Disodium (Z)-4-(9-octadecenylamino)-4-oxo-2(or 3)-sulphonatobutyrate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to GLP and valid testing guidelines, therefore it is considered relevant, adequate and reliable for classification.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Butanoic acid, 4-amino-4-oxo-2(or 3)-sulfo-, N-(C16-C18 (even numbered), C18 unsaturated alkyl), disodium salts

IUPAC Name:

Butanoic acid, 4-amino-4-oxo-2(or 3)-sulfo-, N-(C16-C18 (even numbered), C18 unsaturated alkyl), disodium salts

Test material form:

other: liquid to pasty

Details on test material:

- Name of test material (as cited in study report): Butanoic acid, 4-amino-4-oxo-2(or3)-sulfo-, N-(C16-C18 (even numbered), C18unsaturated alkyl)), disodium salts; Sodium tallow sulphosuccinamates
- Physical state: Liquid to pasty; opaque
- Analytical purity: 25.5% active matter (correction factor: 3.92)
- Impurities (identity and concentrations): See confidential details
- Composition of test material, percentage of components: See confidential details
- Purity test date: February 14, 2012
- Lot/batch No.: MIBX042332
- Expiration date of the lot/batch: December 31, 2013
- Stability under test conditions: Stable
- Storage condition of test material: At +10° to +25 °C
- Other: Manufacturer/supplier: Huntsman Holland BV, Merseyweg 10, 3197 KG Botlek-Rotterdam, The Netherlands

Test animals

Species:

rat

Strain:

other: CD® / Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Research,Models and Services Germany GmbH,Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at start of dosing: Males: 59 days; Females: 69 days
- Weight at start of dosing: Males: 212.4 g to 239.5 g; Females: 191.8 g to 226.6 g
- 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 are cleaned and changed once a week.
- Diet (e.g. ad libitum): Commercial ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany), ad libitum with the exception of the night before the day of blood withdrawal for Iaboratory examination. Food residue was removed and weighed.
- Water (e.g. ad libitum): Tap water was offered daily ad libitum
- Acclimation period: 7 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: From: October 17, 2012 To: Males: November 29, 2012; Females: December 17, 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: tap water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: application volume was 5 mL/kg bw/day.
The test item was dissolved in the vehicle tap water to concentrations of 12, 24 and 60 mg test item/ml tap water. The test item formulations were freshly prepared and adjusted to the animal's current body weight on each administration day.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Tap water

Details on mating procedure:

- 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
- After approx. 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: yes. This mating-procedure was repeated until at least 8 pregnant dams are available for each group.
- After successful mating each pregnant female was caged (how): 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; on the other side of the animal room than the males with each dose group separated by an empty row.

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. 28342 (14-day dose-range-finding).

Duration of treatment / exposure:

Males: Beginning 2 weeks prior to mating lasting up to the day before sacrifice until a minimum dosing period of 28 days was completed.
Females: Beginning 2 weeks prior to mating continuing up to, and including, day 3 post partum or the day before sacrifice.

Frequency of treatment:

Once daily

Details on study schedule:

screening study

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 60, 120 and 300 mg/kg bw/day
Basis:
actual ingested

