2-methylundecanal

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

No treatment-related adverse effects were observed up to the highest dose level.

Based on the results of the reproduction/developmental toxicity screening test, the following No-Observed-Adverse-Effect Levels (NOAELs) of ALDEHYDE C 12 MNA PURE were established:

Parental NOAEL: at least 15000 ppm

Reproduction NOAEL: at least 15000 ppm

Developmental NOAEL: at least 15000 ppm

15000 ppm in the diet corresponded to mean daily test item intake levels of 1093 (pre-mating period) - 991 (post-mating period) mg/kg bw/day in males, and 1005 (pre-mating period), 1303 (post-coitum period) and 2527 (lactation period) mg/kg bw/day in females.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, 2000

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Identification: ALDEHYDE C 12 MNA PURE
Appearance: Colourless to pale yellow liquid
Stable under storage conditions until: 07 Jan 2019
Chemical name (IUPAC, synonym or trade name): 2-METHYLUNDECANAL
EC number: 203-765-0

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive 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.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Receipt
On 14 Feb 2018, female Crl: WI(Han) rats were received and on 28 Feb 2018, male Crl:WI(Han) rats were received from Charles River Deutschland, Sulzfeld, Germany. At initiation of administration, males were 10 weeks old and weighed between 258 and 299 g and females were 13 weeks old and weighed between 198 and 237 g.
A health inspection was performed before the initiation of administration.

Animal Identification
Prior to start of the pre-test period (females) or treatment period (males), each animal was identified using earmark and tattoo. Prior to the pre-test period, reserve females were numbered R1 through R8 at random by indelible marker.
Pups were identified on postnatal day (PND) 1. They were randomized per litter and individually identified by means of subcutaneous injection of Indian ink. When general hair growth blurred the identification, the pups were identified by tattoo on the feet.

Environmental Acclimation
The animals were allowed to acclimate to the Test Facility toxicology accommodation for 5 days prior to start of the pre-test period (females) or 5 days before the commencement of administration (males).

Selection, Assignment, Replacement, and Disposition of Animals
A total of 40 females was selected at randomization before initiation of the pre-test phase. A total of 40 females with regular estrous cycles continued in the study. The supernumerary females were removed from the study, and their estrous cycle results were kept in the raw data but not reported.
Animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20% of the sex mean. Males and females were randomized separately.

Housing
On arrival and following the pre-test (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam.
The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The room in which the animals were kept was documented in the study records.
Animals were separated during designated procedures/activities.
Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.
The housing conditions were maintained unless deemed inappropriate by the Study Director and/or Clinical Veterinarian.

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 22 to 23°C with an actual daily mean relative humidity of 33 to 53%. A 12-hour light/12-hour dark cycle was maintained, except when interrupted for designated procedures. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food
Prepared powder diets were provided ad libitum throughout the study, except during designated procedures. During the acclimatization period, animals had free access to standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
The feed was analyzed by the supplier for nutritional components and contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
It is 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 is performed, and results of these analyses are on file at the Test Facility.
It is 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. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands), except when interrupted by study procedures/activities.

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

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 1 onwards for a minimum of 28 days. Males were exposed for 29 days, up to and including the day before scheduled necropsy. This included 14 days prior to mating and during the mating period. Females were exposed for 14 days prior to mating (to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and 14-16 days after delivery, up to and including the day of scheduled necropsy (females with offspring: 51-56 days (most females) or 63 days (two females of Group 4); females without offspring: 42 days).

The first day test diets were available to the animals was designated as Day 1.

The amount of test item incorporated in the diet was kept at a constant level in terms of ppm (mg test item/kg diet), 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. Each day the remaining diet was replaced with new diet retained from the freezer and acclimated to room temperature conditions for at least 1 hour prior to opening the diet bag.

Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, from exposure to maternal urine/feces, or spilled diet from the food hopper.

The oral route of administration via dietary inclusion was selected because this is the intended route of human exposure.
The dose levels were selected based on the results of a 90-day study with dietary administration of Aldehyde-C12 MNA Pure in rats (Test Facility Study No. 517127), and in an attempt to produce graded responses to the test item

Details on mating procedure:

Cohabitation/Mating Procedure – F0-Generation
After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.

