there at -20 ± 5 °C until analysis. The samples were analyzed by HPLC - ELSD detector. The test item was used as the analytical standard.

Duration of treatment / exposure:

At least 28 days to male rats and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post-partum.

Frequency of treatment:

once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control group (Group 1)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Test group (Group - 2)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Test group (Group - 3)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Test group (Group - 4)

No. of animals per sex per dose:

12 males and 12 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on a previous dose range-finding toxicity study in Wistar rat, using dose levels of 0, 100, 300 and 1000 mg/kg/day, where no test item related effects were observed up to the dose level of 1000 mg/kg/day.

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: for viability and mortality: twice daily, for cage-side clinical observations: once daily, during acclimatization and up to day of necropsy. Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: In males, it was performed once prior to the first administration of the test item and weekly thereafter. In females, it was performed once prior to the first administration of the test item, weekly during the pre-pairing and pairing periods and on days 0, 6, 13 and 20 of the gestation period.

BODY WEIGHT: Yes
- Time schedule for examinations: once during acclimatization and daily from treatment start to day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 15 of the pre-pairing period.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 males and 5 females from each group
- The following parameters were examined:
Complete Blood Cell Count: erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, hemoglobin concentration distribution width, leukocyte count (total), differential leukocyte count, platelet count.
Coagulation: prothrombin time (= thromboplastin time), activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day 15 of the pre-pairing period.
- Animals fasted: Yes
- How many animals: 5 males and 5 females from each group
- The following parameters were examined: glucose, urea, creatinine, bilirubin (total), cholesterol (total), triglycerides, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl-transferase, bile acids, sodium, potassium, chloride, calcium, phosphorus, protein (total), albumin, globulin, albumin/globulin ratio.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: male rats shortly before the scheduled sacrifice and females on day 3 post-partum
- Dose groups that were examined: 5 males and 5 females from each group
- Battery of functions tested: hand-held observations, open field observations, reflexes, hind limb / fore limb grip strength

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 1)
HISTOPATHOLOGY: Yes (see table 1)

Other examinations:

No data

Statistics:

The following statistical methods were used to analyze food consumption, body weights, functional observational battery, locomotor activity, clinical laboratory investigations and reproduction data:
• Means and standard deviations of various data were calculated and included in the report.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Clinical signs:

no effects observed

Description (incidence and severity):

In group 3, female no. 78 was found with prolapsed vagina, ruffled fur and pale eyes on day 2 post partum, therefore the dam and its litter was killed for ethical reasons. This was considered to be incidental and not related to the treatment with the test item.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In group 3, one female (no. 78) was killed for ethical reason two days before scheduled necropsy due to prolapsed vagina and cervix.
All the other animals survived until the scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

MALES:
Pre-Pairing, Pairing and After Pairing Periods : At 1000 mg/kg body weight/day, mean body weights were slightly reduced from about day 10 of pre-pairing period throughout pairing and after pairing periods but without attaining statistical significance. Mean body weight gain was also reduced when compared to controls with statistically significance on days 12 and 13 of the pre-pairing period. Thereafter the body weight gain was comparable with controls in the pairing and after pairing periods. No effects on body weights or body weight gains were observed at 300 and 100 mg/kg body weight/day.

FEMALES:
Pre-Pairing, Pairing, Gestation and Lactation Periods : No effects on body weights and body weight gains were observed during pre-pairing, gestation and early lactation periods for females.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males
In group 4, the absolute values for white blood cell count, neutrophils and lymphocytes for males were statistically significantly lower when compared to the control but the values were within the range of the historical control data.
In groups 2 and 3 no altered parameters were observed.