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 active ingredient/kg bw by oral gavage (LPT Study No. 28342). One of 5 male animals dosed at 300 mg/kg bw and 3 of 5 males and 1 of 5 females dosed at 1000 mg/kg bw died prematurely. Premortal symptoms in form of pilo-erection, reduced motility, increased respiratory rate, cold to touch, decreased respiratory rate and a reduced drinking water consumption. Pilo-erection, a thickened abdomen, pultaceous faeces, an anus soiled with faeces and breathing sounds were noted for the male and female animals treated with 1000 mg /kg bw.
The body weight of the male animals treated with 300 or 1000 mg/kg bw and of the female animals treated with 1000 mg Empimin MK B/kg bw/day was reduced. Body weight gain and body weight at autopsy were reduced accordingly. A reduced food consumption was noted for male and female animals treated at 1000 mg /kg bw.
At macroscopic inspection at necropsy, one of 5 male animals treated with 300 mg/kg bw showed reddened lungs. Furthermore, changes in the gastrointestinal tract (filled with gas, distended, with liquid content, nearly empty, (mucosa) reddened, soft content, haemorrhagic foci, detachment of mucosa), lungs (reddened), spleen (reduced in size) and external observation (thickened/soft abdomen, anus smeared with faeces, pilo-erection) were noted for the male and female animals (including deceased animals) treated with 1000 mg/kg bw.
The relative and absolute kidney weights of the animals were decreased starting at 100 mg/kg bw for the male animals and at 1000 mg/kg bw for the female animals.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Cage side 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.
- Time schedule: Throughout the test period, each animal was observed for clinical signs at least once daily. Individual animals were observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness. 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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure (to allow for within-subject comparisons) and once a week thereafter, detailed clinical observations were made in all animals; in test week 4 these observations were performed prior to any laboratory investigations.
-These observations were made outside the home cage in a standard arena and at the same time, each time. Signs observed included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereo-typies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes.
- 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 1 post-partum) and day 4 post-partum. Body weights were recorded individually for each adult animal.
- The pups were weighed within 24 hours of parturition (day 1 post-partum) and on day 4 post-partum.

FOOD CONSUMPTION: Yes
- The quantity of food left by individual animals was recorded on a weekly or daily basis throughout the experimental period with the exception 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 upon completion of a treatment week. From these data the food consumption (in g/kg bw/day) was determined using the following formula:
Relative food consumption[g/kg b.w./day] = (Total food given [g] - Total food left [g])/ (Number of animal days# x Body weight [kg])
# The term 'animal days' counts one animal day for each animal alive for a whole day; it is assumed that on the day of death an animal does not eat.

FOOD EFFICIENCY: No

WATER CONSUMPTION : Yes
- Water consumption was monitored daily by visual appraisal throughout the study.

HAEMATOLOGY: see Section 7.5.1

CLINICAL CHEMISTRY: See Section 7.5.1

NEUROLOGICAL OBSERVATIONS: see Section 7.5.1

REPRODUCTIVE PARAMETERS:
Number of pregnant females
Pre-coital time
Gestation length calculated from day 0 of pregnancy
Corpora lutea
lmplantation sites
Number of (viable) pups day0/4-

REPRODUCTIVE INDICES:
Gestation Index
Fertility Index
Birth Index
Live Birth Index
Viability Index
Pre-implantation loss [%]
Post-implantation loss [%]

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
testis weight, epididymis weight
At the time of sacrifice or 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.
The following organs or parts of organs of all male adult animals were fixed in 7% formalin; testes and epididymides were fixed in Bouin' s fixative:
Epididymis (2), Gross lesions, Prostate, Seminal vesicle, Testicle (2).
Detailed histopathologic examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure) of all adult males of groups 1 to 4 following H-E and PAS staining.

Litter observations:

STANDARDISATION OF LITTERS
- screening study: Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter, were carefully examined externally for gross abnormalities.

PARAMETERS EXAMINED
Number of pups absolute (total/live)
Number of pups per dam (total/live)
Number of male and female pups (total/live)
Number of stillbirths
Mean pup weight

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Paternal animals animals: All surviving animals: The male animals were sacrificed on test day 37.
- Maternal animals: All surviving animals: Dams with offspring were sacrificed on day 4 postpartum, or shortly thereafter. Females showing no evidence of copulation were sacrificed 24 days after the last day of the mating period.

GROSS NECROPSY
-At the time of sacrifice, or premature death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Special attention was paid to the organs of the reproductive system.
-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.
-The numbers of corpora lutea and implantation sites were recorded in the female adult animals and reported.