From the mating period onwards, the following parameters were recorded for each female:
male number paired with, mating date, confirmation of pregnancy and delivery day.
Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a littr is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed.
Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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

Concentration Analysis
Duplicate sets of samples (approximately 5 g) 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) 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.

Duration of treatment / exposure:

Males were exposed for 29 days, up to and including the day before scheduled necropsy. This included 14 days prior to mating and during the mating period. Females were exposed for 14 days prior to mating (to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and 14-16 days after delivery, up to and including the day of scheduled necropsy (females with offspring: 51-56 days (most females) or 63 days (two females of Group 4); females without offspring: 42 days).

Frequency of treatment:

Daily in the diet ad libitum

Dose / conc.:

1 500 ppm

Dose / conc.:

5 000 ppm

Dose / conc.:

15 000 ppm

Remarks:

corresponded to mean daily test item intake levels of 1093 (pre-mating period) - 991 (post-mating period) mg/kg bw/day in males, and 1005 (pre-mating period), 1303 (post-coitum period) and 2527 (lactation period) mg/kg bw/day in females.

No. of animals per sex per dose:

10 rats/sex/dose level

Control animals:

yes, plain diet

Details on study design:

ALDEHYDE C 12 MNA PURE was administered via the diet to male and female Wistar Han rats at dietary concentrations of 1500, 5000 and 15000 ppm (10 rats/sex/dose level).
Concurrent controls (10 rats/sex) received basal diet without test item. Males were treated for two weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for two weeks prior to mating, during mating, during postcoitum, and 14-16 days of lactation (mostly for 51-56 days). Three females without offspring (non-pregnant) were exposed for 42 days.

Positive control:

none

Parental animals: Observations and 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.

Clinical Observations – F0-Generation
Clinical observations were performed once daily, once prior to first administration and at least once daily from start of administration onwards, up to the day prior to necropsy.
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

Body Weights – F0-Generation
Animals were weighed individually on the first day of treatment, and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A terminal weight was recorded on the day of scheduled necropsy (fasted for males, nonfasted for females).

Food Consumption – F0-Generation
Food consumption was quantitatively measured daily throughout the study up to the day prior to scheduled necropsy, except for males and females which were housed together for mating and for females without evidence of mating

Water Consumption – F0-Generation
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was expected or noted at visual inspection of the water bottles.

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus.

Sperm parameters (parental animals):

The organs identified below were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated.
Organs Weighed at Necropsy: Epididymis, prostate, seminal vesicle, Testes

The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin: Epididymis, thyroid gland, ovaries and testes.
All tissues were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology.
For the testes of all males of Groups 1 and 4, and the Group 2 male that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
A peer review on the histopathology data was performed by a second pathologist.

Litter observations:

Mortality/Moribundity Checks – F1-Generation
Pups were observed daily for general health/mortality. The number of live and dead pups were determined on PND 1 and daily thereafter. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations – F1-Generation
Clinical observations were performed at least once daily for all pups. Only days on which clinical signs were present between the first and last litter check are given in the respective report tables.

Body Weights – F1-Generation
Live pups were weighed individually on PND 1, 4, 7 and 13.

Sex – F1-Generation
Sex was externally determined for all pups on PND 1 and 4.

Anogenital Distance – F1-Generation
Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.

Areola/Nipple Retention – F1-Generation
All male pups in each litter were examined for the number of areola/nipples on PND 13.

Culling – F1-Generation
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

Postmortem examinations (parental animals):

Necropsy – F0-Generation
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
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.
The numbers of former implantation sites were recorded for all paired females.
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 and the number of corpora lutea was recorded in addition.

Organ Weights – F0-Generation
The organs identified in the table below were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated.

Tissue Collection and Preservation – F0-Generation
Representative samples of the tissues identified in the table below were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated

Histopathology – F0-Generation
Tissues were examined by a board-certified toxicological pathologist with training and experience in laboratory animalpathology.
For the testes of all males of Groups 1 and 4, and the Group 2 male that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
A peer review on the histopathology data was performed by a second pathologist.