Females
The assessment of the hematology data did not reveal any test item-related effects in females in any dose group. In groups 4 and 3, statistically significantly lower hemoglobin level was noted (9.5 mmol/l each) but the values were within the historical control data (8.0 - 10.3 mmol/l). Therefore, it was not considered to be related to the treatment. The significantly higher hematocrit value and monocytes count noted in group 2 were considered to be incidental.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males
In group 4, the levels of total cholesterol and total protein were statistically significantly decreased but the values were within the reference data.
The globulin level was also statistically significantly decreased, and the value was slightly below the historical data (20.84 g/l compared to 21.34 g/l in the reference data). These were not considered to be related to the treatment with the test item.
In group 3, the chloride concentration was statistically significantly higher than control, but it was not considered to be related to the treatment.
In group 2 statistically significantly decreased total protein and albumin level was noted. Due to the lack of dose dependency this was considered not to be related to the treatment with the test item.

Females
In group 4, the levels of alanine aminotransferase and potassium were statistically significantly increased but were within the historical background data.
In group 3, the total bilirubin was statistically significantly decreased due to three animals (nos. 73, 74, 75) with zero value. Due to the lack of dose dependency this was considered of no toxicological importance. No changes were noted in group 2.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

None of the parameters under investigation during the functional observational battery gave an indication of a test item-related effect.
Mean values of grip strength for hind paws of lactating females was dose dependently lower in all test item-treated groups when compared to current controls.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no effects on organ weights of males and females observed in any dose group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related macroscopic findings were observed in males and females in any dose group.
The findings noted such as discoloration of the lungs, thymus, liver, mandibular lymph node, mammary gland and eyes, fibrin-like coating in the stomach, reduced size of testes or adrenal glands, prolapsed vagina, foci in thymus and constriction of spleen were found incidentally across the dose groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

In particular, qualitative examination of the stages of spermatogenesis in the testis did not reveal any treatment-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle. No treatment-related microscopic abnormalities were observed in the evaluation of the ovarian follicles and corpora lutea of the ovaries or the evaluation of the uterus.
All other findings were typical of this strain and age of rat and were considered to be incidental.

Histopathological findings: neoplastic:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The highest administered dose

Critical effects observed:

not specified

Conclusions:

Based on the obtained results a general NOAEL (No Observed Adverse Effect Level) was established at 1000 mg/kg bw/day.

Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of FAT #: 93580/A on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provides information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. The study was performed according to OECD TG 422 and EPA OPPTS 870.3650.

FAT 93580/A was administered to male rats for 6 weeks and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post-partum.

The following dose levels were applied: group 1 (control): 0 mg/kg bw/day, group 2: 100 mg/kg bw/day, group 3: 300 mg/kg bw/day, group 4: 1000 mg/kg bw/day.

A standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (corn oil).

In group 3, one female was killed for ethical reason two days before scheduled necropsy due to prolapsed vagina and cervix. All the other parental animals survived until the scheduled necropsy.

No clinical signs related to the treatment with the test item were observed in males or females of P generation during the study. None of the parameters under investigation during the functional observational battery gave an indication of a test item-related effect for P generation.

There was no indication of a test item-related effect observed in males and females of P generation in any treated group.

There was no test item-related effect observed on food consumption of males and females of P generation in any dose group.

At 1000 mg/kg bw/day, mean body weights and mean body gains of males were slightly reduced in the pre-pairing period but it was a transient slight effect as the body weight gain was comparable with controls in the pairing and after pairing periods.

No effects on body weights or body weight gains of males were observed at 300 and 100 mg/kg bw/day.

No effects on body weights and body weight gains were observed during pre-pairing, gestation and early lactation periods for females in any dose group.

The assessment of the haematology data did not reveal any test item-related effects in females and in males of P generation at any dose level.

Clinical biochemistry investigation did not reveal any test item-related effect. No test item-related macroscopic or microscopic findings were noted in P generation.

The mean number of pups at first litter check and its sex ratio was not affected by the treatment with the test item. No abnormal pup was noted at any dose level. Mean pup weights on day 1 and day 4 post-partum were unaffected by treatment with the test

item. No test item-related findings were noted at macroscopic examination of F1 pups.

Based on the obtained results, a NOAEL (No Observed Adverse Effect Level) for males and females was established at 1000 mg/kg bw/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

relative humidity exceeded the given range in the guideline (18.4 - 66.2 % instead of 30 - 70 %)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

relative humidity exceeded the given range in the guideline (18.4 - 66.2 % instead of 30 - 70 %)

GLP compliance:

yes

Limit test:

no

Justification for study design:

Recognized by international guidelines as a recommended test system.

Test material

Test material information

Constituent 1

Test material form:

other: waxy solid

Details on test material:

None

Specific details on test material used for the study:

Identification: FAT 93580/A
CAS Number: 84962-05-0
Active Ingredients: Octadecanoic acid, reaction products with diethylenetriamine and urea, acetates
Description: White-yellowish waxy solid; odour: acetic acid
Batch Number: 0022357000
Purity/Concentration as Supplied: More than 90% of octadecanoic acid, reaction products with diethylenetriamine and urea, acetates
Purity: 94.3 %
Correction for Purity: No
Stability of Test Item in Vehicle: At least 8 days if stored at room temperature
Expiry Date (Retest Date) as handled by Harlan Laboratories Ltd: 31-Dec-2018
Storage Conditions: Room temperature (20 ± 5 °C), in the dark
Safety Precautions: Routine hygienic procedures (gloves, goggles, face mask).

Test animals

Species:

rat

Strain:

other: RccHan : WIST(SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories BV, Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: 339-389 g (males); 223-273 g (females)
- Fasting period before study: no
- Housing: In groups of three to five animals in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding with paper enrichment. During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet (e.g. ad libitum): pelleted standard Harlan Teklad 2018C rodent maintenance diet, ad libitum
- Water (e.g. ad libitum): community tap-water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.9-23.6
- Humidity (%): 18.4-66.2
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
FAT #: 93580/A was weighed, crushed using a mortar and a small amount of vehicle was added to disperse the test item. The suspension was transferred to a glass beaker and the remaining vehicle was added. Using a magnetic stirrer and/or an ultra turrax, a homogeneous suspension was prepared. Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer. Dose formulations were stored at room temperature (20 ± 5 °C) in glass beakers. Based upon the results of stability analyses, dose formulations were stable for at least 8 days.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 0, 25, 75, 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
- Lot/batch no.: 492194511, 103197718

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: maximum 14 days
- Proof of pregnancy: copulation plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. To confirm the stability (8 days) samples of about 2 g of each concentration were taken from the middle of each aliquot used on day 7 of the treatment. During the last week of the treatment, samples were taken from the middle to confirm concentration.
The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to the analytical laboratory and stored there at -20 ± 5 °C until analysis. The samples were analyzed by HPLC - ELSD detector. The test item was used as the analytical standard.

Duration of treatment / exposure:

approximately 53 days

Frequency of treatment:

once daily

Details on study schedule:

Acclimatization: 7 days (males and females)
First test item administration: day 1 of pre-mating (males and females)
Pre-pairing: 15 days (males and females)
Blood sampling: day 15 of pre-mating (males and females)
Pairing: 14 days maximum (males and females)
Gestation: approximately 21 days (females)
Treatment ends: on day before sacrifice (males), on day 3 post-partum (females)
Necropsy: After treatment for 6 weeks, when no longer needed for assessment of reproductive effets (males), dams and pups on day 4 post-partum

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control Group (Group 1)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Test group (Group - 2)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Test group (Group - 3)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Test group (Group - 4)

No. of animals per sex per dose:

12 males and 12 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on a previous dose range-finding toxicity study in Wistar rat, using dose levels of 0, 100, 300 and 1000 mg/kg/day, where no test item related effects were observed up to the dose level of 1000 mg/kg/day.

Positive control:

No

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: for viability and mortality: twice daily, for cage-side clinical observations: once daily, during acclimatization and up to day of necropsy. Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: In males, it was performed once prior to the first administration of the test item and weekly thereafter. In females, it was performed once prior to the first administration of the test item, weekly during the pre-pairing and pairing periods and on days 0, 6, 13 and 20 of the gestation period.

