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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:
2012/2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline 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 1996
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650, adopted July 2000
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Laromer LR 8869
- Physical state: colorless, clear liquid
- Analytical purity: for details see analytical report no. 11L00263
- Purity test date: 2012-01-11
- Lot/batch No.: 110009P040
- Expiration date of the lot/batch: 2012-07-4
- Stability under test conditions: verified for at least 7 days
- Storage condition of test material: at room temperature, light protected, avoid temperatures above 25°C

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany, male and female rats were derived from different litters to rule out sibling mating
- Age at study initiation: (P) 10-11 wks;
- Weight at study initiation: (P) Males: 294 g; Females: 197 g, on average
- Fasting period before study: no
- Housing: individually (except during overnight mating and lactation) in Makrolon type M III cages
- Diet (e.g. ad libitum): Kliba maintenance diet mouse-rat “GLP” meal, ad lib.
- Water (e.g. ad libitum): tap water ad lib.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr):15

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:The test substance was applied as suspension, which was prepared at least once a week. An appropriate amount of the test substance was weighed, and corn oil was added up to the desired volume. The suspension was kept homogenous during administration by stirring with a magnetic stirrer.

The administration volume was 4ml/kg b.w.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test substance is poorly soluble in water.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight for a maximum of 14 days, or until pregnancy was proven
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): single
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in corn oil for a period of 7 days at room temperature was proven before the start of the study (BASF Project No. 11L00386).
For homogeneity and concentration control analyses, each 6 samples of all concentrations was drawn at the start of the study and towards the end of the administration period. Homogeneity was confirmed, all concentrations measured were within 90-110% of the target concentration.
Duration of treatment / exposure:
males: 36 days
females: 51 days
Frequency of treatment:
daily, except to animals being in labor
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000mg/kg (high dose was reduced to 600mg/kg on day 19)
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on results of a range-finding study - no severe signs of toxicity were observed in animals treated with 1000mg/kg of the test substance for 14 days.
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on workdays, daily on weekends and public holidays
- Cage side observations: check for moribund animals, pertinent behavioral changes, signs of overt toxicity, parturation, littering and lactation behavior of the dams

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the first administration, weekly thereafter
- The following parameters were examined: abnormal behavior during “handling”, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), urine, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: day 0, weekly thereafter with the following exceptions for females: During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. Females with litter were weighed on the day of parturition (PND 0) and on PND 4.

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes, except during mating

WATER CONSUMPTION: Monitored by daily visual inspection

OTHER (for details see entry for this study in the repeated dose section):
- Functional observation battery and motor activity measurement of 5 males and females per group 2 days prior to sacrifice
- Urinanalysis in 5 males and females per group one day prior to necropsy
- Clinicochemical and hematological examinations 5 males and females on the day of necropsy after fasting for 16h
Sperm parameters (parental animals):
Parameters examined in male parental animals:
testis weight, epididymis weight, stages of spermatogenesis in the male gonads
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, clinical symptoms, weight gain

GROSS EXAMINATION OF DEAD PUPS:
yes, externally and organs were assessed macroscopically
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals on day 37
- Maternal animals: All surviving animals on day 52

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were weighed
- in all animals: epididymides, testes
- in 5 males and females of each group: adrenal glands, brain, heart, kidneys, liver, spleen, thymus
The uteri of all cohabited female parental animals were examined for the presence and number of implantation sites. The uteri of apparently non-pregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations.
The following tissues were examined histotechnically in at least 5 animals per sex of the control and high dose group unless noted otherwise
adrenal glands, gross lesions (all affected animals in all dosage groups), bone marrow (femur), brain, cecum, cervix, coagulation glands, colon, duodenum, epididymides, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (auxillary and mesenteric), ovaries, oviducts, prostate gland, peyer's patches, rectum, sciatic nerve, seminal vesicles, spinal cord, spleen, forestomach (all animals from all dose groups), glandular stomach, testes, thymus, thyroid glands, trachea, urinary bladder, uterus, vagina
Postmortem examinations (offspring):
NECROPSY
- All surviving pubs were subjected to postmortem examinations: external examination and macroscopic examination of organs.
Reproductive indices:
For the males, mating and fertility indices were calculated for F1 litters according to the following formulas:
Male mating index (%) = number of males with confirmed mating (vaginal sperm detected in females) / number of males placed with females x100
Male fertility index (%) = number of males proving their fertility (implants in utero) / number of males placed with females x100

For the females, mating, fertility and gestation indices were calculated for F1 litters according to the following formulas:
Female mating index (%) = number of females mated (vaginal sperm detected) / number of females placed with males x100
Female fertility index (%) = number of females pregnant (implants in utero) / number of females mated (vaginal sperm or implants in utero) x100
Gestation index (%) = number of females with live pups on the day of birth / number of females pregnant x100
Offspring viability indices:
Live birth index (%) = number of liveborn pups at birth / total number of pups born x100
Viability index (%) = number of live pups on day 4 after birth / number of live pups on the day of birth x100

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
In test group 3 (1000 and 600 mg/kg bw/d) female animal No. 80 was found dead on study day 17 (Mating Day 3), female animal No. 74 was sacrificed moribund on study day 19 (GD 2), and male animal No. 32 was found dead on study day 35. These premature deaths were assessed as being related to the test substance. One female animal of test group 2 (300 mg/kg bw/d) was also found dead on study day 44. The animal died because of a gavage error. Therefore, a relation to treatment was excluded.