ORGAN WEIGHTS: Yes
-The following organs of all adult animals were weighed individually before fixation and identified as left or right:
Epididymis (2), Testicle (2)
- Determination of the organ weights of the following organs was only performed from 20 adult males and 20 adult females, which were randomly selected: Adrenal gland (2), Heart, Liver, Thymus, Brain, Kidney (2), Spleen. Adrenal glands and kidneys were weighed individually and identified as left or right.
- Animals Nos.:
Group 1: 2, 3, 4, 6, 7 12, 14, 15, 16, 18
Group 2: 22, 23, 25, 26, 27 31, 32, 34, 37, 40
Group 3: 42, 44, 45, 47, 49 51, 52, 53, 58, 59
Group 4: 61, 64, 66, 67, 68 72, 73, 75, 79, 80

HISTOPATHOLOGY: Yes
- The following organs or parts of organs of all adult animals were fixed in 7% formalin; testes and epididymides were fixed in Bouin's fixative:
Epididymis (2), Gross lesions, Mammary gland, Ovary (2), Prostate, Seminal vesicle, Testicle (2), Uterus (incl. cervix and oviducts), Vagina.
Detailed histopathological examination was performed on one testicle and one epididymis with special emphasis of the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure of the selected animals of group 1 and 4 following haematoxylin-eosin and PAS staining.
-In addition, the following organs or parts of organs of the selected 20 adult males and 20 adult females (see section above) were fixed in 7% formalin:
Adrenal gland (2)
Bone marrow (os femoris)
Brain (cerebrum, cerebellum, brain stem)
Heart (left and right ventricle, septum)
Intestine, small (duodenum, jejunum, ileum, incl. Peyer's patches, Swiss roll method)
Intestine, large (colon, rectum)
Kidney and ureter (2)
Liver
Lungs (with mainstem bronchi and
bronchioles), preserved by inflation with
fixative and then immersion
Lymph node (1, cervical), Lymph node (1, mesenteric)
Nerve (sciatic)
Oesophagus
Spinal cord (3 sections)
Spleen
Stomach
Thyroid (incl. parathyroids)
Thymus
Tissue masses or tumours (incl. regional lymph nodes)
Tongue (incl. base)
Trachea (incl. larynx)
Urinary bladder
-Only the 10 selected animals from the control group and the high dose group (20 animals in total) were considered for histopathological evaluation.
Group 1: 2, 3, 4, 6, 7 12, 14, 15, 16, 18
Group 4: 61, 64, 66, 67, 68 72, 73, 75, 79, 80

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:
Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter, were carefully examined externally for gross abnormalities.
The animals were sacrificed under ether anaesthesia by cutting the aorta abdominalis, exsanguinated, weighed, dissected and inspected macroscopically. All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition of the thoracic viscera were noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined.
The lungs were removed and all pleural surfaces examined under suitable illumination.
The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

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

Statistics:

Toxicology and Pathology data were captured, whenever possible, using the departmental computerized systems (Provantis® 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 are used:

STUDENT's t-test: All numerical functional tests (≤ 0.05 and p ≤0.01)

Multiple t-test based on DUNNETT, C. W .; New tables for multiple Comparisons with a control; Biometrics, 482-491 (Sept 1964): Body weight I Food consumption IHaematology I Clinical chemistry I Absolute and relative organ weights (≤0.05 and p ≤ 0.01)

For all numerical values (e.g. body weight, food consumption and organ weight data) homogeneity of variances was tested by using the BARTLETT chi-square test. lf the variances were 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 was p≤0.01.

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.

Reproductive indices:

Gestation Index
Fertility Index
Birth Index
Live Birth Index
Viability Index
Pre-implantation loss [%]
Post-implantation loss [%]

Offspring viability indices:

Gestation length calculated from day 0 of pregnancy
Corpora lutea
lmplantation sites
Number of pups absolute
Number of pups per dam
Number of male and female pups
Number of stillbirths
Number of pups with malformations

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

1 male and 1 female died prematurely in the intermediate dose group; 1 male and 1 female died prematurely in the high dose group; slight signs of toxicity in a few animals of the high dose group in form of pilo-erection and increased salivation

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

statistically significant reduction in body weight in male animals of the 120 mg test item/kg bw dose group and in both sexes at 300 mg test item/kg bw/day

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

statistically significant reduction in body weight in male animals of the 120 mg test item/kg bw dose group and in both sexes at 300 mg test item/kg bw/day

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Description (incidence and severity):