BODY WEIGHT: Yes
- Time schedule for examinations: once during acclimatization and daily from treatment start to day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

OTHER:
FUNCTIONAL OBSERVATION BATTERY:
- Time schedule for examinations: At one time during the study (males shortly before the scheduled sacrifice and females on day 3 post-partum) relevant parameters were performed with five P generation males and five P generation females from each group.

CLINICAL LABORATORY INVESTIGATIONS:
- Time schedule for examinations: Blood samples were obtained on day 15 of the pre-pairing period from 5 males and 5 females from each group.

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

spermatogenesis

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: external examinations, sex ratios, body weights, stillbirths, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS:
no data

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after treatment for 6 weeks, when no longer needed for assessment of reproductive effects
- Maternal animals: All surviving animals on day 4 post-partum

GROSS NECROPSY
- no details

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 (Organ weights and preservation) were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGTHS
- no details

Statistics:

The following statistical methods were used to analyse food consumption, body weights, functional observational battery, locomotor activity, clinical laboratory investigations and reproduction data:
• Means and standard deviations of various data were calculated and included in the report.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Reproductive indices:

Corpora lutea, duration of gestation, implantation rate and post-implantation loss, litter size at first litter check, postnatal loss days 0-4 post-partum.

Offspring viability indices:

The viability index was 97.5, 99.3, 98.7 and 100 % in groups 1, 2, 3 and 4, respectively.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

In group 3, female no. 78 was found with prolapsed vagina, ruffled fur and pale eyes on day 2 post partum, therefore the dam and its litter was killed for ethical reasons. This was considered to be incidental and not related to the treatment with the test item.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In group 3, one female (no. 78) was killed for ethical reason two days before scheduled necropsy due to prolapsed vagina and cervix.
All the other animals survived until the scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

MALES:
Pre-Pairing, Pairing and After Pairing Periods : At 1000 mg/kg body weight/day, mean body weights were slightly reduced from about day 10 of pre-pairing period throughout pairing and after pairing periods but without attaining statistical significance. Mean body weight gain was also reduced when compared to controls with statistically significance on days 12 and 13 of the pre-pairing period. Thereafter the body weight gain was comparable with controls in the pairing and after pairing periods.
No effects on body weights or body weight gains were observed at 300 and 100 mg/kg body weight/day.

FEMALES:
Pre-Pairing, Pairing, Gestation and Lactation Periods : No effects on body weights and body weight gains were observed during pre-pairing, gestation and early lactation periods for females.

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:

effects observed, non-treatment-related

Description (incidence and severity):

Males
In group 4, the absolute values for white blood cell count, neutrophils and lymphocytes for males were statistically significantly lower when compared to the control but the values were within the range of the historical control data.
In groups 2 and 3 no altered parameters were observed.

Females
The assessment of the hematology data did not reveal any test item-related effects in females in any dose group.
In groups 4 and 3, statistically significantly lower hemoglobin level was noted (9.5 mmol/l each) but the values were within the historical control data (8.0 - 10.3 mmol/l). Therefore, it was not considered to be related to the treatment.
The significantly higher hematocrit value and monocytes count noted in group 2 were considered to be incidental.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males
In group 4, the levels of total cholesterol and total protein were statistically significantly decreased but the values were within the reference data.
The globulin level was also statistically significantly decreased, and the value was slightly below the historical data (20.84 g/l compared to 21.34 g/l in the reference data). These were not considered to be related to the treatment with the test item.
In group 3, the chloride concentration was statistically significantly higher than control, but it was not considered to be related to the treatment.
In group 2 statistically significantly decreased total protein and albumin level was noted. Due to the lack of dose dependency this was considered not to be related to the treatment with the test item.