Almost all animals of both sexes of the mid and high dose group showed mostly slight, sometimes moderate salivation within 2 hours after the test substance administration on several days of the study. The same was true for individual low dose animals. From the temporary, short appearance immediately after dosing it could be assumed that salivation was induced by a bad taste of the test substance or local affection of the upper digestive tract. The effect was related to the test substance but assessed as being non-adverse.

Animal No. 74 of test group 3 (1000 and 600 mg/kg bw/d) showed poor general condition, piloerection and respiration sounds on GD 1 and GD 2. Therefore, the animal was sacrificed in a moribund condition on GD 19. Animal No. 75 of the same group showed semiclosed eyelids, poor general condition, hunched posture, smeared fur, piloerection (up to GD 7) and gasping on GD 2 and respiration sounds on GD 3. The same animal showed respiration gasping and sounds on LD 1. These findings were assessed as being related to treatment.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No treatment-related changes in body weight data were observed in animals of all test groups.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
One low dose female showed bloody red vaginal discharge and was unable to deliver on GD 22 and 23 as well as LD 0. The finding was assessed as being incidental as no dose-response relationship occurred.
For all except one high dose male (and corresponding female) mating was confirmed. Of these, 2 control males, one low dose did not generate pubs. 2 high dose males also failed to produce offspring due to premature death of the corresponding female. The gestation index varied between 100% (control), 88.9% (low dose), 100% (mid dose), 87.5% (high dose). The rate of liveborn pubs was also unaffected by the test substance as stillborn pubs only occurred in the control and high dose group.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Absolute thymus weights were significantly decreased in high dose male animals when compared to control group animals. This finding was not considered to be treatment related, since no relative organ weights were significantly altered.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Erosions, ulcers, and in one case a thickened forstomach were observed in all high dose male and females (with the exception of one female) and 5 mid dose females and 4 mid dose males. In the animals that did not become pregnant, no gross lesions of reproductive organs were observed.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Five male and 6 female animals of test group 3 (1000 and 600 mg/kg bw/d) as well as each 2 males and females of test group 2 (300 mg/kg bw/d) revealed multifocal erosions/ulcers in the forestomach. Accompanying these erosions/ulcers a mild to extreme diffuse or focal squamous hyperplasia with hyperkeratosis and a mild to severe submucosal edema was present. When the hyperplasia of the squamous epithelium was focal, it was located at the cardia region (stomach entrance). Within the edema a higher number of inflammatory cells were observed. Not all animals with a macroscopically diagnosed erosion/ulcer showed this finding. But the consistency of the accompanying findings (hyperplasia with hyperkeratosis and edema) which were observed in animals with and without erosion/ulcer led to the conclusion that this all belongs to the same complex. As the erosions/ulcer that could be seen microscopically were very small it is possible that they were not on the level of section. But taken all these findings together they point towards an irritating effect of the substance on the squamous epithelium of the forestomach. Especially the cardia region (entrance of the stomach) was affected.
The three animals of test group 3 (1000 and 600 mg/kg bw/d) which were found dead or were sacrificed in a moribund (animal Nos. 32, 74 and 80) did not show any other findings that could explain the death beside the ones in the stomach. Two of them revealed a moderate to severe thymus atrophy which was regarded to be a consequence to the stress caused by the findings in the forestomach. But it is not clear whether these observed findings were the cause of the death although death was regarded to be a consequence to the treatment with the test substance.
All investigated nonpregnant females as well as their male mating partners did not show relevant histopathological findings that could explain infertility.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
>= 300 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: death and poor general condition in high dose animals
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
>= 100 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: irritation in the stomach
Dose descriptor:
NOAEL
Remarks:
reproductive performance
Effect level:
>= 600 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: high dose

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
Stillborn pubs were only observed in the control (1 pub) and low dose group (2 pubs), which were assessed as incidental. One low dose pub was cannibalized, all other pubs from all dose groups survived until scheduled necropsy on PND4.

No difference in sex ratio was observed.

CLINICAL SIGNS (OFFSPRING)
No adverse clinical signs were noted.

BODY WEIGHT (OFFSPRING)
Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the concurrent control values.

GROSS PATHOLOGY (OFFSPRING)
A small liver with a discolored liver lobe (pale) was observed in 1 male pup of test group 1 (100 mg/kg bw/d). These findings were assessed as being spontaneous in nature and without biological relevance.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
>= 600 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: high dose

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion