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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2013
Reference Type:
other: Summary 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 8887
- Physical state: yellowish / clear liquid
- Analytical purity: 78.1g/100g (see BASF study 11L00371)
- Purity test date: 2011-11-03
- Lot/batch No.: 110009P040
- Expiration date of the lot/batch: 2012-09-30
- Stability under test conditions: confirmed for up to 7 days (see BASF study 11L00439)
- Storage condition of test material: room temperature under light exclusion

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: 294-338 g; Females: 201-234 g
- Fasting period before study: no
- Housing: individually (except during overnight mating and lactationin) 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
- Photoperiod (hrs dark / hrs light): 12h/12h

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 daily. 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.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks, only overnight from 3p.m. to 7-9 a.m.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- No replacement of first male after unsuccesluf mating.
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and concentration control analyses of the test-substance preparations were performed in all concentrations at the start of the administration period. Additionally, samples from all concentrations as reverse samples for concentration control analysis were taken at the end of the study. All concentrations were in the expected target range of 90-110% of the nominal concentration.
Duration of treatment / exposure:
31days (males), 52days (females)
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
40, 100, 250 mg/kg b.w./d
Basis:
actual ingested
doses were adjusted to purity of the substance
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Doses were selected based on a 14day test study in female Wistar rats. 4 rats per group were treated daily with 250, 500, and 1000 mg/kg b.w. via gavage. One animal in the high dose group was found dead on day 13, probably due to excessive irritation in the stomach and digestive system. Of the surviving three animals, one showed hyperemia in the cecum, ulceration/erosion in the glandular stomach and discoloration of the jejunum contents, two showed ulcerations / erosion in the forestomach. In the mid dose, one animal also showed erosion / ulceration in the forestomach, which were evaluated as severe enough to likely cause death throughout the much longer treatment time in the OECD422 main study. Thus the lowest dose was selected as the maximum dosage for the main study. Here, lesions in the forestomach were also observed in one animal, but severity was reduced.
Positive control:
none

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 32 (3 days after the maximum mating period)
- Maternal animals: All surviving animals on day 53

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, stomach (also all males from the low and mid dose groups), 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. Animals with notabel findings or abnormalitites were evaluated on a case-by-case basis.
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 animals)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No mortality was observed.
Almost all animals of the high dose group showed salivation within 2 hours after substance administration, which was most likely induced by local irritation of the digestive tract of bad taste of the substance.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
unaffected by the test substance

FUNCTIONAL OBSERVATION BATTERY
All findings were equally distributed between the test and control groups and thus not considered to be treatment related.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Male/female mating index = 100% for all groups
Male/female fertility index = 90% (control and low dose), 100% (mid dose), 70% (high dose), which is in the range of historical control data
Duration of gestation varied between 22.1 and 22.5 days without any significant differences. The gestation index and live birth index were 100% in all groups. Only one female in the low dose group had 3 stillborn pubs.

ORGAN WEIGHTS (PARENTAL ANIMALS)
no test substance related effects.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No test substance related effects were observed. Especially, no gross lesions of reproductive organs were detected in the males and females that did not produce offspring.

HISTOPATHOLOGY (PARENTAL ANIMALS)
In 7 out of 10 males of the high dose group the forestomach showed minimal hyperkeratosis, which is probably due to local irritant properties of the test substance, that were already observed in the pre-study for dose selection. No further differences were observed.
No histopathological correlate was obeserved in females and males which failed to produce offspring. Only one of these males showed minimal testicular degeneration. This is frequently observed in these rats and usually compatible with fertility, but might have been the cause for infertility in this study.

OTHER FINDINGS (PARENTAL ANIMALS)
No test substance related effects on clinical chemistry and urinalysis parameters were observed.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
>= 250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOAEL
Remarks:
reproductive performance
Effect level:
>= 250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
The viability index was 100% (mid and high dose), 99.1% (low dose) and 97.8% (control)

SEX DISTRIBUTION
not affected by the test substance

CLINICAL SIGNS (OFFSPRING)
none observed

BODY WEIGHT (OFFSPRING)
Not affected by the test substance. One female runt was found in a rather large litter (16pubs) of the high dose group. Also 5 male and 9 female runts were found in two litters of the mid dose females. All except one male whose weight was within the historical control data, also belonged to a large litter of 17 pubs. These differences were no longer observed on PND 4.

GROSS PATHOLOGY (OFFSPRING)
No test substance related effects observed.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
>= 250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test the oral administration by gavage to Wistar rats revealed no signs of systemic toxicity at a dose level of 250mg/kg bw/d in animals of both sexes. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 250 mg/kg bw/d in males and in females. The NOAEL for reproductive performance and fertility was set to 250 mg/kg bw/d in male and female Wistar rats. The NOAEL for developmental toxicity was effective 250 mg/kg bw/d.