C18 unsaturated fatty acids, reaction products with 1-aminopropan-2-ol, maleic anhydride and sodium bisulfite

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 422

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-18(even numbered) and C18 unsaturated)alkyl)amino]ethyl]esters, disodium salts

IUPAC Name:

Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-18(even numbered) and C18 unsaturated)alkyl)amino]ethyl]esters, disodium salts

Test material form:

other: liquid

Test animals

Species:

rat

Strain:

other: CD® / Crl:CD(SD)

Details on test animals or test system and environmental conditions:

- Source: Charles River Laboratories Research, Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at start of dosing : Males 55 days; Females: 48 days
- Weight range at start of dosing: Males: 281.0 to 308.0 g; Females: 68.9 to 195.7 g
- Fasting period before study: Ad libitum with exception of the night before the day of blood withdrawal for laboratory examination.
- Housing: With exception of the mating period, the animals were kept singly in MAKROLON
cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt/ Arkeburg, Germany) is used as bedding material in these cages. The cages were cleaned and changed once a week.
- Diet (e.g. ad libitum): ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany, ad libitum with exception of the night before the day of blood withdrawal for laboratory examination.
- Water (e.g. ad libitum): Tap water is offered daily ad libitum.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C (maximum range)
- Humidity (%): 55% ± 15% (maximum range)
- Air changes (per hr): Not provided
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
Males From: August 27, 2012 To: October 2, 2012
Females From: August 27, 2012 To: October 19, 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Application volume: 5 mL/kg bw/day. The test item was dissolved in the vehicle tap water to concentrations of 20, 60 and 200 mg test item /mL tap water and was administered orally at a constant volume once daily. The amount of the test item was adjusted to the animal's current body weight daily. The test item-vehicle mixture was freshly prepared every day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the analysis of the test item-vehicle mixtures samples of approx. 2 x 5 mL were taken at the following time points and stored at ≤ -20°C until analysis at LPT.
*Start of treatment period: Analysis of stability and concentration: Immediately after preparation of the test item-vehicle mixtures as well as 8 and 24 hours after storage of the test item preparations at room temperature: 3 samples/dose level group = 9 samples
*End of treatment period: Concentration: During treatment with the test item always before administration to the last animal/dose level group: 3 samples
The samples were labelled with the study number, test item, test species, type of sample, aliquot number, concentration, test day, sampling time and date.
The validation of the analytical method is part of LPT study No. 28344 (14-day dose-range-finding).

The measured actual concentrations of the test item in the test item vehicle mixtures were between 99.99% and 102.96% of the nominal concentrations (table 26).

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1 (1 male and 1 female animal were placed in one cage during the dark period)
- Length of cohabitation: The female was placed with the same male until pregnancy occurred or 2 weeks had elapsed.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

Males: 2 weeks prior to mating, during the mating period and approx. 2 weeks post mating at least until the minimum total dosing period of 28 days has been completed (up to and including the day before sacrifice).
Females: 2 weeks prior to mating and continuing up to, and including, day 3 post-partum or the day before sacrifice.

Frequency of treatment:

daily

Duration of test:

