2-methylundecanal

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-methylundecanal

EC Number:

203-765-0

EC Name:

2-methylundecanal

Cas Number:

110-41-8

Molecular formula:

C12H24O

IUPAC Name:

2-methylundecanal

Specific details on test material used for the study:

Identification: ALDEHYDE C 12 MNA PURE
Appearance: Colourless to pale yellow liquid
EC: 203-765-0

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

On 02 May 2018 and 04 May 2018, time-mated female Wistar Han Rats were received from Charles River Laboratories Deutschland, Sulzfeld, Germany. The females arrived on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating). They were approximately 10-14 weeks old and weighed between 197 and 244 g at the initiation of administration.
A health inspection was performed upon receipt of the animals

Justification for Test System and Number of Animals
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for developmental toxicity testing by regulatory agencies. Charles River Den Bosch has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants.
The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
This study plan was reviewed and agreed by the Animal Welfare Body of Charles River Laboratories Den Bosch B.V. within the framework of project license AVD2360020172866.

Animal Identification
At study assignment, each animal was identified by indelible ink.

Environmental Acclimation
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of administration.

Selection, Assignment, Replacement, and Disposition of Animals
On the day of receipt, animals were assigned to groups by a computer-generated random algorithm according to body weights. Each set of females mated on the same date (i.e. 4 sets) was distributed as evenly as possible over the dose groups with body weights within ± 20% of the mean for each set of animals.

Housing
On arrival and following randomization females were housed individually in Macrolon plastic cages (MIII type, height 18 cm) containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. The room in which the animals were kept was documented in the study records. Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group and animal number.

Environmental Conditions
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 20 to 21°C with an actual daily mean relative humidity of 49 to 70%. A 12-hour light/12-hour dark cycle was maintained.

Food
Prepared powder diet was provided ad libitum throughout the study, except during designated procedures. Until start of treatment on Day 6 post-coitum, animals had free access to the same diet (without the test item) received from the supplier in powder form (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). The feed (without the test item) was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water
Municipal tap water was freely available to each animal via water bottles.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility.
It was considered that there were no known contaminants in the water that would interfere with the objectives of the study.

Animal Enrichment
For psychological/environmental enrichment and/or nesting material, animals were provided with paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands).

Veterinary Care
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

The test item was administered to the appropriate animals by inclusion in the diet ad libitum from Day 6 to Day 21 post-coitum, inclusive.
The amount of test item incorporated in the diet was kept at a constant level in terms of ppm, throughout the study period. After termination, the actual test item intake was estimated based on the body weight and food consumption values.
The diet was provided in stainless steel containers, covered by a stainless steel grid to prevent spillage.
The same diets remained in the food hopper for a maximum of one day. On the day of weighing the remaining food in the food hopper, was replaced with new diet retained from the freezer (≤-15°C) acclimated to room temperature conditions for at least 1 hour prior to opening the diet bag.

Justification of Route and Dose Levels
The oral route of exposure via dietary inclusion was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
The dose levels were selected based on the results of obtained in the Reproduction/Developmental Toxicity Screening Test of Aldehyde-C12 MNA Pure by
Dietary Administration in Rats (Test Facility Study no. 517133), and in an attempt to produce graded responses to the test item.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical Method
Analyses were performed using a validated analytical procedure (Test Facility Study No. 517126).

Concentration Analysis
Duplicate sets of samples (approximately 5 g) for each sampling time point were used for concentration analysis, the remaining samples were retained at the Test Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration. After acceptance of the analytical results, backup samples were discarded.

Homogeneity Analysis
Duplicate sets of samples (approximately 5 g) for each sampling time point were used for homogeneity analysis, the remaining samples were retained at the Test Facility as backup samples. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was =< 10%. After acceptance of the analytical results, backup samples were discarded.

Stability Analysis
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 517126) demonstrated that the test item is stable in the diet when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Test Facility Study No. 517126.

Details on mating procedure:

Time-mated female Wistar Han Rats were received from Charles River Laboratories Deutschland, Sulzfeld, Germany. The females arrived on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating).

Duration of treatment / exposure:

The test item was administered to the appropriate animals by inclusion in the diet ad libitum from Day 6 to Day 21 post-coitum, inclusive.

Frequency of treatment:

Daily in the diet ad libitum

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Doses / concentrations 2

Dose / conc.:

1 500 ppm

Doses / concentrations 3

Dose / conc.:

5 000 ppm

Doses / concentrations 4

Dose / conc.:

15 000 ppm

Remarks:

corresponding to an actual test item intake of approximately 1350 mg/kg/day

No. of animals per sex per dose:

22 females per dose level

Control animals:

yes, plain diet

Details on study design:

The dose levels were selected based on the results of obtained in the Reproduction/Developmental Toxicity Screening Test of Aldehyde-C12 MNA Pure by Dietary Administration in Rats (Test Facility Study no. 517133), and in an attempt to produce graded responses to the test item.

