Reaction products of amines, dicoco alkyl and glycollic acid

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-07-05 to 2011-12-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 421

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

- Physical state: Clear amber liquid
- Expiration date of the lot/batch: 2011-12-12
- Stability under test conditions: Not specified - analytical verification of formulations
- Storage condition of test material: room temperature, protected from light
- Analytical purity: 13C NMR = 99% active
- Purity test date:: 2011-01-18

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Potage, Michigan, USA
- Age at study initiation: ca 8 weeks
- Weight at study initiation: Male: 249-278g, female: 190-215g
- Fasting period before study: No
- Housing: individually housed in suspended, stainless steel, wire-mesh type cages. During pairing, the animals were randomly cohabitated in the cage of the male. On approximately GD 20, F0 females were individually housed in plastic solid bottom cages containing wood chip bedding. Females were housed in these solid bottom cages with pups during the 4-day lactation period
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26°C
- Humidity (%): 30 to 70%
- Air changes (per hr): Not specified
- Photoperiod (hrs dark / hrs light): 12 hour light/dark period

IN-LIFE DATES: From: 2011-07-05 To: 2011-08-26

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: weekly

VEHICLE
- Justification for use and choice of vehicle (if other than water): test material is highly insoluble in water.
- Concentration in vehicle: 250 mg/mL (nominal)
- Amount of vehicle (if gavage): 0.4, 1.2, 2.4 and 4.0 mL/kg
- Lot/batch no. (if required): ZV0181

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gas Chromatograph (GC) system equipped with a Flame Ionization Detector (FID)
Data Acquisition / Chromatography Data System
GC/FID Column: Rxi-1HT, 30m x 0.32mm x 0.25μm (or equivalent)
Column Flow: Helium @ 1 mL/min
Oven Program: 250°C for 5 minutes, ramp @ 5°C/min to 400°C, hold for 5 minutes
Injector Temperature: 350°C
Injection Mode: Split: 20:1
Injection Volume: 1.0 μL
Detector Temperature: 400°C
Hydrogen Flow: 40 mL/min
Air Flow: 400 mL/min
Makeup Flow (N2): 35 mL/min

A minimum of five of six calibration standards must be used to generate the calibration curve. If the lowest or highest calibration standard is excluded from the regression, sample results outside the acceptable range will not be reported. For quantitation purposes, the response of three peaks will be combined to determine the analyte response. The retention times of the three peaks will be identified in the SST as the third, fourth and fifth peaks.
These retention times will be used to identify the peaks in the other injections for each analytical run.

Acceptance Criterion
The Percent Recovery (PR) of each of the calibration standards must be 100 ±10%, with respect to the calibration standard nominal concentration. The Coefficient of Determination (R2) value must be >= 0.995

System suitability test:
Replicate injections of an independent preparation were used to determine system suitability for each run. The acceptance criteria for SST were as follows: injection repeatability (RSD) ≤5%, tailing factor (T) ≤2, and resolution (R) ≥1.5. The analyte peak retention time was between 11.849 and 12.536 minutes for peak 1; between 14.262 and 14.952 for peak 2; and between 16.565 and 17.261 for peak 3. The retention time and peak area RSD were consistently ≤5% for all peaks in all runs. There were no significant peaks adjacent to the analyte; therefore, the resolution was ≥25.1 for all injections. The tailing factors were consistently ≤1.4 for all peaks in all runs. The SST parameters met the acceptance criteria for all reported runs.

Calibration curve:
A multi-point calibration curve was used for quantification by injecting six standards. The acceptance criterion for the calibration curve was a coefficient of determination (R2) ≥0.995 and the acceptance criterion for the individual standards was a concentration of 100 ±10% recovery of the nominal concentration. All of the standards for each of the reported runs met the acceptance criteria for accuracy and linearity.

Repeatability:
Injections of the performance check standard (same solution as the SST standards) were performed periodically throughout the runs. The acceptance criterion for the performance check standards was 100 ±10% recovery of the nominal concentration. All performance check injections for the reported runs met the acceptance criterion.

