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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19-Dec-2013 to 09-Sep-2014
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
according to guideline
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
according to guideline
other: Japanese Guidelines, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Teratology (2-1-18)
GLP compliance:
yes (incl. QA statement)
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
No molecular formula
Test material form:
Details on test material:
Chemical name: Amines, di-C16-18 (even numbered) alkyl
EC no.: 629-721-4

To the best of knowledge, the sample used is representative to the boundary composition shared and agreed by each registrant.

Test animals

Details on test animals or test system and environmental conditions:

- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Breeder: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Number of Animals: 88 mated females, 22 mated females per group
- Age (Day 0 Post coitum): 11 to13 weeks
- Body Weight Range (Day 0 Post coitum): 197 to 258 g
- Identification: Cage card and individual animal number (ear tattoo).
- Randomization: Computer-generated random algorithm.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.


- Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). Values outside of these ranges occasionally occurred, which were considered not to have any influence on the study. These data were not reported but were retained in the raw data. There was 12-hour fluorescent light / 12-hour dark cycle with music during the light period.
- Accommodation: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J.Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (ISO-BLOX from Harlan Laboratories B.V. / Netherlands).
- Diet: Pelleted standard Harlan Teklad 2018C (batch no. 38/13) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. Results of representative analyses for contaminants are archived at the test facility.
- Water: Community tap-water from Itingen was available ad libitum in water bottles. Results of bacteriological assay, chemical and contaminant analyses of representative samples are archived at the test facility.

Administration / exposure

Route of administration:
oral: gavage
corn oil
Details on exposure:

The dose formulations were prepared daily using the test item as supplied by the Sponsor. The dose formulations were considered stable for at least 4 hours in corn oil stored at room temperature (15 - 25 °C), based on results obtained in a previous non-GLP study (D76437).

Amines, di-C16-18 (evennumbered) alkyl was weighed into a glass beaker on a tared Mettler balance and the vehicle was added. The mixtures were mixed using an Ultra Turrax and stirred with a magnetic stirrer. Separate formulations were prepared for each concentration. For the first day of treatment the test item (a wax-like substance) was broken into large chunks with a spatula and the required amount was then removed from the bottle to be weighed. After stirring, the formulations were examined by eye, to confirm that all test item was dissolved. However, this proved difficult due to the thick, cloudy appearance of the formulations. In light of this, for the second formulation preparation, the substance was instead scraped from the bottle, prior to weighing, thus reducing the possibility to miss lumps of test item in the turbid solution.

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.


- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type of study.
- Frequency of Administration: Once daily (within four hours)
- Target Dose Levels: 0 mg/kg bw/day (Group 1, control group), 100 mg/kg bw/day (Group 2), 300 mg/kg bw/day (Group 3) and 1000 mg/kg bw/day (Group 4)
- Rationale for Dose Level Selection: Based on a dose range-finding study, Harlan Study Number D76437 (non-GLP study).
- Dose Volume: 5 mL/kg body weight
- Dose Concentrations: 0 mg/mL (Group 1), 20 mg/mL (Group 2), 60 mg/mL (Group 3) and 200 mg/mL (Group 4)
- Duration of Acclimatization Period: At least 18 days. In order to start the study immediately in January 2014 the animals were required to be delivered by 19 December 2013.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:

On the first dose formulation day samples from the control group as well as three samples (top, middle and bottom) of about 0.5 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 1 g of each test item concentration were taken from the middle to confirm the stability (4 hours at room temperature (20 ± 5 °C)). Due to the values seen for concentration following analysis, (67.9% group 2, 120.6% group 3, 145% group 4), additional formulation samples were taken from a formulation preparation made during the course of the study to analyze the concentration and homogeneity. As all stability samples taken at the time were within acceptance criteria, no further stability samples were taken. Towards the end of the study, samples were taken from the middle of a third preparation to confirm concentration.

The aliquots for analysis of dose formulations were delivered at ambient temperature or frozen (e.g. on dry ice) to Dr. G. Heinemann and stored there at -20 ± 5 °C until analysis.

The analysis was performed by Harlan Laboratories Ltd. using a LC/MS method provided by the Sponsor. The test item was used as the analytical standard. Analyzed samples were not discarded without written consent from the study director. Duplicate samples were taken of all samples and were stored at -20 ± 5 °C at Harlan Laboratories Ltd., Itingen / Switzerland.

As a result of ongoing development to the method of analysis, the dilution solvent for the first dilution step was changed from acetone to tetrahydrofuran, due to improved solubility. Furthermore, instead of trifluoro acetic acid, formic acid was used, for better ionization. However, all technical parameters or final dilution factors stayed unchanged. As a result of this method development, all reserve samples were analyzed and, where necessary, re-analyzed due to concentrations lower than expected.