Test substance intake: gavage

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

increased pre-implantation and post-Implantation loss at 300 mg test item/kg bw/day, and decreased gestation index, birth index and No. life pups

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Mortality:
Male animals:
One of 10 animals (no. 42) of the intermediate dose group (120 mg test item/kg bw/day) died prematurely on test day 27 and one of 10 animals (no. 62) of the high dose group (300 mg test item/kg bw/day) died prematurely on test day 17. Both animals showed no premortal signs of clinical toxicity.
Female animals:
One of 10 animals (no. 52) of the intermediate dose group (120 mg test item/kg bw/day) died prematurely on gestation day 12 (TD 29), showing piloerection and an anus soiled with faeces one or 2 days before death.
One of 10 animals (no. 79) of the high dose group (300 mg test item/kg bw/day) died prematurely on test day 20 during its mating period without premortal signs of clinical toxicity.

Clinical signs:
Male animals:
Four animals with signs of clinical toxicity were noted in the high dose group (300 mg test item/kg bw/day). Three of them showed slight salivation on one or 2 days, another one breathing sounds on 2 test days.
Female animals:
In the high dose group (300 mg test item/kg bw/day) slight to moderate salivation was noted in 5 animals for 1 day during the lactation period. Piloerection was noted for one animal during the pre-mating period and for another animal during the gestation period on one or 2 test days. Animal no. 77 showed breathing sounds on test days 6 and 7.

BODY WEIGHT (PARENTAL ANIMALS)
BODY WEIGHT
Male animals:
Statistically significant (p≤0.05 or p≤0.01) reductions in body weight between 7.8% and 6.7% compared to the control were noted in the intermediate dose group (120 mg test item/kg bw/day) during the mating and post-mating period.
At the high dose group (300 mg test item/kg bw/day) the body weight of the animals was statistically significantly reduced from test day 8 (p≤0.05, by 9.0%) to test day 36 (p≤0.01; by 13.0%).
Accordingly, statistically significant (p≤0.05 or p≤0.01) reductions in body weight gain were noted in the intermediate and the high dose group (120 and 300 mg test item/kg bw/day).
Female animals:
At the intermediate dose group (120 mg test item/kg bw/day) a slight statistically not significant reduction in body weight was noted by 6.3% compared to the control.
Statistically significant reductions were noted in the high dose group (300 mg test item/kg bw/day), starting at the end of the pre-mating period (by 5.5%; not significant) and more pronounced during the gestation period by 9.2% (p≤0.01) on gestation day 20. During the lactation period body weight was statistically not significant reduced by nearly 11% on lactation day 1 and 4.
Accordingly, a statistically not significant reduction in body weight gain was noted at the end of the pre-mating period and a statistically significant reduction (p≤0.01) at the end of the gestation period in the high dose group (300 mg test item/kg bw/day).

FOOD CONSUMPTION
Male animals:
A statistically significant (p≤0.01) reduction in food consumption by 11.0% was noted in the high dose group (300 mg test item/kg bw/day) compared to the control during the first test week.
Female animals:
At the high dose group (300 mg test item/kg bw/day) reductions in food consumption were noted during the first test week by 11% (p≤0.01) and on gestation day 7 (by 9%, non-significant) and 14 (by 10%; p≤0.05) compared to the control.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): oral gavage