Homogenity
Week 1 dose formulations were analyzed for homogeneity verification. Two samples from the top, middle, and bottom of the 250 mg/mL concentration were injected and analyzed.
Samples for each level met the sample analysis acceptance criterion for accuracy and precision (100 ±15% average recovery; ≤15% RSD).

Concentration
Dose formulations for Weeks 1, 2, 3, 4, and 8 were analyzed for concentration verification. Duplicate samples from the middle stratum at the 0 mg/mL (vehicle control) and 250 mg/mL concentration were injected and analyzed. Only vehicle control samples were submitted for analysis for Week 8 dose formulations. Samples for each level met the sample analysis acceptance criteria for accuracy and precision (100 ±15% average recovery; ≤15% RSD).
Test article was not detected in the vehicle control samples.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: one male: one female
- Length of cohabitation: maximum: 14 days
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

Pre-mating: 15 days
Mating: 14 days
Gestation: ca 22 days
Post-parturition: 5 days
Non-pregnant females: 25 days after last scheduled pairing day
Total number of days dosing:
Males 42 days
Females 54 days

Frequency of treatment:

Once per day

Duration of test:

54 days

No. of animals per sex per dose:

12 per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected by the Sponsor, or in consultation with the Sponsor, on the basis of available data from a previous study. In a 28-day toxicity study in rats, animals were administered the test article by oral gavage at dose levels of 25, 250, and 1000 mg/kg/day. Following exposure to the test article for 28 consecutive days, no toxicologically significant changes were observed and the No-Observed-Effect-Level (NOEL) was considered to be 1000 mg/kg/day. The OECD 421 Guideline specifies the limit dose to be 1000 mg/kg/day, and therefore, this was selected to be the high-dose level. The low-dose level was selected to be ten times lower than the limit dose. Based on the lack of reproductive data, the two mid-dose levels were chosen to be three and six times higher than the low-dose level.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
morbidity, mortality, injury, and the availability of food and water twice

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
Evaluation of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, as well as evaluation of respiration.

BODY WEIGHT: Yes
- Time schedule for examinations:
All P0 animals: weekly
Mated females: GD 0, 7, 14, and 20, and LD 0 and 4
Body weight change was calculated between each weighing interval, over the entire premating period (males and females), and for the following intervals: GD 0-7, 7-14, 14-20, and 0-20, and LD 0-4.

FOOD CONSUMPTION: Yes
Individual food consumption for the P0 animals was measured and recorded weekly, except during the mating period. Food consumption was also measured on GD 0, 7, 14, and 20, and on LD 0 and 4

WATER CONSUMPTION : Yes
Availability checked in cageside observations.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on LD 4
- Organs examined:
- Epididymis (2)
-Gonads:
ovary (2) with oviduct (2)
testis (2)
- Gross lesions
- Prostate
- Seminal vesicle (2)
- Uterus [both horns]/ Cervix
- Vagina

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes

Fetal examinations:

F1 PUP EVALUATIONS:
All pups still born, euthanized in extremis, found dead, and surviving to the scheduled necropsy on LD 4 were externally sexed and examined for external malformations and variations.

Statistics:

Data for each sex within a set were analyzed separately.
The raw data were tabulated within each time interval, and the mean and standard deviation were calculated for each endpoint by sex and group. For each endpoint, treatment groups were compared to the control group using the analysis outlined below:
- Parental in-life data: Group Pair-wise comparisons
- Fertility Indices: Gestation length and copulatory interval - Group Pair-wise comparisons
All other - Fischer's Exact Test
- Uterine examination/litter: Pup sex ratio (% viable males/litter) and pup survival (LD 0-4) - Arcsin-Square-Root Transformation
Pup weight - Covariate Analysis
All other - Group Pair-wise comparisons
- Pathology: Group Pair-wise comparisons

Indices:

Fertility Index - Males
No. males impregnating a female/ No. males paired x 100
Fertility Index - Females
No. females pregnant/No. females paired x 100
Live Birth Index
No. live pups at birth /No. pups born x 100
Stillborn Index
No. stillborn pups /No. pups born
Viability Index – Day 4
No. pups surviving 4 days (preculling)/ No. pups born

Historical control data:

No data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

1) Test material formulation:

Homogeneity

The results confirmed that the prepared formulation at 250 mg/mL on Week 1 from the top, middle, and bottom aliquots was homogeneous based on the average percent relative standard deviation, which was 3%, and within the acceptance criteria established (≤10%). In addition, the average recovery rate was 96%, which was also within the acceptance criteria established (±15% of nominal).