The results obtained from the analysis of the reserve samples were reported. The results of all prior analyses are included in the raw data.

The test item served as analytical standard/reference item.


No test item was detected in the vehicle control formulation.

The mean concentrations of Amines, di-C16-18 (evennumbered) alkyl in the dose formulations ranged from 90.6% to 117.7% with reference to the nominal and were within the accepted range of ±20%, with the exception of the group 2 formulations which were prepared on 13 and
20-Jan-2014 which were determined to be 69.4% and 130.2% with reference to the nominal concentration, respectively.

All dose formulations were considered to be homogeneous with the distribution of Amines, di-C16-18 (evennumbered) alkyl in the preparations approved because single results found did not deviate more than 10.6% from the corresponding mean and met the specified acceptance criterion of ≤15%.

The test item was found to be stable in application formulations of group 2, when kept four hours at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean value. However, application formulations of group 3 and 4 did not meet this criterion. In group 3 the maximum deviation of time-zero mean was found to be 11.1% and deviated only slightly from target and in group 4 the maximum deviation of time-zero mean was found to be 18.2%.
Details on mating procedure:
After acclimatization, females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed.

The day of mating was designated day 0 post coitum.

Male rats of the same source and strain were used only for mating. These male rats are in the possession of Harlan Laboratories and were not considered part of the test system. The fertility of these males had been proven and was continuously monitored.
Duration of treatment / exposure:
15 days (day 6 - 20 post coitum)
Frequency of treatment:
Once daily
Duration of test:
- Acclimatization: at least 18 days of acclimatization (in order to start the study immediately in January 2014 the animals were required to be delivered by 19 December 2013)
- Gestation: 21 days
Doses / concentrations
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
actual ingested
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Details on study design:
The purpose of this study was to detect effects on the pregnant rat and development of the embryo and fetus consequent to exposure of the female to the test item from day 6 post coitum (implantation) to day 20 post coitum (the day prior to Caesarean section).


Maternal examinations:

Twice daily


Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy).


Recorded at 3-day intervals (days 0 - 3, 3 - 6, 6 - 9,9 - 12, 12 - 15, 15 - 18 and 18 - 21 post coitum).


Recorded daily from day 0 until day 21 post coitum.


At the scheduled necropsy on day 21 post coitum, females were sacrificed by CO2 asphyxiation and the fetuses removed by Caesarean section.
Ovaries and uterine content:
Post mortem examination, including gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, corpora lutea count and position of fetuses in the uterus was performed and the data recorded. The uteri (and contents) of all females with live fetuses were weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain.

If no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulfide to accentuate possible hemorrhagic areas of implantation sites.
Fetal examinations:
Fetuses were removed from the uterus, sexed, weighed individually, examined for gross external abnormalities, sacrificed by a subcutaneous injection of sodium pentobarbital and allocated to one of the following procedures:

- Microdissection technique (sectioning/dissection technique). At least one half of the fetuses from each litter were fixed in Bouin's fixative (one fetus per container). They were examined by a combination of serial sections of the head and microdissection of the thorax and abdomen. This included detailed examination of the major blood vessels and sectioning of the heart and kidneys. After examination, the tissue was preserved in a solution of glycerin/ethanol (one fetus per container). Descriptions of any abnormalities and variations were recorded.

- The remaining fetuses were eviscerated and with the exception of over the paws, the skin was removed and discarded. Carcasses were processed through solutions of ethanol, glacial acetic acid with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and aqueous glycerin for preservation and storage. The skeletons were examined and all abnormal findings and variations were recorded. The specimens were preserved individually in plastic vials. The fetal evaluation process followed the Harlan Laboratories Ltd. document “Skeletal Rat User Guide - Version 2”. The assessment included, but was not limited to, all principal skeletal structures including cranium, vertebral column, rib cage and sternum, pectoral and pelvic girdles. The coding of fetal anomalies/variants and identification of skeletal structures was as, Harlan Laboratories Ltd. document “Abbreviations used in Skeletal Assessment of the Rat Fetus at Day 21 post coitum - Version 2”.

After the fixation in Bouin’s fluid for visceral evaluation and the staining of the fetuses for skeletal examination, specimens were sent to the principal investigator and evaluated by Harlan Laboratories Ltd, Shardlow, UK. The principal investigator was notified prior to shipment. After the evaluation, the fetuses were sent back to Harlan Laboratories Ltd, 4452 Itingen / Switzerland and were archived there.

Investigations were performed by personnel appointed by the principal investigator and were conducted according to Harlan Laboratories Ltd. Standard Operating Procedure 189.15 “Evaluation of Fetal Skeletons” and Standard Operating Procedure 189.017 ”Evaluation of Fetal Visceral by Microdissection”. Fetuses were examined under a binocular microscope. Fetuses were examined in mixed group order. Each litter was examined in sequential order.

