Registration Dossier

Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 February 2009 to 19 March 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study data over 12 years old provided by ECHA, on a previously notified substance considered comparable and suitable for read-across use for the substance being registered (see attachments for justification of read-across). Study was performed according to OECD and EU guidelines and according to GLP principles.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Principles of method if other than guideline:
On Day 11 post-coitum animal 71 (Group 3) delivered. Evaluation: This animal’s data were excluded from calculation, because her first mating date was missed and thus was not dosed according to the appropriate schedule. Sufficient data were available from other animals.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): AMIDE #71
- Substance type: White powder
- Physical state: solid
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 10-13 weeks
- Weight at study initiation: 187-250 grams (day 0 post-coitum)
- Fasting period before study: no
- Housing:
Pre-mating During acclimatization, females were housed in groups of 5 animals/cage in Macrolon cages (MIV type, height 18 cm).
Mating Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm). During the weekend, mating procedures were suspended and the animals were housed in groups of maximum 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
Post-coitum Females were individually housed in Macrolon cages (MIII type, height 18 cm).
General Sterilized sawdust as bedding material and paper as cage-enrichment was supplied.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: At least 5 days prior to pairing under laboratory conditions.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 - 21.8
- Humidity (%): 29 - 79
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle (1.036) and not for the test substance.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX
- Concentration in vehicle: 10, 40 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: not specified in terms of days, but until successful pairing
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: not applicable.
- Further matings after two unsuccessful attempts: not applicable
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smear, referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
No analytical method based on gas chromatography (GC), liquid chromatography (HPLC), UV spectrophotometry or total organic carbon analysis (TOC) could be developed for the determination of the test substance in formulations with propylene glycol (determined during NOTOX Project 482838 - 90 day study).
Duration of treatment / exposure:
From Day 6 to Day 19 post-coitum, inclusive
Frequency of treatment:
daily.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 200, 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
24 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on results of a dose range finding study

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes (mortality/viability check)
- Time schedule: twice daily (early morning/late afternoon)


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from Day 0 post-coitum onwards


BODY WEIGHT: Yes
- Time schedule for examinations:Days 0, 3 and 6-20 (daily) post-coitum


FOOD CONSUMPTION: Yes
- Food consumption for each animal determined: Days 0-3, 3-6, 6-9, 9-12, 12-15, 15-17 and 17-20 post-coitum


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: Day 20 post-coitum
- Organs examined: External, thoracic and abdominal organs.

OTHER:
Organ weights: Spleen weights (and terminal body weight) were recorded from 10 pregnant animals of all dose groups on the scheduled day of necropsy. Previous results from a 28 day study indicated spleen parameters should be examined due to macroscopic and histological findings.

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes





Litter observations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [aproximately half per litter]

Other:
- The number and distribution of live and dead fetuses
- The number and distribution of embryo-fetal deaths
- The weight of each fetus
- The sex of each fetus from the ano-genital distance (during necropsy)
and also from gonadal inspections (during further fetal examination)
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher 1950) was applied to frequency data.

Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral, skeletal and combined), and each particular external, visceral and skeletal malformation or variation was subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis 1952) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test (Dunn 1964) was used to compare the compound-treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Reproductive indices:
For each dose group reproduction parameters were expressed in two ways:
-As a mean (with standard deviation) of the number observed for each litter
-As a mean litter proportion calculated on a total group basis
Offspring viability indices:
For each litter the following calculations were performed:

Pre-implantation loss (Number of corpora lutea - number of implantation sites) / Number of corpora lutea x 100

Post-implantation loss (Number of implantation sites - number of live fetuses) / Number of implantation sites x 100


The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis.

Where:

Viable Fetuses Affected/Litter (%) = (No. Viable Fetuses Affected/Litter) / No. Viable Fetuses/Litter x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

Maternal pregnancy data:
There were 23, 20, 16 and 22 females pregnant and used for calculation in Groups 1, 2, 3 and 4 respecitvely. Once control female had resorptions only.

Effect levels (P0)

Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no toxicologically signficant effects

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

Litter size:
There were no treatment related effects on litter size for any group.

Sex ratio:
The male:female sex ratios of each treatment group were unaffected by treatment.

Fetal body weight:
The was no effecty on fetal body weights.

External malformations and variations:
No test substance related effects on fetal external morphology, visceral morphology, skeletal morpholgy.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no toxicologically signficant effects

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Maternal findings
No maternal toxicity was observed in the 50, 200 and 1000 mg/kg/day groups.
 
Developmental findings
No developmental toxicity was observed in the 50, 200 and 1000 mg/kg/day groups.

Based on the results in this prenatal developmental toxicity study the maternal, reproductive and developmental No Observed Adverse Effect Level (NOAEL) for AMIDE #71 was established as being at least 1000 mg/kg body weight/day.
Executive summary:

Summary

A prenatal developmental toxicity study of AMIDE#71 in rats by oral gavage.

Ninety-six mated female Wistar Han rats were assigned to four dose groups. The test item was administered once daily by gavage from Day 6 to 19 post-coitum inclusive, at doses of 50, 200 and 1000mg/kg/day(Groups 2, 3 and 4 respectively). The rats of the control group received the vehicle, propylene glycol, alone. Females were checked daily for the presence of clinical signs. Food consumption of females was determined at periodic intervals; body weight was determined daily during treatment and at periodic intervals in the other periods.

All animals surviving to Day 20 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanised. One half of the fetuses were decapitated and the heads were fixed in Bouin's fixative, all fetuses were dissected and examined for visceral anomalies and subsequently fixed in 96% aqueous alcohol and stained with Alizarin Red S for skeletal examinations.

Results

Accuracy, homogeneity and stability of formulations were not demonstrated by analyses as no analytical method could be developed for gas chromatography (GC), liquid chromatography (HPLC), UV spectrophotometry or total organic carbon analysis (TOC).

Maternal findings

No maternal toxicity was observed in the 50, 200 and 1000 mg/kg/day groups.

Development findings

No developmental toxicity was observed in the 50, 200 and 1000 mg/kg/day groups.

Conclusion

Based on the results in the prenatal developmental toxicity study the maternal, reproductive and developmental No Observed Adverse Effect Level (NOAEL) for AMIDE#71 was established as being at least 1000 mg/kg body weight/day.