Postmortem examinations (offspring):

Unscheduled Deaths - F1-Generation
The single pup (Group 4, litter no. 79) that died before scheduled termination were was examined externally and sexed (both externally and internally). The stomach of this pup was examined for the presence of milk.

Scheduled Euthanasia - F1-Generation
On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible. All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.
In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation.

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 comparison 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

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs were noted during the observation period.
Findings were limited to alopecia in two females (treated at 5000 or 15000 ppm). These incidental findings occurred within the range of background findings in rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these findings were regarded as unrelated to treatment.

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:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights and body weight gain were not affected by treatment.
An isolated and slight, but statistically significant difference from controls noted in females at 15000 ppm (higher mean body weight on post-coitum Day 14) was regarded as unrelated to treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Females at 15000 ppm consumed less food than controls (absolute and relative to body weight) from Day 2 of the lactation period onwards. The differences were statistically significant on several occasions and amounted to about 15 and 20% for absolute and relative food consumption, respectively. During the pre-mating and gestation periods, food consumption of 15000 ppm females was comparable to that of controls.
No treatment-related changes in food consumption (absolute or relative to body weight) were observed in males treated up to 15000 ppm and females treated up to 5000 ppm.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related microscopic observations.
All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Reproductive performance
One control female (no. 49, mated with male no. 09), one female at 1500 ppm (no. 54, mated with male no. 14) and one female at 15000 ppm (no. 71, mated with male no. 31) were not pregnant despite evidence of mating. Histopathology did not reveal any changes in the reproductive organs of these animals that could explain their lack of pregnancy.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item, and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid Hormone
Serum levels of T4 in F0-males were considered not to be affected by treatment.
The statistically significantly lower mean T4 value noted at 5000 ppm was regarded as unrelated to treatment due to the lack of a dose-related trend.

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to be affected by treatment.
All treated females had regular cycles, generally of four days.
From start mating until positive pairing continuous di-estrus was noted in two females at 15000 ppm (nos. 78 and 79). The disturbance from their regular cyclicity that was observed during the pre-test and pre-mating period was considered to have occurred as a results of the change in circumstances at start mating and not reflecting an effect of the test item.
One control female (no. 47) showed an irregular cycle at the end of the pre-mating period. She had a normal precoital interval (evidence of mating after one day of cohabitation) and a delivered a healthy litter.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Mating Index
Mating index was not affected by treatment. All females showed evidence of mating (mating index 100% for all groups).

Precoital Time
Precoital time was considered not to be affected by treatment. Most females showed evidence of mating within five days.

Number of Implantation Sites
Number of implantation sites was not affected by treatment.

Fertility Index
Fertility index was not affected by treatment. Except for one control female, one 1500 ppm female and one 15000 ppm female (nos. 49, 54 and 71, respectively), all females were pregnant. Fertility indices were 100% for the 5000 ppm group and 90% for the other groups.
The non-pregnancies, without related histopathology changes in reproductive organs, were regarded as unrelated to treatment due to the incidental occurrence and lack of a dose-related
trend.

Parental results
No parental toxicity was observed up to the highest dose level tested (15000 ppm).
The only treatment-related finding in parental rats was a reduction in food consumption of females treated at 15000 ppm from Day 2 of the lactation period onwards. The magnitude of this effect was modest (about 15% difference from control values) and body weight (gain) of 15000 females was similar to that of controls throughout the study. Therefore, the lower food consumption in the lactation period was regarded as non-adverse.
There were no treatment-related changes in the other parameters examined (i.e. survival, clinical appearance, body weight (gain), serum level of thyroid hormone T4 (males), organ weights (thyroid in both sexes and male reproductive organs), macroscopic examination, and microscopic examination of the thyroid and reproductive organs).

Reproductive results
No reproduction toxicity was observed up to the highest dose level tested (15000 ppm).
No treatment-related changes were noted in any of reproductive parameters examined (i.e. mating and fertility indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

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 observed up to the highest dose tested

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment.
The clinical signs observed incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Live Birth Index:
No pups were found dead at first litter check. Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was 100% for all groups.