On the day of receipt, animals were assigned to groups by a computer-generated random algorithm according to body weights. Each set of females mated on the same date (i.e. 4 sets) was distributed as evenly as possible over the dose groups with body weights within ± 20% of
the mean for each set of animals.

Examinations

Maternal examinations:

Mortality/Moribundity Checks – F0-Generation
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Mortality/Moribundity Checks – F0-Generation
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Body Weights – F0-Generation
Animals were weighed individually on Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

Food Consumption – F0-Generation
Food consumption was quantitatively measured for Days 2-6 and daily from Day 6 onwards (start of treatment) up to and including Day 21 post-coitum.

Water Consumption – F0-Generation
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

Unscheduled Deaths – F0-Generation
No animals died during the course of the study.

Necropsy – F0-Generation
All animals were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No organs (except for the uterus) were weighed.
Each ovary and uterine horn of all animals were dissected and examined as quickly as possible to determine:

The number of corpora lutea.
- The weight of the (gravid) uterus.
- The number of implantation sites.
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths.
- The sex of each fetus based on the ano-genital distance.

In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.

Ovaries and uterine content:

Each ovary and uterine horn of all animals were dissected and examined as quickly as possible to determine:

The number of corpora lutea.
- The weight of the (gravid) uterus.
- The number of implantation sites.
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths.
- The sex of each fetus based on the ano-genital distance.

In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.

Fetal examinations:

Fetal Examinations – F1-Generation
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

External Examinations – F1-Generation
Each viable fetus was sexed, examined in detail to detect macroscopic visible abnormalities and their weight.

Visceral Examinations– F1-Generation
The sex of all fetuses was confirmed by internal examination and approximately one-half of the fetuses (live and dead) in each litter (all groups) were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar.
The heads were removed from this one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination using the Wilson sectioning technique. After examination, the tissues without variation or malformations were discarded. Tissues with variations or malformations were stored in 10% formalin.
Fetuses selected for skeletal examination for which visceral malformations were suspected, were also subjected to visceral examination.
All carcasses, including the carcasses without heads, were eviscerated, labeled and fixed in 96% aqueous ethanol for subsequent examination of skeletons.

Skeletal Examinations– F1-Generation
All eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson.
Subsequently, skeletal examination was done for one-half of the fetuses (i.e. the fetuses with heads). Fetuses selected for visceral examination for which skeletal malformations were suspected were also subjected to skeletal examination.
All specimens were archived in glycerin with bronopol as preservative.
A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and Study Director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons
were conducted using two sided tests and were reported at the 1% and 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity were noted during the observation period up to treatment at 15000 ppm (Group 4).
At the check of the animals at receipt, one animal missed a piece of one of its ears and a scab was observed on the edge. This injury explained the scabs on the ear of female no.11 (Group 1) at the start of the study. No further clinical signs were observed during the pretreatment phase.
Temporary signs of piloerection were noted in female no.72 (Group 4 at 15000 ppm) on Days 3 and 4 of treatment. As body weight gain and food consumption in this animal were normal over these days, these clinical signs were considered of no toxicological significance. Alopecia was also noted in this female from Day 3 of treatment onwards and persistent until termination. This finding is regularly observed and was within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study, and was therefore considered to be unrelated to treatment.
No further clinical signs were observed during the treatment phase in the other Group 4 females and in all females of Groups 1 to 3.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weights, body weight gain and weight gain corrected for gravid uterus of treated animals remained in the same range as controls over the treatment period.
The statistical significances (p<0.05) apparent for body weight gain at Days 15 and 21 postcoitum were considered to have occurred by chance, based on the fact that they occurred in the absence of a dose response relationship.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A lower consumption of the test-diet, absolute and relative to body weight, was observed in Group 4 females (at 15000 ppm) over the first day of treatment (Days 6-7), reaching levels of statistical significance when compared to controls. From Day 7 onwards test-diet consumption in Group 4 females was within normal limits, but was always a little lower in comparison with that in the other groups. Incidentally, the difference reached a level of statistical significance consumption (absolute and relative to body weight) when compared to controls. i.e. over Days 15-16 and 20-21. The overall mean of the test-diet consumption in Group 4 females was approximately 10% lower than in the other groups.
A lower test-diet consumption at start of treatment, reaching a level of statistical significance for the mean relative to body weight value when compared to controls, was also observed in Group 3 females (at 5000 ppm). However, the test-diet consumption in Group 3 females over the remaining treatment period and the overall mean was similar to the control consumption values.
Group 2 female test-diet consumption, absolute and relative to body weight, was similar to the control level over the study period.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
Incidental findings in female no.26 (Group 2) and female no.69 (Group 4) included isolated, reddish foci in the thymus and enlarged iliac lymph nodes and a yellowish, firm nodule in the uterine adipose tissue, respectively. These findings are occasionally seen among rats used in these types of study and in the absence of correlated microscopic findings they were considered changes of no toxicological significance.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

Maternal Pregnancy Data
The numbers of pregnant females, corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation

Details on results:

No maternal toxicity was observed in the 1500, 5000 and 15000 ppm groups. Slightly decreased test diet consumption over the treatment period was noted in animals at 15000 ppm, which was considered to be related to the taste of the test item rather than a sign of toxicity

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

The numbers of pregnant females, corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The numbers of pregnant females, corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

The numbers of pregnant females, corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.

