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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 Aug 2012 - 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline and GLP compliant study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 22. March, 1996
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650, July 2000
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Fatty acids, C18-unsatd., dimers, ethoxylated (NIO AD1)
- Physical state: amber liquid
- Analytical purity: ~ 94%
- Lot/batch No.: 0007876143
- Expiration date of the lot/batch: 22-Aug-2013
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., Horst, Netherlands (RccHanTM:WIST(SPF)
- Age at study initiation: 11 weeks
- Weight at study initiation: weight range: male 293 to 335 g; female 193 to 229 g
- Housing: in groups of three to four in Makrolon type-4 cages with wire mesh tops up to the day of randomization, afterwards individually in Makrolon type-3 cages
- Diet: Pelleted standard Harlan Teklad 2018C rodent maintenance diet, ad libitum
- Water: Community tap-water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- 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: Fatty acids, C18-unsatd., dimers, ethoxylated (NIO AD1) was weighed into a glass beaker on a tared precision balance and the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Separate formulations were prepared for each concentration.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 5 days
- Proof of pregnancy: vaginal smear sperm positive or a copulation plug was observed referred to as day 0 post coitum
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken on the first day of treatment and towards the end of the study. The samples were analyzed by GC coupled to an FID detector following an analytical procedure provided by the Sponsor and adapted at Harlan Laboratories. The test item was used as the analytical standard. Duplicates were taken of all samples and were stored at Harlan Laboratories Ltd., Füllinsdorf / Switzerland. The samples were discarded after approval of the data.
Duration of treatment / exposure:
Males: 28 days (14 days pre-pairing, 5 days pairing, sacrifice after treatment of 28 days)
Females: 6 weeks (14 days pre-pairing, 5 days pairing, 21 days gestation, 4 days lactation)
Frequency of treatment:
daily, 7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
11
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a 14-day range-finding study (D57190, BASF project number 01R0072/12X185) the dose levels of 100, 300, and 1000 mg/kg bw per day were selected for the present study.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily
Once prior to the first administration of the test item (day 6 of acclimatization) and weekly thereafter (in the gestation period on day 0, 6, 13 and 20 post coitum), detailed clinical observations were performed outside the home cage in a standard arena. Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.

BODY WEIGHT: Yes
- Time schedule for examinations: daily, from treatment start to day of necropsy

FOOD CONSUMPTION:
- Food consumption for each animal determined: Males: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 – 14, after pairing period days 1 – 7 and 7-10. Females: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; gestation days 0 – 7, 7 14 and 14 – 21 and days 1 - 4 of the lactation. No food consumption was recorded during the pairing period.
Sperm parameters (parental animals):
Parameters examined in male parental generation:
testis weight, epididymis weight
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
litter size, live births, still births, sex ratio, individual weights on days 1 and 4 post partum

GROSS EXAMINATION OF DEAD PUPS:
yes, for gross anomalies
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after 28 days of treatment
- Maternal animals: All surviving animals on day 5 post partum

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

HISTOPATHOLOGY / ORGAN WEIGHTS
Testes, epididymides, prostate, seminal vesicles, ovaries, oviduct, vagina and uterus from all animals of the control and high-dose group were examined. The same applied to all occurring gross lesions. The remaining organs/tissues of 5 randomly selected males and females of the control and high-dose group, respectively, were examined histopathologically. The following tissues were preserved: Prostate, Seminal vesicles with coagulating gland, Testes, Epididymides (males); Ovaries, Uterus (females); Gross lesions, Brain, Spinal chord (cervical, thoracic, lumbar), Small and large intestines (incl. Peyer’s patches), Stomach (forestomach and glandular stomach), Liver
Kidneys, Adrenals, Lymph nodes (axillary and mesenteric), Urinary bladder, Aorta, Heart, Thymus, Thyroids and parathyroids, Trachea and lungs (preserved by inflation with fixative and then immersion), Pituitary gland, Spleen, Peripheral nerve (sciatic), Bone marrow (femur), Femur with knee joint, Mammary gland (male and female), Pancreas
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at day 4 post partum.

GROSS NECROPSY
- Gross necropsy consisted of examination for gross anomalies.

Statistics:
The following statistical methods were used to analyze food consumption, body and organ weights, clinical laboratory and reproduction data and macroscopical findings:
• Means and standard deviations of various data were calculated.
• The Dunnett-test [C.W. Dunnett: A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Statist. Assoc. 50, pp. 1096-1121 (1955)] (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 [R.G. Miller: Simultaneous Statistical Inference, Springer Verlag, New York (1981)] (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 [R.A. Fisher: Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950)] was applied if the variables could be dichotomized without loss of information.
Reproductive indices:
Percentage mated = (Females mated/Females paired)*100
Fertility index = (Females achieving a pregnancy/Females paired) *100
Conception rate = (Females achieving a pregnancy/Females mated)*100
Gestation index = (Number of females with living pups/Females pregnant)*100
Offspring viability indices:
Birth index = (pups born alive/number of implantations)*100
Viability index = (pups alive before cullig on day 4 post partum/pups born alive)*100