Females
In group 4, the levels of alanine aminotransferase and potassium were statistically significantly increased but were within the historical background data.
In group 3, the total bilirubin was statistically significantly decreased due to three animals (nos. 73, 74, 75) with zero value. Due to the lack of dose dependency this was considered of no toxicological importance. No changes were noted in group 2.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

None of the parameters under investigation during the functional observational battery gave an indication of a test item-related effect.
Mean values of grip strength for hind paws of lactating females was dose dependently lower in all test item-treated groups when compared to current controls.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In particular, qualitative examination of the stages of spermatogenesis in the testis did not reveal any treatment-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle. No treatment-related microscopic abnormalities were observed in the evaluation of the ovarian follicles and corpora lutea of the ovaries or the evaluation of the uterus.
All other findings were typical of this strain and age of rat and were considered to be incidental.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performance and fertility did not give any indication to a test item-related effect in any dose group.
All females in groups 1, 2 and 3 mated within the first pairing period. In group 4, one female (no. 87) was mated during the second pairing period.
In group 2, for one female (no. 63) the mating was not detected when mated with male 16 and therefore was allocated to mate with male 15, where no mating happened because it was noted that she was already pregnant. This animal delivered viable pups but data of the dam and its litter were excluded from the summary tables of this report due to missing data of body weights and
food consumption in the gestation period.
Two females each in the control (nos. 53, 59) and group 2 (nos. 71, 72) and one female in group 4 (no. 85) were not pregnant.
The median and mean precoital times were unaffected by treatment with the test item. Mean precoital times were 3.8, 2.8, 2.6 and 3.6 days in groups 1, 2, 3 and 4, respectively. The median precoital time was 3, 3, 2 and 3 days in order of ascending dose level. For the female in group 4 that mated during the second pairing period.
The fertility index and conception rates were 83.3%, 83.3%, 100% and 91.7% in groups 1, 2, 3 and 4, respectively.
In group 4, female no. 93 delivered five pups but they were found dead at first litter check (for 3 out of 5 the sex was undetermined due to cannibalism). Due to this the gestation index was 90.9% in group 4 and 100% in all other dose groups. This finding was considered to be incidental.

Details on results (P0)

Corpora Lutea Count:
Mean number of corpora lutea per dam (determined at necropsy) was comparable with controls (15.0, 16.3, 15.7 and 15.3 in order of ascending dose level) and gave no indication of a test itemrelated effect.

Duration of Gestation:
The mean duration of gestation was unaffected by exposure to the test item. Mean duration of gestation was 21.7, 21.4, 21.7 and 21.8 days, in order of ascending dose level.

Implantation Rate and Post-Implantation Loss :
The implantation rate and post-implantation loss did not reveal any test item-related effects. The mean numbers of implantations per litter were 14.1, 15.4, 15.2 and 12.0 in order of ascending dose levels. The mean incidence of post-implantation loss as a percentage of total implantations was 16.3, 7.8, 8.3 and 15.9%, in order of ascending dose levels. The slightly lower mean number of implantations in the high dose group was considered to be not related to the treatment with the test item due to the lack of statistical significance and that they were in the historical background range (11.0 - 13.8). The post-implantation loss % of implantations was slightly exceeded the historical background range (3.7 - 14.4%) but it was lower than current control, therefore it was considered to be unrelated to treatment.

Litter Size at First Litter Check:
The litter size at first litter check and the birth index were not affected by the treatment with the test item. In groups 1, 2, 3 and 4 birth index was 83.7, 92.2, 91.1 and 84.1 % and mean litter size at first litter check was 11.8, 14.2, 13.8 and 10.1, respectively. In group 4, the mean litter size was slightly lower than current control but was within the historical control range (9.5 - 13.2).