REPRODUCTIVE FUNCTION:
-Pre-coital time:
No test item-related influence was noted.
-Gestation length:
No test item-related influence was noted on the gestation length of the females in the low and intermediate dose group (60 and 120 mg test item/kg bw/day) compared to the control group.
In the high dose group (300 mg test item/kg bw/day), a prolonged gestation length of 23 days was noted in comparison to 22.3 day in the control group.
-Evaluation of reproduction parameters of the dams:
No test item-related differences were noted for the low and intermediate dose group (60 and 120 mg test item/kg bw/day).
The statistically significant decrease in the percentage of pre-implantation loss, noted in the intermediate dose group, was without relevance, as the decrease is not an adverse effect.
In the high dose group (300 mg test item/kg bw/day) a non-statistically significant decrease in the gestation index was noted due to 3 (nos. 71, 76, 77) of 8 pregnant dams without pups. In detail, the gestation index of the high dose group was reduced to 62.5% in comparison to 100% in the control and the low and intermediate dose groups.
The pre-implantation loss index was statistically significantly increased to 33.6% per dam, due to 3 dams without live pups, which showed pre-implantation losses between 53.6 and 94.4%.
The post-implantation loss index was statistically significantly (p≤0.01) increased to a mean value of 68.0% per dam. With the exception of the dams nos. 74 and 75, all the other 6 pregnant dams showed a high percentage of post-implantation loss between 58.8% and 100%. The highest post-implantation losses were noted for animal nos. 72, 77, 78 and 80 with a total loss of between 10 and 16 implantation sites per animal. Hence, the high loss of implantation sites was not restricted to the 3 dams without viable pups (nos. 71, 76, 77).
Correlating the birth index was also statistically significantly reduced.
No test item-related influence was noted on the live birth index, as no stillbirth was noted in the high dose group

ORGAN WEIGHTS (PARENTAL ANIMALS)
Male animals:
At the high dose group (300 mg test item/kg bw/day) an increase was noted in the relative organ weights of the left gonad (by 12.6%; non-significant), the right gonad (by 13.0%; p≤0.05) and the liver (by 19.8%; p≤0.05).
The absolute organ weights were statistically significantly decreased for the left adrenal gland (by 25%; p≤0.01), the right adrenal gland (by 22.9%: p≤0.01), the left epididymis (by 15.6%; p≤0.01), the right epididymis (by 11.7%; p≤0.01), the heart (by 17.0%; p≤0.05), the left kidney (by 14.1%; p≤0.05) and the right kidney (by 16.0%; p≤0.05).
Female animals:
No test item related findings were noted.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No test item related findings were noted.

HISTOPATHOLOGY: NON-NEOPLASTIC (restricted to the control and the high dose group)
No test item related findings were noted in male and female animals.
No microscopic changes were noted for the reproductive organs of the male and female animals.
No test item-related influence was noted on the stages of spermatogenesis.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: reproductive effects

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

In the high dose group (300 mg test item/kg bw/day) the total litter weight per dam of the 5 dams with live born pups was non-statistically significantly reduced on lactation day 1 (by 35.2%) and on lactation day 4 (by 28.2%).

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
No test item-related differences were noted between the survival index of the pups of the control group and the pups of the treatment groups (60, 120 and 300 mg test item/kg bw/day).

BODY WEIGHT (OFFSPRING)
No test item-related influence was noted on the mean litter weight of the pups in any of the treatment groups.
No test item-related influence was noted on the total litter weight per dam in the low and intermediate dose group (60 and 120 mg test item/kg bw/day). In the high dose group (300 mg test item/kg bw/day) the total litter weight per dam of the 5 dams with live born pups was non-statistically significantly reduced on lactation day 1 (by 35.2%) and on lactation day 4 (by 28.2%). This was due to the lower number of pups per dam, noted in the high dose group.

GROSS PATHOLOGY (OFFSPRING)
The external examinations of the pups at sacrifice on day 4 of lactation revealed no test item-related external visible changes in any of the treatment groups.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1. Fertility and Reproductive parameters Parental generation