Viability Index:
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. Viability indices were 99% for the 15000 ppm group and 100% for the other groups.
The incidental death of one pup at 15000 ppm (litter no. 79, pup found dead on PND 1) was unrelated to treatment.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights of pups were considered not to be adversely affected by treatment.
It was noted that mean weights of male and female pups at 15000 ppm were slightly (6-9%) lower than those of control pups from PND 1 onwards. Statistical significance was not achieved and the magnitude of the differences did not increase in the course of the lactation period. Therefore, the lower pup body weights noted at 15000 ppm were considered not to reflect an adverse effect of the test item.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Serum T4 levels in male and female PND 14-16 pups were not affected by treatment.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment up to 15000 ppm had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of the few findings noted remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Gestation Index and Duration
Gestation index and duration of gestation were not affected by treatment. All pregnant females had live offspring (gestation index 100% for all groups).

Parturition/Maternal Care
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Post-Implantation Survival Index
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was considered not to be affected by treatment. The survival indices were 91, 93, 90 and 89% for the control, 1500, 5000 and 15000 ppm groups, respectively.

Litter Size
The number of living pups at first litter check (live litter size) was not affected by treatment.
Mean live litter sizes were 10.7, 10.7, 10.0 and 11.4 for the control, 1500, 5000 and 15000 ppm groups, respectively

Live Birth Index
No pups were found dead at first litter check. Live birth index (number of live offspring on
PND 1 as percentage of total number of offspring born) was 100% for all groups.

percentage of total number of offspring born) was 100% for all groups.

Viability Index
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. Viability indices were 99% for the 15000 ppm group and 100% for the other groups.
The incidental death of one pup at 15000 ppm (litter no. 79, pup found dead on PND 1) was unrelated to treatment.

Lactation Index
Lactation index (number of live offspring on PND 13 as percentage of number of live offspring after culling on PND 4) was not affected by treatment. No pups died after PND 4, resulting in a lactation index of 100% for all groups.

Sex Ratio
Sex ratio was considered not to be affected by treatment.

Anogenital Distance
Anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment.

Areola/Nipple Retention
Treatment up to 15000 ppm had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Macroscopy
No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of the few findings noted remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

No developmental toxicity was observed up to the highest dose level tested (15000 ppm).
No treatment-related or toxicologically relevant changes were noted in the developmental parameters examined (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, live litter size, maternal care, clinical signs, body weight, anogenital distance (PND 1), areola/nipple retention (PND 13 males), serum level of T4 thyroid hormone (PND 14-16), and macroscopic examination.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

15 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no test item related effect up to the highest dose tested

Key result

Critical effects observed:

no

Reproductive effects observed:

no

Lowest effective dose / conc.:

15 000 ppm

Treatment related:

no

Conclusions:

In conclusion, based on the results of this reproduction/developmental toxicity screening test, the following No-Observed-Adverse-Effect Levels (NOAELs) of ALDEHYDE C 12 MNA PURE were established:
Parental NOAEL: at least 15000 ppm
Reproduction NOAEL: at least 15000 ppm
Developmental NOAEL: at least 15000 ppm

15000 ppm in the diet corresponded to mean daily test item intake levels of 1093 (pre-mating period) - 991 (post-mating period) mg/kg bw/day in males, and 1005 (pre-mating period), 1303 (post-coitum period) and 2527 (lactation period) mg/kg bw/day in females.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 093 mg/kg bw/day

Species:

rat

Quality of whole database:

GLP and OECD Guideline compliant study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

No treatment-related adverse effects were observed up to the highest dose level.

Based on the results in the 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.

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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

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.

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

Dose / conc.:

0 ppm

Dose / conc.:

1 500 ppm

Dose / conc.:

5 000 ppm

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.

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

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

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

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

Key result

Abnormalities:

no effects observed

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

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

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

15 000 ppm

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).

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 350 mg/kg bw/day

Species:

rat

Quality of whole database:

GLP and Guideline compliant study

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Aldehyde C12 MNA was assessed for reproductive and pre-natal oral toxicity according to OECD 421 and 414 respectively. The No Observed Adverse Effect Level (NOAEL) for the rat was considered to be 15000 ppm,corresponding to dose levels of 1093 mg/kg (reproductive toxicity study) and 1350 mg/kg (pre-natal study).

Based on these results, no classification is required for reproductive or developmental toxicity.