Postnatal Loss Days 0 - 4 Post Partum:
There was no effect on post-natal loss between days 0 and 4 post partum. In group 4, female no. 93 delivered five pups but they were found dead at first litter check (for 3 out of 5 pups the sex was undetermined due to cannibalism). This was considered to be In group 3, one litter (no. 78) was killed for ethical reason since the mother had prolapsed vagina and showed severe clinical symptoms on day 2 post partum. Mean postnatal loss was 0.3, 0.1, 0.2 and 0.0 and the viability index was 97.5, 99.3, 98.7 and 100 % in groups 1, 2, 3 and 4, respectively.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

general

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: the highest administered dose

2

Dose descriptor:

NOEL

Remarks:

for reproduction

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: the highest administered dose

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No abnormal findings were noted at first litter check or during the first 4 days post partum.
In group 3, the pups of dam no. 78 with prolapsed vagina and one pup in group 2 (litter 67) were cold to touch and a wound was found of a group 4 pup (litter 94) on the posterior dorsum on day 1 post partum. These findings were considered not be related to the treatment with the test item

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean pup weights on day 1 and day 4 post partum were unaffected by treatment with the test item. On day 1 post partum mean pup weights were 6.3, 6.0, 6.1 and 6.3 g and on day 4 post partum were 9.6, 8.6, 9.2 and 9.7 g in order of ascending dose level.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related findings were noted at macroscopic examination of F1 pups.
Discoloration of the lung for one male group 1 and 3 pup each and one group 2 female pup was observed and one control female pup was reduced in size. Additionally, sex was undetermined for three pups in group 4.

Histopathological findings:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

VIABILITY (OFFSPRING): No effects

CLINICAL SIGNS (OFFSPRING): No data

BODY WEIGHT (OFFSPRING): No effects

SEXUAL MATURATION (OFFSPRING): Not examined

ORGAN WEIGHTS (OFFSPRING): No data

GROSS PATHOLOGY (OFFSPRING): No effects

HISTOPATHOLOGY (OFFSPRING): No data

Effect levels (F1)

Key result

Dose descriptor:

NOEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: the highest administered dose

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The NOEL for reproduction/developmental toxicity was concluded to be 1000 mg/kg bw/day.

Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of FAT #: 93580/A on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provides information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. The study was performed according to OECD TG 422 and EPA OPPTS 870.3650.

FAT 93580/A was administered to male rats for 6 weeks and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post-partum.

The following dose levels were applied:

group 1 (control): 0 mg/kg bw/day,

group 2: 100 mg/kg bw/day,

group 3: 300 mg/kg bw/day,

group 4: 1000 mg/kg bw/day.

A standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (corn oil).

Observation:

In group 3, one female was killed for ethical reason two days before scheduled necropsy due to prolapsed vagina and cervix. All the other parental animals survived until the scheduled necropsy. No clinical signs related to the treatment with the test item were observed in males or females of P generation during the study. None of the parameters under investigation during the functional observational battery gave an indication of a test item-related effect for P generation. There was no indication of a test item-related effect observed in males and females of P generation in any treated group. There was no test item-related effect observed on food consumption of males and females of P generation in any dose group.

At 1000 mg/kg bw/day, mean body weights and mean body gains of males were slightly reduced in the pre-pairing period but it was a transient slight effect as the body weight gain was comparable with controls in the pairing and after pairing periods. No effects on body weights or body weight gains of males were observed at 300 and 100 mg/kg bw/day. No effects on body weights and body weight gains were observed during pre-pairing, gestation and early lactation periods for females in any dose group. The assessment of the haematology data did not reveal any test item-related effects in females and in males of P generation at any dose level. Clinical biochemistry investigation did not reveal any test item-related effect. There were no effects observed on reproduction and breeding data which could be related to the treatment with the test item. There were no effects on organ weights of males and females of P generation observed in any dose group. No test item-related macroscopic or microscopic findings were noted in P generation. The mean number of pups at first litter check and its sex ratio was not affected by the treatment with the test item. No abnormal pup was noted at any dose level. Mean pup weights on day 1 and day 4 post-partum were unaffected by treatment with the test item. No test item-related findings were noted at macroscopic examination of F1 pups.

Based on the findings of the study, a general NOAEL (No-Observed-Adverse-Effect-Level) was considered to be 1000 mg/kg bw/day. The reproductive/developmental NOEL (No Observed Effect Level) was considered to be 1000 mg/kg bw/day.