Males: 2 weeks prior to mating, during the mating period and approx. 2 weeks post mating at least until the minimum total dosing period of 28 days has been completed (up to and including the day before sacrifice).
Females: 2 weeks prior to mating and continuing up to, and including, day 3 post-partum or the day before sacrifice.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels have been selected in agreement with the Sponsor based on the results of a 14-day dose-range-finding study in rats dosed at 100, 300 and 1000 mg act. ingr./kg bw by oral gavage (LPT Study No. 28344). None of the animals died prematurely. None of the male and female rats treated orally with 100 or 300 mg act. ingr./kg bw/day revealed any changes in behaviour, external appearance or faeces. Salivation was noted for 2 of 5 male animals treated at 1000 mg/kg bw/day on 1 or 3 test days starting on test day 9 and increased faeces was noted for 3 of 5 male and 2 of 5 female high dosed animals on 6 or 9 test days starting on test day 5. No test item-related changes on body weight and body weight gain were noted for the male and female rats up to 1000 mg act. ingr./kg bw/day. No test item-related changes on food consumption were noted for the male and female rats treated orally with 100 or 300 mg act. ingr./kg bw/day. The food consumption of
the male and female animals treated with 1000 mg act. ingr./kg bw/day was slightly increased by 9% for the males and by 10% for the females in test week 2 (statistically significant at p ≤ 0.01 for both sexes). No test item-related influence was noted for the drinking water consumption at any of the tested dose levels. None of the male and female rats treated orally with 100, 300 or 1000 mg act. ingr ./kg bw/day revealed changes at macroscopic inspection at necropsy or organ weights.
- Rationale for animal assignment (if not random):
The animals are randomly allocated to the test groups.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the test period, each animal was observed for clinical signs at least once daily. Mortality was recorded twice daily. In addition, animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals were checked regularly from 7:00 a.m. to 11 :00 a.m. with a final check performed at approximately 3:30 p.m.
- Individual animals were observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness. Cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Additionally, once before the first exposure (to allow within-subject comparisons) and once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena and at the same time, each time preferably by observers unaware of the treatment. Signs noted included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes. Body weights were recorded individually for each adult animal.
- Time schedule for examinations:
Males and females were weighed on the first day of dosing, weekly thereafter and at termination. During gestation, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum) and day 4 post-partum.

FOOD CONSUMPTION: Yes
The quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period with the execution of the mating period. Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment week.

WATER CONSUMPTION:
- Time schedule for examinations: Water consumption was monitored daily by visual appraisal throughout the study.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: Dams with offspring were sacrificed on day 4 post-partum, or shortly thereafter.
- Organs examined: At the time of sacrifice or premature death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.

OTHER:
REPRODUCTIVE PARAMETERS:
Number of pregnant females
Pre-coital time
Gestation length calculated from day 0 of pregnancy
Corpora lutea
lmplantation sites

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes . Apparently non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI.
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter. Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter, were carefully examined externally for gross abnormalities.
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: No
- Head examinations: No

Statistics:

Toxicology and Pathology data were captured, whenever possible, using the departmental computerized systems (Provantis®8 Integrated preclinical software, Instem LSS Ltd.). Raw data not fully compatible with the computerized systems were maintained on paper according to appropriate SOPs.
The test item-treated groups (2 - 4) were compared with the control group (1 ).
The following statistical methods were used:
STUDENT' s t-test All numerical functional tests
(p ≤0.05 and ≤0.01)


Multiple t-test based on Body weight I Food consumption I
DUNNETT, C. W. Haematology I Clinical chemistry I
New tables for multiple Absolute and relative organ weights
Comparisons with a control (p ≤ 0.05 and ≤0.01)
Biometrics,
482-491 (Sept 1 964)

For all numerical values (e.g. body weight, food consumption and organ weight data) homogeneity of variances was tested by using the BARTLETT chi2-test. lf the variances are homogeneous, the DUNNETT test (p ≤ 0.01) was used to compare the experimental groups with the control group.
In case of heterogeneity of variances, the STUDENT's t-test was carried out; limit of significance is p ≤ 0.01.
Exact test of R. A. FISHER Histopathology, if applicable
(p≤0.05)
For the comparison of classification measurements (for example the fertility index) the FISHER's exact test, n < 100 or chi2-test with Yates' correction for continuity, n ≥ 100 (p ≤0.05 and p ≤0.01) were employed.
These statistical procedures were used for all data. Significantly different data were indicated in the tables of the report.
The mean values and standard deviations were calculated to the highest possible degree of accuracy and then rounded to the reported number of decimal places. Hence, deviations to the last decimal place of up to ± 1 may occur caused by rounding.