Peer review of 10% of the fetal examinations was performed according to Harlan Laboratories Ltd. Standard Operating Procedure 189.15 and 189.17. All findings were recorded on the appropriate proforma. These findings were collated, tabulated and a phase report produced. This phase report was audited by Harlan Laboratories Ltd. Quality Assurance Department and dispatched together with the raw data to the study director.
The following statistical methods were used to analyze food consumption, body weights and reproduction data:

- Means and standard deviations of various data were calculated and included in the report.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

For skeletal or visceral findings, the data was first assessed using Bartlett’s test for homogeneity of variance. If the data were homogeneous, parametric assessment, one way analysis of variance and, if significant, Dunnett’s multiple comparison test were employed. Where the data was found to be non-homogeneous, non-parametric assessment, Kruskal-Wallis and, if significant, pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test were used. Due to the preponderance of non-normal distributions, non-parametric assessment could be routinely used for fetal data.
From the on-line recorded reproduction data, the following parameters were calculated: pre- and post-implantation losses, embryonic and fetal deaths, live and dead fetuses, abnormal fetuses, fetal sex ratios and fetal body weights.

For reproduction data, group mean values were calculated both on a litter basis and on a percentage per group basis. Mean fetal weights were calculated from the individual weights both on a per group and on a per litter basis.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects. Remark: effects seen on body weight and food consumption but deemed not to be adverse

Details on maternal toxic effects:


All females survived until the scheduled necropsy.


No clinical symptoms or signs were observed during the study that were considered to be related to administration of the test item at any dose level. Vaginal bleeding was observed in group 4 female 75 on day 14 of gestation. However, as this observation was seen in only one female, on one day of gestation and at necropsy this animal was observed to be non-pregnant, it was considered that the vaginal bleeding seen was unrelated to administration of the test item.


No test item-related changes were observed in the food consumption for the duration of the dosing period in animals given 100 or 300 mg/kg/day. Although statistically significant increases in the amount of food consumed were seen on occasion, these increases were both sporadic in nature and were not dose related and therefore considered not to be as a result of treatment with the test item.

For animals given 1000 mg/kg/day, a statistically significant decrease in the amount of food consumed was seen between days 18 and 21 of gestation. This was considered to be a result of test item administration.


There was no effect on group mean absolute body weight, body weight gain (%) or corrected body weight gain (corrected for the gravid uterus weight) in animals given 100 or 300 mg/kg/day.

From day 20 of gestation, a slight decrease in group mean body weight gain was apparent in animals given 1000 mg/kg/day, achieving statistical significance on day 21 of gestation, when compared to control animals. This decrease was also evident in group mean absolute body weights, however in this instance no statistical significance was seen. A marked, statistically significant decrease in group mean corrected body weight gain (corrected for the gravid uterus weight) was also apparent at 1000 mg/kg/day, with a gain of 6.8 g (2.7%) compared to 31.1 g (12.6%) for the controls.


The relevant reproduction data (post implantation loss and number of fetuses per dam) were not affected by treatment with the test item.



There were no treatment-related macroscopic findings seen at any dose level.

Effect levels (maternal animals)

Dose descriptor:
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:

No findings were observed during external examination of the fetuses in groups given 100, 300 or 1000 mg/kg/day.


No test item-related effects on the sex ratio of the fetuses were noted in any group.


No test item-related effects on fetal body weights were noted in dams at any dose level.

For dams administered 1000 mg/kg/day, the mean body weight of live fetuses, calculated on both an individual and litter basis, were marginally decreased when compared to the controls, achieving statistical significance on an individual basis. However, this decrease in fetal weight was within historical background data ranges and was considered to be due to the higher number of fetuses per litter seen at this dose level, than seen in the controls and not due to toxicity of the test item.


During visceral examination of the fetuses, findings were noted in:

- 21% examined fetuses (in 79% litters) in group 1
- 23% examined fetuses (in 59% litters) in group 2
- 23% examined fetuses (in 75% litters) in group 3
- 30% examined fetuses (in 83% litters) in group 4

At 1000 mg/kg body weight/day there was one fetus (dam 80, fetus 385) with a significant structural malformation which was described as Anophthalmia. Due to the isolated nature of this finding and the fact that this finding can be seen at a low incidence as a spontaneous event, this finding was considered not to be a consequence of treatment. All other observations of differences in the viscera of fetuses within all treated groups show no treatment related changes when compared to control fetuses. The incidence of visceral variations were either not dosage related, seen at low incidence or provided any pattern of change that is indicative of treatment related structural change. The type and incidence of fetal observations are those commonly observed in the rodent fetus in developmental toxicity studies.