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no unplanned deaths during the course of this study. No clinical signs were noted in males of all dose levels during the whole course of the study and in females at 0, 100 and 1000 mg/kg bw/day.
Incidentally, in one female treated at 300 mg/kg bw/day, a reddish sore and scab in the neck were noted from day 15 of gestation onwards, resulting in a slight hairloss. While the reddish sore disappeared within 6 days, the scab and hairloss persisted until the day of scheduled necropsy.
No treatment-related findings at detailed weekly clinical observation were noted in males and females at any dose level.
Findings in males were limited to day 13 of the pre-pairing period and consisted of slightly to moderately slowed righting reflex in two males at 100 and three males at 300 mg/kg bw/day. On three occasions (once at 100 mg/kg bw/day and twice at 300 mg/kg bw/day), this finding was accompanied by a slightly decreased activity. Due to the absence of a dose-dependency, these findings were considered to be incidental.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males: As an isolated finding, body weight gains were slightly, but statistically significantly lower at 1000 mg/kg bw/day on Day 5 of the pre-pairing period. From day 4 of the after pairing period onwards, a higher body weight gain was noted at 100 mg/kg bw/day up to study termination. This difference reached statistical significance on most occasions from this day onwards. No statistically significant difference was noted in absolute body weights in this group. The higher mean body weight gains were considered to be a result of biological variability, especially in the light of absent dose-dependency. The overall differences in mean body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were: +13%, +14%, +13% and +13% during the pre-pairing period and +1%, +1%, +2% and +2% during the pairing period and +1%, +3%, +1% and +2% during the after pairing period (percentages refer to the body weight gain within the period).
Females: No test item-related effects on mean body weight and mean body weight gain were present at any dose level. As an isolated finding, body weight gains were slightly, but statistically significantly lower at 1000 mg/kg bw/day on Day 9 of the pre-pairing period. The overall differences in mean body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were: +8%, +8%, +9% and +7% during the pre-pairing period, +60%, +57%, +56% and +57% during the gestation period and +4%, +3%, +3% and +2% during the lactation period (percentages refer to the body weight gain within the period).
There were no effects on mean food consumption at any dose level. In males treated at 1000 mg/kg bw/day, food consumption was slightly statistically significantly increased between days 11 and 14 of the pre-pairing period. In general, in this group, there was a trend towards a higher food consumption (+3.8% over pre-pairing and +9.8% during after pairing). However, since no effect on body weights was noted in males treated at 1000 mg/kg bw/day, this finding is considered to be due to biological variability rather than test item related.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No effects on mating performance and fertility were observed at any dose level. All females were mated within the first pairing period. The median and mean precoital times were unaffected by treatment with the test item. Mean (median) precoital times calculated for the first pairing period were 2.9 (3), 3.3 (3), 2.7 (3) and 2.7 (3) days in order of ascending dose levels. One female at 100 mg/kg bw/day was not pregnant. As a result the fertility index in this group was 90.9% and 100.0% at 0, 300 and 1000 mg/kg bw/day. Gestation index (number of females with living pups as a percentage of females pregnant) was 100% at all dose levels.
Mean number of corpora lutea per dam (determined at necropsy) was not affected by treatment with the test item (16.7, 16.8, 13.3 and 13.8 in order of ascending dose level). At 300 mg/kg bw/day, the number of corpora lutea was statistically significantly lower when compared with controls. In the absence of a dose-relationship, this difference was considered to be incidental.
The mean duration of gestation was unaffected by exposure to the test item. Mean duration of gestation was 21.6, 21.4, 21.5 and 21.7 days, in order of ascending dose level.
No effects on implantation rate or post-implantation loss were observed at any dose level. The mean number of implantations per dam was 14.0, 13.5, 11.7 and 11.9 in order of ascending dose levels. The mean incidence of post-implantation loss as a percentage of total implantations was 10.4%, 8.9%, 3.9% and 16.0%, in order of ascending dose level. The high (but not statistically significant different) value at 1000 mg/kg bw/day was based mainly on female no. 79, which had 13 implantation sites but gave birth to only 4 pups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Absolute liver weights and liver to body weight ratios were statistically significantly higher in males at 1000 mg/kg bw/day (approximately 15% and 13%, respectively). This measurement correlated with the microscopic findings in the liver.
In males at 100 and 300 mg/kg bw/day as well as in females at all dose levels, no significant differences were noted in organ weights or organ weight ratios.