Early or late resorptions:

no effects observed

Description (incidence and severity):

The numbers of pregnant females, corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.

Dead fetuses:

no effects observed

Description (incidence and severity):

The numbers of pregnant females, corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 15 000 ppm

Based on:

test mat.

Basis for effect level:

other: No test item related effects at the highest dose tested

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no toxicologically relevant effects on fetal body weights (both sexes) noted by treatment up to 1000 mg/kg.
Mean combined (male and female) fetal body weights were 5.3, 5.1, 5.3 and 5.2 grams for the control, 1500, 5000 and 15000 mg/kg groups, respectively.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment up to 1000 mg/kg.
Mean sex ratios (males:females) were 52:48, 53:47, 56:44 and 50:50 for the control, 1500, 5000 and 15000 mg/kg groups, respectively.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on litter size of any group.
Mean litter sizes were 11.7, 11.5, 10.5 and 11.1 fetuses/litter for the control, 1500, 5000 and 15000 mg/kg groups, respectively.

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on external morphology following treatment up to 15000 ppm.
The only fetus affected externally was control fetus A012-05. This fetus had an absent tail (confirmed skeletally) and anal atresia, which was as such considered to be of spontaneous origin.
External variations were not seen in any group.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on skeletal morphology following treatment up to 15000 ppm.
Only three different malformations were revealed at skeletal examination. A vertebral anomaly occurred in Group 3 fetuses A051-07, A064-11 (the latter one also viscerally malformed) and in Group 4 fetus A079-03. The Group 4 fetus also had a sternal anomaly and sternoschisis.
All these malformations were also previously noted among historical control fetuses and due to the low incidences, they were not considered to be treatment related.
Skeletal variations occurred at an incidence of 66.1%, 74.7%, 70.7% and 73.2% per litter in Groups 1, 2, 3 and 4, respectively. All the ones that were noted occurred in the absence of a dose-related incidence trend, infrequently and/or at frequencies that were within the range of available historical control data. Therefore, they were not considered treatment related.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on visceral morphology following treatment up to 15000 ppm.
Three viscerally malformed fetuses were observed in this study. In both Group 2 and 3 was a fetus whereby situs inversus went along with abnormal lobation of the liver (nos. A025-07 and A064-11, respectively). The third affected was control fetus A002-09 who had two aorta anomalies. The low incidence and group distribution of the above malformations do not indicate a treatment relationship and three malformations (situs inversus, abnormal lung lobation and dilatation of the aorta) were also in the list of historical control data. Therefore, all were considered to be chance findings.
Only one visceral variation was observed in this study and the single occurrence of this finding in a Group 3 fetus does not indicate any treatment relationship.

Other effects:

no effects observed

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 15 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test item related effects oberved up to the highest dose tested

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

15 000 ppm

Any other information on results incl. tables

Time-mated female Wistar Han rats were treated with ALDEHYDE C 12 MNA PURE from Day 6 to 21 post-coitum, inclusive by dietary administration at dose levels of 1500, 5000 and 15000 ppm. The rats of the control group received the control diet, alone.

Test-diets prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

A lower consumption of the test-diet was observed in the animals at 15000 ppm, and to a lesser extend in the animals at 5000 ppm, over the first day of administration of the test-diets to the animals, i.e. Day 6-7 post-coitum. In the animals at 5000 ppm the test-diet consumption had increased to normal levels over the second day and until the end of treatment on Day 21. In the animals at 15000 ppm the test-diet consumption the test-diet consumption had also increased from the second day of administration onwards, but remained slightly lower over the treatment period when compared to control levels. The overall test-diet consumption in these latter animals was approximately 10% lower than in the other groups, including controls. Despite these effects in food consumption, predominantly in animals at 15000 ppm, no effects were observed in any of the other maternal parameters investigated in this study (i.e. clinical appearance, body weight and macroscopic examination). It was considered that the effects in consumption of the test-diet were related to the taste of the (test item in the) diet rather than signs of toxicity. Furthermore, the absence of changes in body weight in the animals at 15000 ppm, might indicated some nutritional value of the test item to compensate for the slightly lower food consumption.

No toxicologically significant changes were noted in any of the developmental parameters investigated in this study (i.e. fetal body weights, external, visceral (including sex) and skeletal malformations and developmental variations).

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for ALDEHYDE C 12 MNA PURE was established as being 15000 ppm in the test-diet, corresponding to an actual test item intake of approximately 1350 mg/kg/day.