Indices:

Reproductive indices:
Gestation Index
Fertility Index
Pre-implantation loss [%]
Post-implantation loss [%]

Offspring viability indices:
Birth Index
Live Birth Index
Viability Index

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No signs of clinical toxicity were noted in the investigated female treatment groups (100, 300 or 1000 mg/kg bw/day) during the whole study.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A slight reduction in body weight was noted for the female rats of the high dose group (1000 mg/kg bw/day). The reduction in body weight was noted from gestation day 0 (7.6%) until lactation day 4 (9.5%). The body weight at autopsy was reduced accordingly.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

oral gavage study
A slightly statistically significant (p≤0.05) decrease in relative food consumption by 7.4% was noted in the high dose group (1000 mg/kg bw/day) during the first test week.

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):

Increased number of eosinophils in intermediate dose females on test day 15 lacking dose de
pendence (p≤0.05).
No test item-related influence was noted.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In the female high dose group (1000 mg/kg bw/day) a statistically significant (p≤0.01) increase (+46%)) was noted for the plasma activity of ALAT.
In the female intermediate dose group (300 mg/kg bw/day) an increase in bile acids on test day 15 (p≤0.05) was noted but not considered test-item related (lacking dose dependence).

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No test item-related influence was noted for the fore- and hindlimb grip strength in any female treatment group (100, 300 or 1000 mg/kg bw/day).
No test item-related influence was noted on the spontaneous motility of the rats in any of the treatment groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related influence on relative and absolute organ weights was noted for the rats treated with 100, 300 or 1000 mg/kg bw/day in comparison to the control group.
Statistically significant differences in the relative organ weights compared to the control, which are not considered to be test item-related were:
- increased relative weight of right kidney in high dose females (slight alteration in comparison to controls without biological relevance and not accompanied by an increase in absolute organ weight)
-decreased absolute heart weight by 16.6% in high dose females (p≤0.05).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test-item related macroscopic changes were noted in any treatment group (100, 300 and 1000 mg/kg bw/day) in females.
In 2 of the 3 non-pregnant rats (nos 16 and 54) from the control and the intermediate dose group (300 mg/kg bw/day) a thickened uterus was noted.
These findings were considered to be not test item-related but spontaneous due to the low number of occurrence.

Neuropathological findings:

no effects observed

Description (incidence and severity):

No test item-related influence in observational screening was noted for any treatment group (100, 300 or 1000 mg/kg bw/day).

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

No test item-related microscopic changes were seen in the reproductive organs for females.
A test item-related squamous cell hyperplasia was noted in the forestomachs from 5/5 female animals of the high dose group (1000 mg/kg bw/day). These test item-related stomach changes were localized in the zone adjoining the glandular stomach mucosa (forestomach or non-glandular mucosa) and attained statistical significance (p≤0.01). Occasionally the squamous cell hyperplasia with subsequent hyperkeratinization was associated with acute inflammation of the submucosa in the non-glandular stomach (1/5 females). Examination of the stomachs from the animals (5 per group) of the low- and intermediate dose groups (100 and 300 mg/kg bw/day) did not reveal any test
item-related changes. As humans lack a fore-stomach, the relevance of these changes for humans is questionable.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No premature deaths were noted in the female rats treated with 100, 300 and 1000 mg/kg bw/day.
No signs of clinical toxicity were noted for the female rats treated with 100, 300 and 1000 mg/kg bw/day.

BODY WEIGHT AND FOOD CONSUMPTION
A slight reduction in body weight was noted for the female rats of the high dose group (1000 mg/kg bw/day).
For the high dose female rats a slight, not statistically significant, reduction in body weight was noted of 3.7% and 3.4% was noted on test days 8 and 15 (pre-mating). Body weight gain was 6.6% on test day 8 and 10.6% on test day 15 in the high dose group in comparison to 10.7% (test day 8) and 14.6% (test day 15) in the control group. Body weight was decreased during the whole gestation period between 7.6% on gestation day 0 and 6.5% on gestation day 20, statistically significant (p≤0.05) on gestation days 7 and 14 with 9.7% and 7.9% decrease, respectively. A small reduction in body weight gain was noted in the high dose group on gestation day 0 (15% in comparison to 18%. On gestation day 20 body weight gain was nearly identical in the high dose group (59.7%) and the control group (59.4%). During lactation a slight but not significant reduction in body weight was noted in the intermediate dose female rats by 4.4 % on lactation day 0 and 5.6% on lactation day 4. In the high dose group the body weight was statistically significantly (p≤0.05) reduced on lactation days 1 and 4 by 9.1% and 9.5%. No noticeable differences were noted in body weight gain during the lactation period with 5.6% in the high dose group and 6.1% in the control group.
A slightly statistically significant (p≤0.05) decrease in relative food consumption by 7.4% was noted in the high dose females during the first test week.

HAEMATOLOGICAL FINDINGS
No test item-related influences were noted between the control group and the treatment groups for the haematological parameters, i.e. the haemoglobin content, the number of erythrocytes, leucocytes, reticulocytes and platelets, the haematocrit value, the thromboplastin time (TPT, aPTT), the mean corpuscular volume (MCV), the mean corpuscular haemoglobin (MCH) and the mean corpuscular haemoglobin concentration (MCHC). No test item-related changes were noted in the relative and absolute differential blood counts.
Statistically significant increased number of eosinophils (p≤0.05) was noted in the intermediate dose females on test day 15. This was considered to be not test item related ( lacking dose dependence).

CLINICAL BIOCHEMISTRY
In the high dose group (1000 mg/kg bw/day) a statistically significant (p≤0.01) increase was noted for the plasma activity of ALAT.
In females no test item related influence was noted for the plasma levels of albumin, of globulin, the albumin/globulin ratio, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), urera in blood, sodium, potassium, calcium, chloride, the activity of the alkaline phosphatase (aP) and the serum levels of the bile acids. Increased bile acids in the intermediate dose females (p≤0.05) was considered to be not test item related (lacking dose dependence).
BEHAVIOUR (FUNCTIONAL FINDINGS)
The functional neurological screenings were performed between test days 40-50 for the female rats, 2 hours after dosing.
No test item-related influence was noted for the fore- and hindlimb grip strength in any treatment group (100, 300 or 1000 mg/kg bw/day).
No test item-related influence was noted on the spontaneous motility of the rats in any of the treatment groups.

ORGAN WEIGHTS(MATERNAL)
No test item-related influence on relative and absolute organ weights was noted for the rats treated with 100, 300 or 1000 mg/kg bw/day in comparison to the control group.
Statistically significant differences in the relative organ weights compared to the control, which are not considered to be test item-related were:
- increased relative weight of right kidney in high dose females (p≤0.05)
Statistically significant differences in the absolute organ weights compared to the control, which are not considered to be test item-related were:
-decreased absolute heart weight by 16.6% in high dose females (p≤0.05)

GROSS PATHOLOGY (MATERNAL)
Macroscopic inspection at autopsy for the females was performed between test days 43 and 54.
No test-item related macroscopic changes were noted in any treatment group (100, 300 and 1000 mg/kg bw/day) in females.
In 2 of the 3 non-pregnant rats (nos 16 and 54) from the control and the intermediate dose group (300 mg/kg bw/day) a thickened uterus was noted.
These findings were considered to be not test item-related but spontaneous due to the low number of occurrence.

NEUROPATHOLOGY (OBSERVATIONAL FINDINGS)
The observational screenings were performed between test days 40-50 for the female rats, 2 hours after dosing.
No test item-related influence was noted for any treatment group (100, 300 or 1000 mg/kg bw/day).

HISTOPATHOLOGY (MATERNAL)
No test item-related microscopic changes were seen in the reproductive organs for females.
A pulmonary congestion was noted in the lungs from 4/5 male (control 0/5) and 5/5 female rats (control 4/5) of the high dose group (1000 mg/kg bw/day). As pulmonary congestion is occasionally seen in rats as a background finding, this change was not considered to be test item-related.
A test item-related squamous cell hyperplasia was noted in the fore-stomachs from 5/5 male and 5/5 female animals of the high dose group (1000 mg/kg bw/day). These test item-related stomach changes were localized in the zone adjoining the glandular stomach mucosa (forestomach or
non-glandular mucosa) and attained statistical significance for both sexes (p≤0.01). Occasionally the squamous cell hyperplasia with subsequent hyperkeratinization was associated with acute inflammation of the submucosa in the non-glandular stomach (for 2/5 males and 1/5 females).
Examination of the stomachs from the animals (5 per group) of the low- and intermediate dose groups (100 and 300 mg/kg bw/day) did not reveal any test item-related changes. As humans lack a fore-stomach, the relevance of these changes for humans is questionable.
All other microscopic changes observed were either coincidental, or lie within the normal background alterations which may be seen in untreated rats of this age and strain.

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

No statistically significant differences were noted in the total number of born pups (alive and dead) or in the number of pups born alive between the control group and the treatment groups of P0 dams.No statistically significant differences were noted in the number of implantation sites between the control and the treatment groups of P0 dams.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Parameters with statistically significant differences in comparison to the control group which were considered as to be not test item related:
-Increased Pre-implantation loss in low dose females (p≤0.05)
-Decreased Post-implantation loss in low and intermediate dose females (p≤0.05)

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No test item-related influence was noted on the gestation length of the females in any of the treatment groups compared to the control group.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): No test item-related influence was noted on the gestation length of the females in any of the treatment groups (100, 300 and 1000 mg/kg bw/day) compared to the control group.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

All animals were successfully mated as determinated by positive sperm detection. No statistically significant differences were noted in the length of the pre-coital time between the control group and the treatment groups.
No test item-related differences were noted between the control group and the treatment groups (100, 300 and 1000 mg act. ingr./kg bw/day) with respect to the fertility index.

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: Slight reduction in body weight in male and female rats dosed at 1000 mg/kg bw/day; other changes were noted as well for clinical biochemistry, macroscopic and microscopic examination

Details on maternal toxic effects:
A slight reduction in body weight was noted for female rats of the high dose group (1000 mg test item/kg bw/day). For the female rats the reduction in body weight was noted from gestation day 0 (7.6%) until lactation day 4 (9.5%). The body weight at autopsy was reduced accordingly.

The laboratory examinations revealed an increased ALAT activity for female rats of the high dose group (1000 mg test item/kg bw/day).

Microscopic examination revealed the occurrence of squamous cell hyperplasia in the non-glandular mucosa of the forestomach from female rats of the high dose group (1000 mg test item/kg bw/day).

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Basis for effect level:

changes in number of pregnant

changes in pregnancy duration

early or late resorptions

effects on pregnancy duration

number of abortions

pre and post implantation loss

total litter losses by resorption

Remarks on result:

other: developmental toxicity

Remarks:

highest dose tested

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Description (incidence and severity):

No test item related influence was noted on the mean and the total litter weight of the pups on lactation day 1 and 4.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Pups were sacrificed on day 4 of lactation.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No statistically significant differences were noted in the total number of born pups (alive and dead) or in the number of pups born alive between the control group and the treatment groups of P0 dams.

Changes in sex ratio:

not examined

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No test item related influence was noted on the mean and the total litter weight of the pups on lactation day 1 and 4.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

No test item-related differences were noted between the survival index of the control group and the treatment groups (100, 300 and 1000 mg act. ingr./kg bw/day).

External malformations:

no effects observed

Description (incidence and severity):

No visible gross abnormalities were noted between the control and the treatment groups.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects. Remark: No test item related influence was noted on the survival rate and the mean and total body weights of the pups.

Details on embryotoxic / teratogenic effects:
No test item related influence was noted on the survival rate and the mean and total body weights of the pups.
Effects on the development of the F1 offsprings (pups) NOAEL (no-observed-adverse-effect level): above 1000 mg/kg bw/day, p.o.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

other: highest dose tested

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Table 1.Developmental parameters