GROSS PATHOLOGY (PARENTAL ANIMALS)
At necropsy, there were no treatment-related macroscopical findings. All gross lesions recorded were considered to be within the range of normal background alterations. All findings were recorded only once in isolated animal across all groups. In males, these findings consisted of foci in the lungs or thymus, pelvic dilation (kidneys), and testes or epididimydes reduced in size. In females, foci either on the lungs, esophagus, kidneys or harderian glands were noted. Furthermore, discoloration of the ovaries or mesenteric lymph nodes as well as constriction of the spleen was recorded.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Minimal centrilobular hepatocellular hypertrophy in the liver of high dose group males was found that correlated with increased liver weights. There were no indicators of liver injury (Kupffer cell proliferation, necrosis, apoptosis, fibrosis, other degenerative changes, etc.), hence, this lesion was considered to be of metabolic nature.
A further microscopic finding that could be attributed to treatment with the test item was recorded in the lungs and consisted of slight aspiration pneumonia in low dose group animals and slight to moderate aspiration pneumonia in animals from the intermediate and high dose groups. This finding was considered to be most likely due to accidental uptake by regurgitation of the test item. The slightly higher incidence and/or severity grade of pneumonia recorded in animals of groups 3 and 4 may be explained by an increased viscosity of the test item/vehicle with increasing concentration of the test item. Subsequently, gavage technique needs higher pressure to push out the test item/vehicle off the probe. By drawing back the gavage probe counter pressure is reduced at the laryngeal orifice, and hence, droplets may be set at this place, and thereafter, inspired.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
reproduction/developmental toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
NOEL
Remarks:
histopathological
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: minimal centrilobular hepatocellular hypertrophy in the liver of high dose group males

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
No effects on litter size were observed at any dose level. Mean litter size at first litter check was 12.7, 12.3, 11.4 and 10.0 pups in order of ascending dose levels. One dead male pup and 4 dead female pups at first litter check were recorded at 1000 mg/kg bw/day.
No effects on postnatal loss were observed at any dose level. Between days 0 and 4 post partum, one pup was lost at 100 and 300 mg/kg bw/day and at 1000 mg/kg bw/day three pups from three different litters.
The higher incidence of dead pups in the highest dose group was mainly due to female 79 which had 13 implantation sites but gave birth to 4 live pups only. This effect is considered to be incidental and not substance specific, since all other females showed numbers within normal ranges.

CLINICAL SIGNS (OFFSPRING)
No test item-related findings were noted for pups during first litter check and lactation at any dose level. In the control group, on day 1 post partum, one pup showed a wound on the abdomen and a second pup showed a wound on the tail. These findings occurred in two different litters.

BODY WEIGHT (OFFSPRING)
Mean pup weights on day 1 and 4 post partum were unaffected by treatment with the test item. On day 1 post partum mean pup weights were 6.2, 5.8, 6.3 and 6.4 g in order of ascending dose level. Also mean body weight gain was similar in all groups. On day 4 post partum no effects on mean body weights were observed.

SEXUAL MATURATION (OFFSPRING)
Sex ratios at first litter check and on day 4 post partum were unaffected by exposure to the test item. The proportion of males at first litter check was 59, 61, 48 and 52%, in order of ascending dose level.

GROSS PATHOLOGY (OFFSPRING)
No findings were noted at macroscopic examination of F1 pups at any dose level.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the microscopic findings recorded in the liver, a histopathological NOEL (No Observed Effect Level) was established at 300 mg/kg/day.
The NOAEL (No Observed Adverse Effect Level) for systemic toxicity was considered to be 1000 mg/kg bw/day.
The NOEL for reproduction/developmental toxicity was considered to be 1000 mg/kg body weight/day.
Executive summary:

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item Fatty acids, C18-unsatd., dimers, ethoxylated (NIO AD1) to rats according to OECD guideline 422. Fatty acids, C18-unsatd., dimers, ethoxylated (NIO AD1) was administered in olive oil as vehicle at dosages of 100, 300 and 1000 mg/kg body weight/day, and controls received the vehicle only. Fatty acids, C18-unsatd., dimers, ethoxylated (NIO AD1) was administered to male rats for 28 days 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.

 

There were no unplanned deaths and no treatment related macroscopical findings. In-life observations of the animals showed decreased rearings in one male at 300 mg/kg bw/day and two males at 1000 mg/kg bw/day, and a slight decrease of body temperature in males and females at 1000 mg/kg bw/day. In the absence of any other changes, these findings were considered to be not adverse.

 

Food consumption and body weights were generally unaffected by treatment.

At histopathological examination, minimal centrilobular hepatocellular hypertrophy in the liver of high dose group males was noted that correlated with increased liver weights. This lesion was considered to be of metabolic nature. Furthermore, slight aspiration pneumonia in low dose group animals and slight to moderate aspiration pneumonia in animals from the intermediate and high dose groups could be attributed to treatment, most likely due to accidental uptake by regurgitation of the test item. A slightly higher incidence and/or severity grade of pneumonia was recorded in animals at a dose level of 300 and 1000 mg/kg bw/day, may be explained by an increased viscosity of the test item/vehicle with increasing concentration of the test item.

 

The NOAEL(No Observed Adverse Effect Level)for systemic toxicity was considered to be 1000 mg/kg bw/day.

The NOEL for reproduction/developmental toxicity was considered to be 1000 mg/kg body weight/day.