During skeletal examination of the fetuses, findings were noted in:

- 96% examined fetuses (in 100% litters) in group 1
- 94% examined fetuses (in 100% litters) in group 2
- 93% examined fetuses (in 95% litters) in group 3
- 95% examined fetuses (in 100% litters) in group 4

The observations recorded at skeletal evaluation are indicative of those findings commonly observed in the rat fetus for developmental toxicity studies. These findings can be described as either precocious or delayed development of the skeleton and reflect the fact that individual variation in fetal skeletal development is a natural event. Whilst intergroup differences in extent of bone ossification/development for different structures can be seen, there is no pattern of skeletal change that can be apportioned to an effect of treatment. There is no pattern of change in skeletal development to indicate an effect of treatment.

Effect levels (fetuses)

Dose descriptor:
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: fetotoxicity

Fetal abnormalities

not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables




Dose (mg/kg)









Female numbers

1 - 22

23 - 44

45 - 66

67 - 88

Number of mated females





Not pregnant (A)





Number of females with live fetuses at termination*





* Only dams with at least one live fetus at Caesarean section were used for the calculations of food consumption, body weight gain and corrected body weight gain data.

(A) Females nos. 2, 11, 15 (group 1), 24, 25, 27, 28, 35 (group 2), 46, 60 (group 3), 67, 75, 76, and 79 (group 4) were not pregnant.


Applicant's summary and conclusion

Amines, di-C16-18 (evennumbered) alkyl (CAS No. 308062-60-4) was tested in the oral OECD 414 prenatal developmental toxicity study in rats. The maternal NOAEL (No Observed Adverse Effect Level) for Amines, di-C16-18 (evennumbered) alkyl was considered to be 1000 mg/kg/day based on the effects seen on body weight and food consumption. Due to the lack of findings seen, the fetal NOEL (No Observed Effect Level) for Amines, di-C16-18 (even numbered) alkyl was considered to be 1000 mg/kg/day.

Executive summary:

The purpose of this study was to detect effects on the pregnant rat and development of the embryo and fetus consequent to exposure of the female to the test item from day 6 post coitum (implantation) to day 20 post coitum (the day prior to Caesarean section).

From the dose formulation analysis, the decreased results seen from the first preparation at 20 mg/mL indicate that the animals were only dosed with 73.3% of the desired concentration, between the 13 and 19 of January. However, in light of the change in formulation preparation method employed (with regards to the removal of the test item from the bottle for weighing) and the subsequent formulation analysis results, it is considered that the decreased concentration was likely only administered on 13-Jan-2014. Also of note is the increased concentration of the 20 mg/mL formulation (125%) prepared the following week on 20-Jan-2014. If one assumes that for the first 7 days all animals were administered a concentration of 73.3% the nominal concentration, then were dosed with 125.7% the nominal concentration for the next 8 days, this would equate to an average dose of 101.2% over the 13 day period. This combined with the formulation preparation on 28 January 2014, with a concentration of 101.8%, would mean all animals received an average dose of at least the desired dose level of 20 mg/mL over the course of their treatment. However, due to the nature and extent of the effects seen at 300 or 1000 mg/kg/day, all of which were of acceptable formulation concentration, it is also considered that any differences in formulation concentrations, if administered for a longer duration, would have no impact on the results or the study outcome.

With regards to the issue of stability of the samples at concentrations of 60 and 200 mg/mL, it should be noted that although both samples fell outside the standard acceptance criteria defined by Harlan Laboratories (10%), the magnitude of the decreases in concentration seen were relatively slight (11.1% and 18.2% for 60 and 200 mg/mL respectively) and within the percentage difference from nominal concentration (20%) that is required for all samples to be considered as acceptable with regards to the achieved concentration. For both groups, sample reanalysis was performed on the stability samples and the mean values are presented, indicating that values higher than those expressed in the table were also recorded. Furthermore, the results of the stability assessment performed with the original method for analysis, using the original stability samples yielded results that were within the acceptance criteria (10% and 3%, respectively). In light of these results and those obtained in the non-GLP range finding study (D76437), it is considered that the formulations of Amines, di-C16-18 (even numbered) alkyl in corn oil were considered stable for up to 4 hours at room temperature.

Although a decreases in both body weight gain and body weight gain corrected for gravid uterus weight were seen, as there were no effects seen on any of the pregnancy or fetal parameters and no further effects were noted in the dams, this finding, although test item related, was not considered to be adverse.

Based on the effects seen on body weight and food consumption, a maternal NOAEL (No Observed Adverse Effect Level) for Amines, di-C16-18 (evennumbered) alkyl was considered to be 1000 mg/kg/day.

Due to the lack of findings seen, the fetal NOEL (No Observed Effect Level) for Amines, di-C16-18 (even numbered) alkyl was considered to be 1000 mg/kg/day.