Concentration

The results of the concentration evaluation from Weeks 1, 2, 3, and 4 confirmed that the animals received the appropriate doses, based on the prepared concentration, as compared to nominal. The mean recovery rates ranged from 95 to 104%. These were within the acceptance criteria established (±15%), and the percent relative standard deviation ranged from 0 to 3%. The control samples were devoid of test article.

2) Parental in-life observations

Mortality:

One female at 1000 mg/kg/day (animal number 318) was found dead on Study Day 11. Macroscopic observations included moderate amount of red fluid in the thoracic cavity and severe laceration/perforation of the esophagus. There were no relevant microscopic

observation for this animal but based on macroscopic observations, the cause of death was confirmed to be the result of a dosing related event and not treatment related.

Detailed clinical observations:

There were no clinical observations that followed a definitive dose response pattern to indicate a relationship to treatment. A number of different findings were noted; however, they were either low in incidence or comparable to the controls and therefore were not

considered test article related.

Body weights and body weight changes:

Mean body weight in the treatment groups (100, 300, 600, and 1000 mg/kg/day) was not statistically different from the controls in either male or female animals during the course of the study. There were some statistically significant alterations in mean body weight change; however, there were no patterns to indicate a relationship to test article administration across the treatment groups during the appropriate premating, pairing, and postmating periods in the males, and during the premating, gestation, and lactation periods in the females. In males, body weight change was significantly decreased at 100 and 1000 mg/kg/day during the postmating period from Days 5-6. As the mean body weight at these dose levels was unaffected by treatment and as body weight change at the 300 and 600 mg/kg/day groups was comparable to controls, the decreases in body weight change at the low and high-dose levels were not considered toxicologically meaningful. In females, mean body weight change was statistically increased at 300 and 1000 mg/kg/day during gestation intervals 14-20 and 0-20. The increase in body weight change was not considered adverse.

Food consumption

Mean food consumption showed some statistically significant increases and decreases in the treatment groups during the course of the study, as compared to controls. However, there were no definitive dose responsive changes that could be attributed to exposure with the test article.

3) Reproductive performance

Parental Reproductive and Fertility Indices

The reproductive and fertility indices evaluated in the treatment groups at 100, 300, 600, and 1000 mg/kg/day did not result in any significant test article-related changes. The male and female mating, fertility, and fecundity indices for the treatment groups ranged from 91.7 to 100%, which was identical to the range of values noted in the controls. In addition, there were no statistically significant changes in the copulatory interval measured between the treatment groups, which ranged from 2.4 to 3.3 days, and the controls at 3.3 days. All values were also within the historical control range for this laboratory (2.4 to 4.2 days).

4) Litter data

Parturition data

There were no test article-related changes in the parturition or F1 litter data for the animals treated with the test article. These parameters included the number of females with at least one stillborn pup, the number of females with all stillborn pups, gestation length, the total number of pups born per litter, gestation index, stillborn index, total implantation scars per litter, and the number of corpora lutea. At 300 mg/kg/day five females were noted with at least one stillborn pup and at 1000 mg/kg/day one female was noted with at least one stillborn pup. As there were fewer females affected at the high-dose level, as compared to the 300 mg/kg/day

group animals, these stillborn rates were considered to be spurious in nature and not toxicologically meaningful.

Pup survival

There were no treatment-related changes in the F1 pup survival during the preculling period as compared to controls. These parameters included the number of liveborn pups per litter on LD 0 and 4 during the preculling period, and the viability index.

Pup sex ratios

The F1 pup sex ratio (percent males per litter) in the treatment groups (ranging from 47.90 to 55.75%) was comparable to that seen in the controls (48.02%).