Parameter	Group 1 Control	Group 2 100 mg/kg	Group 3 300 mg/kg	Group 4 1000 mg/kg
Number of pregnant dams	8/10	9/10	9/10	10/10
Number of pups at birth (total)	96	125	123	127
Number of pups at birth (mean)	12.0	13.9	13.7	12.7
No. of live pups at birth (total)	96	125	123	126
No. of live pups at birth (mean)	12.0	13.9	13.7	12.6
No. of live pups on day 4 (total)	96	125	123	123
No. of live pups on day 4 (mean)	12.0	13.9	13.7	13.7
Viability index day 1-4 (mean %)	100.0	100.0	100.0	90.0
Viability index day 1-4 (total^# %)	100.0	100.0	100.0	97.6
Mean body weight day 0/1 male and female pups (g)	6.23	6.34	6.29	6.11
Mean body weight day 4 male and female pups (g)	8.51	9.07	9.11	8.73
Mean total litter weight day 0/1, male and female pups (g)	75.00	87.38	85.91	77.09
Mean total litter weight day 4, male and female pups (g)	102.54	124.08	123.28	118.94
External examinations of pups (No. of litters/pregnant dams) - no externally visible  abnormalities      - stomach without milk	8/8 0/8	9/9 0/9	9/9 0/9	10/10 1/10 (3/3)*

#: total number of live pups on the respective lactation day

* 3 live born pups from dam no. 77 were found dead without milk on lactation day 2.

Applicant's summary and conclusion

Conclusions:

NOAEL (no-observed-adverse-effect level) of the F1 offspring: above 1000 mg/kg bw/day, p.o.

Executive summary:

The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development according to OECD guideline 422. The test item was administered orally by gavage to rats at dose levels of 100, 300 and 1000 mg active ingredient/kg bw/day. The application started two weeks before mating on test day one and ended on the day or one day before sacrifice. Day of sacrifice was on test day 37 for the male rats and between lactation day 4 and 7 for the female rats.

Effects on the parental generation (general toxicity)
No test item-related premature death was noted in any treatment group (100, 300 and 1000 mg/kg bw/day).

No signs of clinical toxicity were noted for the male and female rats of the low and intermediate dose groups (100 and 300 mg/kg bw/day).

A slightly increased salivation was noted in one male rat, no further signs of clinical toxicity were noted for the male and female rats of the high dose group (1000 mg/kg bw/day).

No test item related influence was noted for the male and female rats of all treatment groups (100, 300 and 1000 mg/kg bw/day) during the observational and functional (grip strength and spontaneous motility) neurological screenings.

A slight reduction in body weight was noted for the male and female rats of the high dose group (1000 mg/kg bw/day). For the male rats the reduction in body weight was noted from test day 8 (4.4%) until test day 36 (5.5%) and for the female rats from gestation day 0 (7.6%) until lactation day 4 (9.5%). The body weight at autopsy was reduced accordingly.

The laboratory examinations revealed an increased ALAT activity for the male and female rats of the high dose group (1000 mg/kg bw/day) and a decrease in the albumin concentration for the male rats of the high dose group.

No test item-related changes were noted during the macroscopic inspection at autopsy with the exception of changes in the stomach from 2 male animals from the high dose group (1000 mg/kg bw/day).

Microscopic examination revealed the occurrence of squamous cell hyperplasia in the non-glandular mucosa of the forestomach from the male and female rats of the high dose group (1000 mg/kg bw/day). Further microscopic findings occurred in form of pulmonary congestion in the male rats from the high dose group.

Effects on reproduction parameters and organs

No test item-related influence was noted on the reproduction parameters in any treatment group (100, 300 and 1000 mg/kg bw/day).

Microscopic examination revealed no changes in the reproductive organs from the male and female rats of the high dose group (1000 mg/kg bw/day).

Effects on the development of the F₁offsprings (pups)

No test item related influence was noted on the survival rate and the mean and total body weights of the pups.

External examination of the pups revealed no visible changes related to the test item.

The following no-observed-effectlevelswere established:

Effects on the F₀-generation

NOAEL (no-observed-adverse-effect level): 300 mg/kg bw/day, p.o.

Effects on reproductive toxicity

NOAEL (no-observed-adverse-effect level): above 1000 mg/kg bw/day, p.o.

Reproductive indices:

Effects on the development of the F₁offsprings (pups)

NOAEL (no-observed-adverse-effect level): above 1000 mg/kg bw/day, p.o.