Parameter	Group 1 Control	Group 2 60 mg/kg	Group 3 120 mg/kg	Group 4 300 mg/kg
No. females evaluated	10	10	10#1	9#2
Mean precoital time (days)	2.2	5.3	3.0	2.2
No.pregnant females	10	8	9#1	8#2
Fertility index (%)	100	80	90	89
No. dams with pups (life + dead)	10	8	8	5
Gestation length (days)	22.3	22.4	22.5	23.0^vv
No. dams with live pups	10	8	8	5
Gestation Index (%)	100	100	100	63
No.Corpora lutea(total)	165	131	119#1	142
No.Corpora lutea(mean)	16.5	16.4	14.9	17.8
No. Implantation sites (total)	139	116	112#1	94
No. Implantation sites (mean)	13.9	14.5	14.0	11.8
No. life pups at birth (total)	125	108	98	37^vv
No. life pups at birth (mean)	12.5	13.5	12.3	4.6^vv
Birth Index (mean %)	88.7	93.9	89.5	32.0^2
Birth Index (total %)	89.9	93.1	87.5	39.4^2
Number of stillbirths	1	5	0	0
No. dams with stillborn pups	1	1	0	0
No. live born pups (total)	124	103	98	37^vv
No. live born pups (mean)	12.4	12.9	12.3	4.6^vv
Live birth index (mean %)	99.3	96.3	100.0	100.0
Live birth index (total %)	99.2	95.4	100.0	100.0
Pre-implantation loss (mean %)	14.0	9.7	9.3	33.6
Pre-implantation loss (total %)	15.8	11.5	5.9^a	33.8^b
Post-implantation loss (mean %)	12.0	9.6	10.5	68.0
Post-implantation loss (total %)	10.8	11.2	12.5	60.6^b

¹ p≤0.05 Chi²-test

² p≤0.01 Chi²-test

*  p≤0.05 Fisher test

**p≤0.01 Fisher test

^v p≤0.05 Dunnett test or Student’s t-test

^vvp≤0.01 Dunnett test or Student’s t-test

^ap< 0.05 Chi²-test

^bp<0.01 Chi²-test

#1: Including female no. 52 which died on gestation day 12 (post-mating)

#2: Female no. 79 was excluded due to premature death on test day 20 (pre-mating)

Applicant's summary and conclusion

Conclusions:

The following no-observed adverse-effect (NOAEL) levels were established:
Paternal and maternal toxicity: NOAEL= 120 mg/kg bw/day, p.o.
Reproductive toxicity: NOAEL=120 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 containing 25.5% active ingredient was administered orally by gavage to rats at dose levels of 60, 120 or 300 mg act.ingr./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 on lactation day 4 or shortly thereafter for the female rats.

                                                                

Effects on the parental generation (general toxicity)

One of 10 male and one of 10 female animals of the intermediate and one of 10 male and one of 10 female animals of the high dose group (120 and 300 mg act. ingr./kg bw/day) died prematurely. In the high dose group (300 mg act. ingr./kg bw/day) clinical signs in form of salivation, piloerection and/or breathing sounds were noted in a few male and female animals for 1 or 2 test days. A statistically significant reduction in body weight was noted for both sexes in the high dose group (300 mg act. ingr./kg bw/day). Accordingly, body weight at autopsy was statistically significantly reduced in the high dose group (300 mg act. ingr./kg bw/day) for the male and female animals. Statistically significant reductions in food consumption were noted for the male and female animals of the high dose group (300 mg act. ingr./kg bw/day).

For the haematological parameters statistically significant changes were noted in the high dose group (300 mg act. ingr./kg bw/day) for the MCH value and the aPTT time (male animals), the number of white blood cells and lymphocytes (female animals).

Changes in the relative or absolute organ weights of several organs were noted for the male animals of the high dose group (300 mg test item/kg bw/day), most remarkably for the relative liver weight which increased for almost 20%. The macroscopic and microscopic examinations revealed no test item related changes.

 

Effects on reproduction

A statistically significant increase was noted for the pre-implantation loss and post-Implantation loss index in the high dose group (300 mg act. ingr./kg bw/day). Correlating, the gestation index, the birth index and the mean number of pups per dam were statistically significantly reduced in the high dose group (300 mg act. ingr./kg bw/day).

 

Effects on the development of the F1offsprings (pups)

No adverse effect was noted on the development of the pups from the dams of the high dose group (300 mg act. ingr./kg bw/day), as there was no reduction in the survival index or the litter weight of the pups.

 

The following no-observed adverse-effect levels were established:

Paternal and maternal toxicity: NOAEL= 120 mg act. ingr./kg bw/day, p.o.

Reproductive toxicity: NOAEL=120 mg act. ingr./kg bw/day, p.o.

Developmental toxicity: NOAEL= 300 mg act. ingr./kg bw/day, p.o.