Pup body weight

There were no definitive test article-related changes in F1 pup weight for the treated groups. Some slight decreases in pup weight were noted at the low- (100 mg/kg/day) and high-dose (1000 mg/kg/day) levels that were statistically different from controls (in males and both sexes combined). However, as the values in the low- and high-dose groups were similar in magnitude and as the values for the 600 mg/kg/day treated animals were comparable to the controls, a clear dose response relationship could not be established.

5) Pup macroscopic

During the macroscopic, external evaluation of the F1 pups (unscheduled stillborn, unscheduled found dead, and scheduled), there were no test article-related findings noted in the treatment groups, as compared to controls. One male pup at 100 and 300 mg/kg/day each were identified with a scabbed area of the hindlimb and discolored skin, respectively. One female pup at 300 and 600 mg/kg/day each were found with a scabbed face and discolored skin, respectively. The remaining pups were all within normal limits.

6) Postmortem study evaluations

Parental macroscopic evaluation

There were no treatment-related macroscopic findings; all macroscopic findings were considered to be incidental because of the low incidence, lack of a dose response relationship, observed in respective control groups, and/or are typical of background changes seen in rats of this strain and age.

Parental organ weights

There were no test article-related organ weight changes.

Parental microscopic evaluations

There were no treatment-related microscopic findings; all microscopic findings were considered to be incidental because of low incidence, lack of a dose response relationship, observed in respective control groups, and/or are typical of background changes seen in rats of this strain and age.

Applicant's summary and conclusion

Conclusions:

Based on the results obtained from this oral reproductive/developmental toxicity screening study in rats, a No-Observed-Adverse-Effect-Level (NOAEL) for general, reproductive, and developmental toxicity was considered to be 1000 mg/kg/day, the highest level tested and the limit dose designated by the guideline.

Executive summary:

Test Guidance

OECD Guideline No. 421

Method and material

This study was conducted to generate limited information concerning the effects of the test article on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus, and parturition. Four treatment groups of twelve CD® [Crl:CD®(SD)] rats/sex/group were administered the test article at dose levels of 100, 300, 600, or 1000 mg/kg/day at dose volumes of 0.4, 1.2, 2.4, or 4.0 mL/kg, respectively. One additional group of twelve animals/sex served as the control and received the vehicle, peanut oil (arachis oil NF) at a dose volume of 4.0 mL/kg. The vehicle or test article was administered to all groups once daily via oral gavage. The males were dosed for 43 days, beginning 14 days prior to pairing. Dosing of the females began 14 days prior to pairing, through the mating period, up to and including Lactation Day (LD) 3.

Observations of the parental (P0) animals included clinical signs, body weights and body weight change, and food consumption during the premating/mating, gestation, and lactation periods, and parturition and litter data. Observations of the offspring (F1) included survival at birth and during lactation, individual pup body weights, and gross abnormalities. At study termination, necropsy examinations were performed on all P0 animals, and organs and tissues were collected, weighed and examined for select groups. On LD 4, surviving F1 pups were examined externally, euthanized, and discarded.

The analytical evaluation of the formulation samples confirmed that they were homogenous and at the targeted concentration required. Control samples were devoid of test article.

 

Results

Oral administration of the test article to male and female rats at 100, 300, 600, and 1000 mg/kg/day in males during the premating/mating and postmating periods did not reveal any test article-related changes. This included parameters consisting of P0 clinical findings, body weight, body weight change, and food consumption. In addition, reproductive, fertility, parturition, and F1 litter data, including external pup observations, as well as P0 gross necropsy findings, organ weights, and microscopic findings did not reveal any changes that could be considered treatment related.

 

Based on the results obtained from this oral reproductive/developmental toxicity screening study in rats, a No-Observed-Adverse-Effect-Level (NOAEL) for general, reproductive, and developmental toxicity was considered to be 1000 mg/kg/day, the highest level tested and the limit dose designated by the guideline.

 

Conclusions

In accordance with CLP EC Regulation No. 1272/2008, the test material is not classified for reproductive toxicity.